Alkyne compounds, vitamin D compounds, analytical methods, and manufacturing methods

Abstract

To provide a compound useful for the analysis of a vitamin D compound.SOLUTION: For example, an alkyne compound 10 prepared by the following reaction is illustrated.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an alkyne compound and a vitamin D compound. The present invention also relates to an analytical method using the vitamin D compound and a method for producing the vitamin D compound. [Background technology]

[0002] There is a great demand for detection and quantification of vitamin D compounds in living organisms in clinical and food nutritional fields. For example, detection and quantification of vitamin D metabolites in biological samples such as blood, urine, body fluids, and cerebrospinal fluid have been performed. Several techniques for the analysis of vitamin D compounds have been proposed.

[0003] For example, Non-Patent Document 1 below describes the synthesis of deuterated analogs of 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3. Also, Non-Patent Document 2 below describes the differential diagnosis of vitamin D-associated hypercalcemia using serum vitamin D metabolite profiling. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Rahul Ray et al., Synthesis of 25-hydroxy-[6,19,19'-2H3vitamin D3 and 1α,25-dihydroxy-[6,19'-2H3]vitamin D3, Steroids, 1992, 57, 142-146. [Non-Patent Document 2] Martin Kaufmann et al., Differential diagnosis of vitamin D-related hypercalcemia using serum vitamin D metabolite profiling, J. Bone Miner. Res., 2021, 36, 1340-1350. [Non-Patent Document 3] Aileen Mendoza et al., Controlled lipid β-oxidation and carnitine biosynthesis by a vitamin D metabolite, Cell Chem. Biol., 28, Published: September 09, 2021 [Non-Patent Document 4] Masahiko Seki et al., A novel caged Cookson-type reagent toward a practical vitamin D derivatization method for mass spectrometric analyses, Rapid Commun. Mass Spectrom., 2020, 34, e8648. Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide compounds useful for the analysis of vitamin D compounds. [Means for solving the problem]

[0006] The present inventors have found that a specific vitamin D compound labeled with a stable isotope is useful for the analysis of vitamin D metabolites, and that the vitamin D compound can be produced by a specific alkyne compound.

[0007] That is, the present invention provides the following. [1] An alkyne compound represented by the following formula (100): [C1-1] JPEG0007636752000001.jpg8474 [In formula (100), A represents a linear carbon chain having an alkynyl group and a vinyl group at each of its two ends, X 1 CH or13 CH, X 21 , X 22 , X 23 , and X 24 are, independently of each other, CHY, CDY, 13 C.H.Y., 13 C.D.Y., C.O. 13 CO, C 18 O, and 13 C 18 O, Y is H, D, NH 2 , 15 NH 2 , O.H., 18 OH, SH, OR, O(CO)R, OSO 3 H, O.S.O. 3 Na, and a sugar substituent, and when two or more Y's are included in formula (100), the Y's may be the same or different from each other; R is an alkyl or alkenyl group. ,a Reel group or an alcohol protecting group selected from t-butyldimethylsilyl, triisopropylsilyl, and triethylsilyl and X 3 is C or 13 C, X 1 , X 21 , X 22 , X 23 , X 24 , and X 3 At least one of the following is a stable isotope: D, 13 C. 18 O, or 15 It is modified by N, The sugar substituent has the structure: [Case 1-2] JPEG0007636752000002.jpg52129 With regard to Wo in the sugar substituent represented by this structure, W represents a substituent of the ring carbon of the sugar, and o represents the number of the substituents and is an integer of 3 to 5. W is H, OH, CH 2 OH, COOH, NH 2 and NHAc, which may be the same or different from each other. (However, the following alkyne compounds: [Case 1-3] (Except for JPEG0007636752000003.jpg6058.) [2] An alkyne compound represented by the following formula (100): [Case 2-1] JPEG0007636752000004.jpg8474 [In formula (100), A represents a linear carbon chain having an alkynyl group and a vinyl group at each of its two ends, X 1 CH or 13 CH, X 21 , X 22 , X 23 , and X 24 are, independently of each other, CHY, CDY, 13 C.H.Y., 13 C.D.Y., C.O. 13 CO, C 18 O, and 13 C 18 O, Y is H, D, NH 2 , 15 NH 2 , O.H., 18 OH, SH, OR, O(CO)R, OSO 3 H, O.S.O. 3 Na, and a sugar substituent, and when two or more Y's are included in formula (100), the Y's may be the same or different from each other; Where X 21 , X 22 , X 23 , and X 24 At least one of the following is CHY, CDY, 13 CHY, and 13 CDY, where Y is NH 2 , 15 NH 2 , O.H., 18 OH, SH, OR, O(CO)R, OSO 3 H, O.S.O. 3 Na, and a sugar substituent; R is an alkyl or alkenyl group. ,a Reel group or an alcohol protecting group selected from t-butyldimethylsilyl, triisopropylsilyl, and triethylsilyl and X 3 is C or 13 C, X 1 , X 21 , X 22 , X 23 , X 24 , and X 3 At least one of the following is a stable isotope: D, 13 C. 18 O, or 15 It is modified by N, The sugar substituent has the structure: [Case 2-2] JPEG0007636752000005.jpg52129With regard to Wo in the sugar substituent represented by this structure, W represents a substituent of the ring carbon of the sugar, and o represents the number of the substituents and is an integer of 3 to 5. W is H, OH, CH 2 OH, COOH, NH 2 and NHAc, which may be the same or different from each other. [3] In equation (100), X 21 , X 22 , X 23 , and X 24 At least one of the above is CHY or CDY. [4] A vitamin D compound represented by the following formula (200): [C3-1] JPEG0007636752000006.jpg8963 [In formula (200), A' represents a cyclic carbon chain corresponding to the A ring of vitamin D compounds; X' 1 is C or 13 C, X' 21 , X' 22 , X' 23 , and X' 24 are, independently of each other, CHY', CDY', 13 CHY', 13 C.D.Y.', C.O. 13CO, C 18 O, and 13 C 18 O, Y' is H, D, NH 2 , 15 NH 2 , O.H., 18 OH, SH, OR', O(CO)R', OSO 3 H, O.S.O. 3 Na, and a sugar substituent, and when two or more Y' are included in formula (200), Y' may be the same or different from each other; R' is an alkyl group or an alkenyl group. ,a Reel group or an alcohol protecting group selected from t-butyldimethylsilyl, triisopropylsilyl, and triethylsilyl and X' 3 is C or 13 C, X' 1 , X' 21 , X' 22 , X' 23 , X' 24 , and X' 3 At least one of the following is a stable isotope: D, 13 C. 18 O, or 15 It is modified by N, P is a C substituted or unsubstituted with a hydroxy group. 1 ~C 10 C substituted or unsubstituted with alkyl or hydroxy groups 2 ~C 10 an alkenyl group, wherein the alkyl group or the alkenyl group may have a lactone group substituted with a hydroxy group; The sugar substituent has the structure: [C3-2] JPEG0007636752000007.jpg52129 With regard to Wo in the sugar substituent represented by this structure, W represents a substituent of the ring carbon of the sugar, and o represents the number of the substituents and is an integer of 3 to 5. W is H, OH, CH 2 OH, COOH, NH 2and NHAc, which may be the same or different from each other. (However, the following vitamin D compounds: [C3-3] (Except for JPEG0007636752000008.jpg6888.) [5] A vitamin D compound represented by the following formula (200): [C4-1] JPEG0007636752000009.jpg8963 [In formula (200), A' represents a cyclic carbon chain corresponding to the A ring of vitamin D compounds; X' 1 is C or 13 C, X' 21 , X' 22 , X' 23 , and X' 24 are, independently of each other, CHY', CDY', 13 CHY', 13 C.D.Y.', C.O. 13 CO, C 18 O, and 13 C 18 O, Y' is H, D, NH 2 , 15 NH 2 , O.H., 18 OH, SH, OR', O(CO)R', OSO 3 H, O.S.O. 3 Na, and a sugar substituent, and when two or more Y' are included in formula (200), Y' may be the same or different from each other; R' is an alkyl group or an alkenyl group. ,a Reel group or an alcohol protecting group selected from t-butyldimethylsilyl, triisopropylsilyl, and triethylsilyl and X' 3 is C or 13 C, X' 1 , X' 21 , X' 22 , X' 23 , X' 24 , and X' 3 At least one of the following is a stable isotope: D,13 C. 18 O, or 15 N, where X' 21 , X' 22 , X' 23 , and X' 24 At least one of the isotopes is stable 13 C. 18 O, or 15 Modified with N or X' 21 , X' 22 , X' 23 and X' 24 At least two of X' are modified with a stable isotope D, or 21 , X' 22 , X' 23 , and X' 24 At least one of the following is CDD: P is a C substituted or unsubstituted with a hydroxy group. 1 ~C 10 C substituted or unsubstituted with alkyl or hydroxy groups 2 ~C 10 an alkenyl group, wherein the alkyl group or the alkenyl group may have a lactone group substituted with a hydroxy group; The sugar substituent has the structure: [C4-2] JPEG0007636752000010.jpg52129With regard to Wo in the sugar substituent represented by this structure, W represents a substituent of the ring carbon of the sugar, and o represents the number of the substituents and is an integer of 3 to 5. W is H, OH, CH 2 OH, COOH, NH 2 and NHAc, which may be the same or different from each other. [6] The vitamin D compound according to the above [3], wherein in formula (200), P has any one of the following structures: [C5] JPEG0007636752000011.jpg33170 [In the structure relating to P, Q, Q', Q'', Q''', and Q'''' are each independently H, CH 2 , C.H. 3 , C 2 H 5 , O, OH, and sugar substituents. [7] An analytical method using the vitamin D compound described in [4] or [5] above. [8] The method of analysis includes analyzing the sample by mass spectrometry; In the analysis, the vitamin D compound is used as an internal standard. The analytical method described in [7] above. [9] The analytical method according to [8], wherein the mass spectrometry is LC-MS / MS or LC / MS.

[10] The method according to [7] above, wherein vitamin D compounds not having a hydroxy group at position 1 of the A ring are analyzed separately from vitamin D compounds having a hydroxy group at position 1 of the A ring.

[11] The method for analyzing according to

[10] above, wherein the vitamin D compound not having a hydroxy group at position 1 of the A ring includes at least one of a 2-hydroxyvitamin D compound and a 4-hydroxyvitamin D compound.

[12] The method for analysis according to

[10] above, wherein the vitamin D compound not having a hydroxy group at position 1 of the A ring includes at least one of a 4β,25-dihydroxyvitamin D3 compound and a 4-hydroxyvitamin D compound.

[13] A method for producing a vitamin D compound according to the above [4] or [5], wherein an alkyne compound represented by the following formula (100) is used as a material for forming the A ring of the vitamin D compound. [C6-1] JPEG0007636752000012.jpg8474 [In formula (100), A represents a linear carbon chain having an alkynyl group and a vinyl group at each of its two ends, X 1 CH or 13 CH, X 21 , X 22 , X 23, and X 24 are, independently of each other, CHY, CDY, 13 C.H.Y., 13 C.D.Y., C.O. 13 CO, C 18 O, and 13 C 18 O, Y is H, D, NH 2 , 15 NH 2 , O.H., 18 OH, SH, OR, O(CO)R, OSO 3 H, O.S.O. 3 Na, and a sugar substituent, and when two or more Y's are included in formula (100), the Y's may be the same or different from each other; Where X 21 , X 22 , X 23 , and X 24 At least one of the following is CHY, CDY, 13 CHY, and 13 CDY, where Y is NH 2 , 15 NH 2 , O.H., 18 OH, SH, OR, O(CO)R, OSO 3 H, O.S.O. 3 Na, and a sugar substituent; R is an alkyl or alkenyl group. ,a Reel group or an alcohol protecting group selected from t-butyldimethylsilyl, triisopropylsilyl, and triethylsilyl and X 3 is C or 13 C, X 1 , X 21 , X 22 , X 23 , X 24 , and X 3 At least one of the following is a stable isotope: D, 13 C. 18 O, or 15 It is modified by N, The sugar substituent has the structure: [C6-2] JPEG0007636752000013.jpg52129With regard to Wo in the sugar substituent represented by this structure, W represents a substituent of the ring carbon of the sugar, and o represents the number of the substituents and is an integer of 3 to 5. W is H, OH, CH 2 OH, COOH, NH 2 and NHAc, which may be the same or different from each other. Effect of the Invention

[0008] The vitamin D compound according to the present invention can be used to detect and / or quantify vitamin D metabolites, and the vitamin D compound can be produced by the alkyne compound according to the present invention. The effects of the present invention are not limited to those described herein, and may be any of the effects described in this specification. [Brief description of the drawings]

[0009] [Figure 1A] FIG. 1 shows NMR spectrum data. [Figure 1B] FIG. 1 shows NMR spectrum data. [Figure 1C] FIG. 1 shows NMR spectrum data. [Figure 1D] FIG. 1 shows NMR spectrum data. [Figure 1E] FIG. 1 shows NMR spectrum data. [Figure 1F] FIG. 1 shows NMR spectrum data. [Figure 1G] FIG. 1 shows NMR spectrum data. [Figure 1H] FIG. 1 shows NMR spectrum data. [Figure 2A] This is an SRM chromatogram of a 25OHD3-26R,23S-lactone-d3 preparation. [Figure 2B] This is an SRM chromatogram of a 1α,25(OH)2D3-26R,23S-lactone-d3 preparation. [Figure 2C] This is a calibration curve for 25OHD3-26R,23S-lactone standard. [Figure 2D] This is a calibration curve for 1α,25(OH)2D3-26R,23S-lactone standard. [Figure 2E] SRM chromatogram of 25OHD3-26R,23S-lactone in pooled serum. [Figure 2F] SRM chromatogram of 1α,25(OH)2D3-26R,23S-lactone in pooled serum. [Figure 2G] FIG. 2 shows a reaction scheme for derivatization. [Figure 2H] FIG. 2 shows a reaction scheme for derivatization. [Figure 3A] This is a calibration curve for 25OHD3 standard. [Figure 3B] This is a calibration curve for the 24R,25(OH)2D3 standard. [Figure 3C] SRM chromatogram of 25OHD3 in pooled serum. [Figure 3D] SRM chromatogram of 24R,25(OH)2D3 in pooled serum. [Figure 4A] The SRM chromatogram of the antibody column non-adsorbed fraction is shown. [Figure 4B] The SRM chromatogram of the antibody column adsorption fraction is shown. [Figure 5A] The calibration curve for 4β,25-(OH)2D3 standard is shown. [Figure 5B] 1 shows an SRM chromatogram of the antibody column non-adsorbed fraction of pooled serum. [Figure 5C] The SRM chromatogram of the antibody column adsorption fraction of pooled serum is shown. [Figure 6] FIG. 1 is a diagram illustrating derivatization of vitamin D compounds and cleavage of the derivatives. [Figure 7] FIG. 1 shows the chemical structures of vitamin D compounds having a hydroxy group at position 1 of the A ring and vitamin D compounds not having a hydroxy group at position 1 of the A ring. [Figure 8]FIG. 1 shows the chemical structures of 1α,25-dihydroxyvitamin D3 and 4β,25-dihydroxyvitamin D3. [Figure 9A] An example of a chemical reaction scheme for producing the vitamin D compound of the present invention is shown below. [Figure 9B] An example of a chemical reaction scheme for producing the vitamin D compound of the present invention is shown below. [Figure 10] FIG. 1 shows examples of vitamin D compounds. [Figure 11] FIG. 1 shows examples of compounds used to produce the alkyne compound of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The following describes preferred embodiments of the present invention. However, the present invention is not limited to the following preferred embodiments and can be freely modified within the scope of the present invention. The present invention will be described in the following order. 1. Description of the Invention 2. First embodiment (alkyne compound) 2-1. Structure of the alkyne compound of formula (I) 2-2. Structure of the alkyne compound of formula (100) 2-3. How to use alkyne compounds 2-4. Method for producing alkyne compounds 3. Second embodiment (vitamin D compounds) 3-1. Structure of Vitamin D Compound of Formula (III) 3-2. Structure of Vitamin D Compound of Formula (200) 3-3. How to Use Vitamin D Compounds 3-4. Manufacturing method of vitamin D compounds 4. Third embodiment (analysis method using vitamin D compound) 4-1. Examples of analysis methods 4-2. Modifications of analysis method 5. Fourth embodiment (method of producing vitamin D compound) 6. Working Example 6-1. Example 1: Preparation of alkyne compounds and vitamin D compounds 6-2. Example 2: 25OHD in pooled serum 3 -26R,23S-lactone and 1α,25(OH) 2 D 3 LC-MS / MS analysis of -26R,23S-lactone 6-3. Example 3: 25OHD in pooled serum 3 , 24R,25(OH) 2 D 3 LC-MS / MS analysis of 6-4. Example 4: 4β,25-(OH) 2 D 3 Separation and measurement of 6-5. Example 5: 4β,25-(OH) in pooled serum 2 D 3 LC-MS / MS analysis of

[0011] 1. Description of the Invention

[0012] Various vitamin D metabolites have been discovered so far. For example, 1α,25(OH) is one of the most important vitamin D metabolites in terms of biological activity. 2 D 3 This is also called active vitamin D. Another example of vitamin D metabolites is vitamin D lactone, which has been reported to inhibit the metabolism of fatty acids (Non-Patent Document 3). Subtle structural changes in the side chain structure of vitamin D metabolites can affect the physiological activity of vitamin D metabolites. To clarify the various physiological activities of each vitamin D metabolite, it is necessary to accurately quantify each vitamin D metabolite.

[0013] 1α,25(OH) 2 D 3Currently, vitamin D is quantified by immunochemical measurement, but it is difficult to individually detect or quantify other trace vitamin D metabolites by immunochemical measurement. LC-MS / MS using a derivatization reagent is expected to be a method for detecting or quantifying vitamin D metabolites with high sensitivity (Non-Patent Document 4 mentioned above). For detection or quantification by LC-MS / MS, vitamin D metabolites labeled with stable isotopes are essential.

[0014] As described in Non-Patent Document 1, a technique for labeling with deuterium by HD exchange using heavy water is known. However, while a vitamin D compound labeled by this method is stored in a solution, HD exchange with light water in the solvent may occur. In other words, vitamin D compounds labeled with deuterium by HD exchange have storage stability problems.

[0015] Vitamin D compounds have an A ring, a seco-B ring, a C ring, and a D ring (herein, the C ring and the D ring are collectively referred to as "CD ring"). The CD ring has different side chains depending on the type of vitamin D compound, i.e., it has structural diversity. The A ring portion of vitamin D compounds also has structural diversity, but the structural diversity is smaller than that of the CD ring side chain. The present invention provides an alkyne compound labeled with a stable isotope. The alkyne compound can be used as a substance for forming the A ring portion. The vitamin D compound produced using the alkyne compound has its A ring portion labeled with a stable isotope. Since a stable isotope is introduced into the A ring portion by using the alkyne compound, various vitamin D metabolites labeled with a stable isotope can be produced without introducing a stable isotope into the CD ring side chain. Here, as described above, the structural diversity of the A ring portion of the vitamin D compound is smaller than that of the CD ring side chain. In other words, various vitamin D compounds can be produced by using fewer variations of alkyne compounds. Therefore, various vitamin D compounds labeled with a stable isotope can be efficiently produced by the alkyne compound according to the present invention.

[0016] Furthermore, the vitamin D compound according to the present invention can be produced by using the alkyne compound according to the present invention as a material for forming the ring A. The vitamin D compound does not need to be a compound labeled by HD exchange using heavy water, and therefore has excellent storage stability.

[0017] The vitamin D compounds according to the present invention can be used in various analytical methods, particularly mass spectrometry, more particularly isotope dilution mass spectrometry. That is, the present invention also provides an analytical method using the vitamin D compounds. In one embodiment, the present invention provides an analytical method comprising detecting or quantifying vitamin D metabolites. The detection or quantification may be performed by a mass spectrometer, particularly a liquid chromatography mass spectrometer, even more particularly LC-MS / MS. The vitamin D compounds according to the present invention can be used regardless of the manufacturer of the mass spectrometer.

[0018] In addition, the vitamin D compound according to the present invention can be used, for example, in the field of clinical testing or food testing. There is a great demand for measuring vitamin D metabolites in the field of clinical testing or food testing. It is considered that the present invention can efficiently produce stable isotope-labeled compounds not only for currently known vitamin D metabolites, but also for vitamin D metabolites that may be discovered in the future. Therefore, the present invention also contributes to the rapid construction of a measuring environment for various vitamin D metabolites.

[0019] The invention will now be described in more detail.

[0020] 2. First embodiment (alkyne compound)

[0021] The present invention provides an alkyne compound represented by the following formula (I). The alkyne compound is labeled with a stable isotope as described below. The alkyne compound can be used to produce various vitamin D compounds, and more specifically, the alkyne compound may be used as a compound forming the A ring portion of various vitamin D compounds. Since the alkyne compound is labeled with a stable isotope, various vitamin D compounds labeled with a stable isotope can be produced by the alkyne compound. The vitamin D compound produced by using the alkyne compound has excellent storage stability.

[0022] 2-1. Structure of the alkyne compound of formula (I)

[0023] The alkyne compound of formula (I) is described below. [ka]

[0024] In formula (I), A represents a linear carbon chain having an alkynyl group and a vinyl group at each of the two ends. That is, the alkyne compound is a compound having an enyne structure. Therefore, it can be said that the present invention provides an enyne compound represented by formula (I). It should be noted that A is merely a symbol given for the sake of convenience in describing the constituent parts of the vitamin D compound described below, and does not represent the actual elements or other entities that constitute the alkyne compound of formula (I).

[0025] In formula (I), X 1 CH or 13 CH. That is, X 1 forms the alkene portion of the enyne structure.

[0026] X 1 When is CH, the one or more X 2 and X 3 At least one of the following is a stable isotope: D, 13 C. 18 O, or15 It is modified by N. X 1 but 13 If CH, the above 1 or more X 2 and X 3 None of the X may be modified with a stable isotope, or even in this case, the one or more X 2 and X 3 At least one of the following is a stable isotope: D, 13 C. 18 O, or 15 It may be modified by N.

[0027] In formula (I), X 2 CHY, CDY, 13 C.H.Y., 13 C.D.Y., C.O. 13 CO, C 18 O, and 13 C 18 m is any one of X, O, and the alkyne compound. 2 m is an integer of 1 to 4. The alkyne compound of formula (I) is a compound having m X's connected in a straight chain. 2 , X 1 and X 3 It is something that exists between. When m is 2 or more, X 2 may be the same or different from each other. That is, the alkyne compound has two or more X 2 In the case where the two or more X 2 CHY, CDY, 13 C.H.Y., 13 C.D.Y., C.O. 13 CO, C 18 O, and 13 C 18 O may be selected independently of each other. Preferably, m is 2, 3, or 4, more preferably, m is 3 or 4, and even more preferably, m is 4. The embodiment where m is 4 is further described below with reference to formula (100).

[0028] The X that is 1 or more2 At least one of the following is a stable isotope: D, 13 C. 18 O, or 15 When modified by N, the X 1 and X 3 None of the X may be modified with a stable isotope, or even in this case, 1 and X 3 Either or both of the above may be modified with a stable isotope. The X that is 1 or more 2 Both are stable isotopes D, 13 C. 18 O, or 15 When not modified by N, the X 1 and X 3 Either or both may be modified with a stable isotope.

[0029] In formula (I), Y is H, D, NH 2 , 15 NH 2 , O.H., 18 OH, SH, OR, O(CO)R, OSO 3 H, O.S.O. 3 Na, and a sugar substituent. When two or more Ys are included in formula (I), the Ys may be the same or different from each other. That is, when the alkyne compound has two or more Ys, the two or more Ys are H, D, NH 2 , 15 NH 2 , O.H., 18 OH, SH, OR, O(CO)R, OSO 3 H, O.S.O. 3 The substituents may be independently selected from among Na, Na, and sugar substituents.

[0030] In formula (I), R represents an alcohol protecting group, an alkyl group, an alkenyl group, or an aryl group. When two or more R are included in formula (I), R may be the same or different from each other. That is, when the alkyne compound has two or more R, the two or more R may be independently selected from an alcohol protecting group, an alkyl group, an alkenyl group, and an aryl group.

[0031] The alcohol protecting group of the alkyne compound as R may be, for example, a silyl alcohol protecting group or a benzyl alcohol protecting group. Examples of the silyl alcohol protecting group include, but are not limited to, t-butyldimethylsilyl (also referred to as TBS or TBDMS), triisopropylsilyl (also referred to as TIPS), and triethylsilyl (also referred to as TES). Examples of the benzyl alcohol protecting group include, but are not limited to, p-methoxybenzyl (also referred to as PMB), benzyloxymethyl (also referred to as BOM), and benzyl (also referred to as Bn). The structures of these exemplary alcohol protecting groups are shown in Table 1 below.

[0032] [Table 1]

[0033] The alkyl group that the alkyne compound has as R may be a substituted or unsubstituted linear or branched alkyl group having 1 to 10 carbon atoms. The number of carbon atoms in the linear or branched alkyl group may be, for example, 1 to 8, 1 to 6, or 1 to 4. The carbon number does not include the number of carbon atoms of the substituent. The alkenyl group contained in the alkyne compound as R may be a linear or branched alkyl group having 2 to 10 carbon atoms. The number of carbon atoms in the linear or branched alkenyl group may be, for example, 2 to 8, 2 to 6, or 2 to 4. The carbon number does not include the number of carbon atoms of a substituent. The aryl group contained as R in the alkyne compound may be a substituted or unsubstituted aryl group having 6 to 20 carbon atoms. The number of carbon atoms in the aryl group is preferably 6 to 18, and may be, for example, 6 to 16, 6 to 14, 6 to 12, or 6 to 10. The number of carbon atoms does not include the number of carbon atoms of the substituent. The aryl group may be an unsubstituted aryl group, and may be, for example, a phenyl group or a naphthyl group.

[0034] In formula (I), the sugar substituent may, for example, have the structure: [ka] With respect to Wo in the sugar substituent represented by this structure, W represents a substituent of the ring carbon of the sugar, and o represents the number of the substituents and is an integer of 3 to 5. W is H, OH, CH 2 OH, COOH, NH 2 , and NHAc, which may be the same or different from each other. That is, the 3 to 5 Ws of the sugar substituent are each independently H, OH, CH 2 OH, COOH, NH 2 , and NHAc.

[0035] In one embodiment, the one or more X 2 At least one of the following is CHY, CDY, 13 CHY, and 13 For example, one or more of X 2 All of these are CHY, CDY, 13 CHY, and 13 It may be either CDY. In this embodiment, preferably, Y may represent any of H, D, OH, and OR, for example, any of H, D, and OR. For example, when two or more Y are included in the alkyne compound, the two or more Y may be independently selected from H, D, OH, and OR.

[0036] In a particularly preferred embodiment, the one or more X 2 At least one of the X is CHY or CDY, for example, 2 Any of the above may be CHY or CDY. In this embodiment, preferably, Y may represent any of H, D, OH, and OR, for example, any of H, D, and OR. For example, when two or more Y are included in the alkyne compound, the two or more Y may be independently selected from H, D, and OR. In this embodiment, preferably, the one or more X 2 comprises at least one CDD and at least one CDOR, where the at least one CDOR may be CDO-(alcohol protecting group), where the alcohol protecting group is as described above.

[0037] In formula (I), X 3 is C or 13 C. That is, X 3 forms the alkyne portion of the enyne structure.

[0038] In formula (I), the X 1 , the 1 or more X 2 and the X 3 At least one of the following is a stable isotope: D, 13 C. 18 O, or 15 That is, the alkyne compound of formula (I) is a compound having at least one stable isotope, and the at least one stable isotope is the X 1 , the 1 or more X 2 and the X 3 may be included in one or more of the following:

[0039] 2-2. Structure of the alkyne compound of formula (100)

[0040] In a particularly preferred embodiment, in formula (I), m is 4. In this embodiment, the alkyne compound may be a compound represented by the following formula (100). The compound represented by formula (100) is described below. [ka]

[0041] In formula (100), A represents a linear carbon chain having an alkynyl group and a vinyl group at each of its two ends.

[0042] In formula (100), X 1 CH or 13 It may be CH.

[0043] X 1 When is CH, the X 21 , X 22 , X 23 , and X 24 And the above X 3 At least one of the following is a stable isotope: D, 13 C. 18 O, or 15 It is modified by N. X 1 but 13 When the X is CH, 21 , X 22 , X 23 , and X 24 And the above X 3 None of the X may be modified with a stable isotope, or even in this case, 21 , X 22 , X 23 , and X 24 And the above X 3 At least one of the following is a stable isotope: D, 13 C. 18 O, or 15 It may be modified by N.

[0044] In formula (100), X 21 , X 22 , X 23, and X 24 are, independently of each other, CHY, CDY, 13 C.H.Y., 13 C.D.Y., C.O. 13 CO, C 18 O, and 13 C 18 In a preferred embodiment, X 21 , X 22 , X 23 , and X 24 At least one of X is CHY or CDY, for example, 21 , X 22 , X 23 , and X 24 Any of the above may be CHY or CDY. X 21 , X 22 , X 23 , and X 24 At least one of the following is a stable isotope: D, 13 C. 18 O, or 15 When modified by N, the X 1 and X 3 None of the X may be modified with a stable isotope, or even in this case, 1 and X 3 Either or both of the above may be modified with a stable isotope. X 21 , X 22 , X 23 , and X 24 Both are stable isotopes D, 13 C. 18 O, or 15 When not modified by N, the X 1 and X 3 Either or both may be modified with a stable isotope.

[0045] In formula (100), Y and R contained in Y may be as described above in formula (I). That is, Y is H, D, NH 2 , 15 NH 2 , O.H., 18OH, SH, OR, O(CO)R, OSO 3 H, O.S.O. 3 R may be any of Na, and a sugar substituent, and R may represent an alcohol protecting group, an alkyl group, an alkenyl group, or an aryl group.

[0046] In formula (100), X 3 is C or 13 It's C.

[0047] In formula (100), X 1 , X 21 , X 22 , X 23 , X 24 , and X 3 At least one of the following is a stable isotope: D, 13 C. 18 O, or 15 The alkyne compound of formula (100) is a compound having at least one stable isotope, and the at least one stable isotope is the X 1 , X 21 , X 22 , X 23 , X 24 , and X 3 may be included in one or more of the following:

[0048] 2-3. How to use alkyne compounds

[0049] The alkyne compounds according to the present invention may be used to produce vitamin D compounds as described above, and more specifically, may be used as materials for forming the A-ring portion of vitamin D compounds.

[0050] In one embodiment, a vitamin D compound can be produced by reacting a bromoolefin compound having a CD ring of a vitamin D compound with an alkyne compound according to the present invention. In this reaction, the Br of the bromoolefin compound reacts with the alkyne moiety of the alkyne compound. This produces a vitamin D compound. For a specific example of this reaction, see the Examples below.

[0051] The bromoolefin compound may be any compound known in the art. The type of the bromoolefin compound (particularly the side chain portion of the D ring) may be appropriately selected by those skilled in the art depending on the type of vitamin D compound to be produced.

[0052] The hydroxy group of the alkyne compound to be subjected to the reaction may be protected by an alcohol protecting group, and in this case, the hydroxy group of the vitamin D compound after the reaction is also protected by an alcohol protecting group. Therefore, after the reaction, a deprotection reaction may be performed. The reagent used for the deprotection reaction may be appropriately selected by those skilled in the art depending on factors such as the type of the alcohol protecting group.

[0053] 2-4. Method for producing alkyne compounds

[0054] The alkyne compound of the present invention can be produced, for example, by using a linear polyhydric alcohol compound as a starting material. The linear polyhydric alcohol compound may be, for example, a polyhydric alcohol compound having at least one hydroxyl group at each of both ends of the linear chain. In addition, the linear polyhydric alcohol compound may have one or more hydroxyl groups at one or more carbons other than the both ends. These hydroxyl groups may be protected, for example, by a protecting group known in the art (for example, the above-mentioned alcohol protecting group, etc.).

[0055] For example, the method for producing an alkyne compound of the present invention may include a labeling step of labeling a linear polyhydric alcohol compound with deuterium by a deuterium labeling reaction, an alkyne group introduction step of introducing an alkyne structure to one end of the deuterium-labeled polyhydric alcohol, and an alkenyl group introduction step of introducing an alkene structure to the other end of the polyhydric alcohol after the alkyne group introduction step. In this manner, the alkyne compound of the present invention can be produced by carrying out deuterium labeling of a linear polyhydric alcohol compound, introduction of an alkyne group, and introduction of an alkenyl group. For specific examples of the production method, see the Examples described below.

[0056] The linear polyhydric alcohol compounds are known in the art, and the carbon number and the position and number of hydroxyl groups of the linear polyhydric alcohol compounds may be appropriately selected depending on the carbon number of the alkyne compound to be produced and the position and number of hydroxyl groups in the alkyne compound. By adjusting the carbon number, the position and the number, various alkyne compounds can be produced.

[0057] The labeling step may be carried out using a reaction known in the art. The labeling step may include, for example, a deuteration reaction using a specific catalyst (e.g., a ruthenium-based catalyst, etc.). FIG. 11 shows natural materials malic acid and aspartic acid as examples of compounds used to obtain the linear polyhydric alcohol compound. As shown in the figure, a deuterated linear polyhydric alcohol can be obtained by performing a deuteration reaction on malic acid or aspartic acid. Also, as shown in the figure, 13 C. 15 N, and 18 A deuteration reaction may be carried out on malic acid or aspartic acid labeled with any one or more of O. By such a deuteration reaction, 13 C. 15 N, and 18A linear polyhydric alcohol labeled with any one or more of O can be obtained. By carrying out the above-mentioned production method using the linear polyhydric alcohol, 13 C. 15 N, or 18 Alkyne compounds labeled with any one or more of O can be obtained. In addition, multiple 13 C. Multiple 15 N or multiple 18 A deuteration reaction may be carried out on the malic acid or aspartic acid labeled with O. By such a deuteration reaction, a plurality of 13 C. Multiple 15 N or multiple 18 A linear polyhydric alcohol labeled with O can be obtained. By carrying out the above-mentioned production method using the linear polyhydric alcohol, a plurality of 13 C. Multiple 15 N or multiple 18 An O-labeled alkyne compound can be obtained. As described above, various types of stable isotope-labeled starting materials can be obtained, and an alkyne compound corresponding to each starting material can be obtained.

[0058] The alkyne group introduction step may be carried out using a reaction known in the art. For example, the alkyne group introduction step may include a terminal alkyne introduction reaction in which an alkyne group is introduced at one end using trimethylsilylacetylene.

[0059] The alkenyl group introduction step may be carried out using a reaction known in the art. For example, the alkenyl group introduction step may include a cyano group introduction reaction for introducing a cyano group to the other end, and a reaction for converting the cyano group into an alkenyl group.

[0060] 3. Second embodiment (vitamin D compounds)

[0061] The present invention provides a vitamin D compound represented by the following formula (III). The vitamin D compound is labeled with a stable isotope as described below. The vitamin D compound may be used, for example, in mass spectrometry, for example for mass spectrometry in which quantification is performed by isotope dilution. The mass spectrometry may be for the detection and / or quantification of vitamin D metabolites. The vitamin D compound according to the present invention also has the advantage of excellent storage stability.

[0062] 3-1. Structure of Vitamin D Compound of Formula (III)

[0063] The vitamin D compounds of formula (III) are described below. [ka]

[0064] In formula (III), A' represents a cyclic carbon chain, which may represent the cyclic carbon chain corresponding to the A ring of a vitamin D compound. It should be noted that A', seco-B, C, and D are merely symbols given for the sake of convenience to explain the constituent parts of the vitamin D compound, and do not represent the actual elements or other constituent parts of the vitamin D compound of formula (III).

[0065] In formula (III), X' 1 is C or 13 When the vitamin D compound of formula (III) is prepared using an alkyne compound of formula (I), X of formula (I) may be C. 1 is X' of formula (III) 1 of carbon.

[0066] X' 1 is C, then the one or more X' 2 and X' 3 At least one of the following is a stable isotope: D, 13 C. 18 O, or 15 It is modified by N. X' 1 but 13 If C, the one or more X' 2 and X' 3 None of X' may be modified with a stable isotope, or even in this case, the one or more X' 2 and X' 3 At least one of the following is a stable isotope: D, 13 C. 18 O, or 15 It may be modified by N.

[0067] In formula (III), X' 2 , CHY', CDY', 13 CHY', 13 C.D.Y.', C.O. 13 CO, C 18 O, and 13 C 18 The vitamin D compound of formula (III) is a linear combination of m X' 2 , X' 1 and X' 3 and m is an integer of 1 to 4. When m is 2 or more, X' 2 may be the same or different from each other, i.e., the vitamin D compound has two or more X' 2 In the case where the two or more X' 2 , CHY', CDY', 13 CHY', 13 C.D.Y.', C.O. 13 CO, C 18 O, and 13 C 18 O may be selected independently of each other. Preferably, m is 2, 3, or 4, more preferably, m is 3 or 4, and even more preferably, m is 4. The embodiment where m is 4 is further described below with reference to formula (300). When the vitamin D compound of formula (III) is prepared using the alkyne compound of formula (I), X of formula (I) 2 is X' of formula (III)2 of carbon.

[0068] The one or more X' 2 At least one of the following is a stable isotope: D, 13 C. 18 O, or 15 When modified by N, the X' 1 and X' 3 None of X' may be modified with a stable isotope, or even in this case, 1 and X' 3 Either or both of the above may be modified with a stable isotope. The one or more X' 2 Both are stable isotopes D, 13 C. 18 O, or 15 When not modified by N, the X' 1 and X' 3 Either or both may be modified with a stable isotope.

[0069] In formula (III), Y' is H, D, NH 2 , 15 NH 2 , O.H., 18 OH, SH, OR', O(CO)R', OSO 3 H, O.S.O. 3 In the case where two or more Y' are included in formula (III), the Y' may be the same or different from each other. That is, in the case where the alkyne compound has two or more Y', the two or more Y' may be H, D, NH 2 , 15 NH 2 , O.H., 18 OH, SH, OR, O(CO)R, OSO 3 H, O.S.O. 3 The substituents may be independently selected from among Na, Na, and sugar substituents. When the vitamin D compound of formula (III) is prepared using the alkyne compound of formula (I), Y in formula (I) corresponds to Y' in formula (III).

[0070] In formula (III), R' represents an alkyl group, an alkenyl group, or an aryl group. When two or more R' are included in formula (III), the R' may be the same or different from each other. That is, when the alkyne compound has two or more R', the two or more R' may be independently selected from an alkyl group, an alkenyl group, and an aryl group.

[0071] When a vitamin D compound of formula (III) is prepared using an alkyne compound of formula (I) containing OR as Y and an alcohol protecting group as R of the OR, the alcohol protecting group of the OR of the alkyne compound of formula (I) may be deprotected and exist as OH in the vitamin D compound of formula (III).

[0072] The alkyl group that the vitamin D compound has as R' may be a substituted or unsubstituted linear or branched alkyl group having 1 to 10 carbon atoms. The number of carbon atoms in the linear or branched alkyl group may be, for example, 1 to 8, 1 to 6, or 1 to 4. This carbon number does not include the carbon atoms of any substituents. The alkenyl group contained in the alkyne compound as R' may be a linear or branched alkyl group having 2 to 10 carbon atoms. The number of carbon atoms in the linear or branched alkenyl group may be, for example, 2 to 8, 2 to 6, or 2 to 4. The number of carbon atoms does not include the number of carbon atoms of the substituent. The aryl group contained as R' in the alkyne compound may be a substituted or unsubstituted aryl group having 6 to 20 carbon atoms. The number of carbon atoms in the aryl group is preferably 6 to 18, and may be, for example, 6 to 16, 6 to 14, 6 to 12, or 6 to 10. The number of carbon atoms does not include the number of carbon atoms of the substituent. The aryl group may be an unsubstituted aryl group, and may be, for example, a phenyl group or a naphthyl group.

[0073] In formula (III), the sugar substituent may have, for example, the structure: [ka] Wo in the sugar substituent represented by this structure is the same as Wo described above in relation to formula (I), and the same description also applies to Wo that may be included in formula (III).

[0074] In one embodiment, the one or more X' 2 At least one of the following is CHY', CDY', 13 CHY', and 13 CDY', for example, the one or more X' 2 Both are CHY', CDY', 13 CHY', and 13 CDY'. In this embodiment, preferably, Y' may represent any of H, D, OH, and OR', for example, any of H, D, OH, and OR'. For example, when two or more Y' are included in the vitamin D compound, the two or more Y' may be independently selected from H, D, OH, and OR'.

[0075] In a particularly preferred embodiment, the one or more X' 2 At least one of the X' is CHY' or CDY', for example, 2 Any of the may be CHY' or CDY'. In this embodiment, preferably, Y' may represent any of H, D, and OH, for example, any of H, D, and OH. For example, when two or more Y' are included in the alkyne compound, the two or more Y' may be independently selected from H, D, and OH. In this embodiment, preferably, the one or more X' 2 contains at least one CDD and at least one CDOH.

[0076] In formula (III), X' 3 is C or 13When the vitamin D compound of formula (III) is prepared using the alkyne compound of formula (I), X of formula (I) is 3 is X' of formula (III). 3 This corresponds to carbon.

[0077] As shown in formula (III), X' 1 and X' 3 may be bonded, i.e., the X' 1 , the one or more X' 2 and X' 3 The ring structure may be formed by: This ring structure corresponds to the A ring in vitamin D compounds.

[0078] In formula (III), the X' 1 , the one or more X' 2 and X' 3 At least one of the following is a stable isotope: D, 13 C. 18 O, or 15 That is, the vitamin D compound of formula (III) is a compound having at least one stable isotope, and the at least one stable isotope is modified by the X' 1 , the one or more X' 2 and X' 3 may be included in one or more of the following:

[0079] The vitamin D compound has a seco-B ring, a C ring, and a D ring, as shown in formula (III). The D ring may have P attached thereto, which represents a side chain.

[0080] In formula (III), P is a C substituted or unsubstituted with a hydroxy group. 1 ~C 10 C substituted or unsubstituted with alkyl or hydroxy groups 2 ~C 10 The alkyl group or the alkenyl group may have a lactone group substituted with a hydroxy group.

[0081] In one embodiment, P in formula (III) is [ka] It may be expressed as: where Q, Q', Q'', Q''', and Q'''' are each independently H, CH 2 , C.H. 3 , C 2 H 5 , O, OH, and any of the sugar substituents.

[0082] More specific examples of P include the following: P may be any one of these. [ka]

[0083] The structure of P may not be limited to those listed above, and any of the D-ring side chains of vitamin D compounds known in the art may be adopted as the structure of P.

[0084] 3-2. Structure of Vitamin D Compound of Formula (200)

[0085] In a particularly preferred embodiment, in formula (III), m is 4. In this embodiment, the alkyne compound may be a compound represented by the following formula (200). The compound represented by formula (200) is described below. [ka]

[0086] In formula (200), A' represents a cyclic carbon chain. A' is the portion that corresponds to the A ring of vitamin D compounds.

[0087] In formula (200), X' 1 is C or 13C. When the vitamin D compound of formula (200) is prepared using an alkyne compound of formula (100), X in formula (100) may be C. 1 The carbon atom of formula (200) is X' 1 of carbon.

[0088] X' 1 is C, then the one or more X' 2 and X' 3 At least one of the following is a stable isotope: D, 13 C. 18 O, or 15 It is modified by N. X' 1 but 13 If C, the one or more X' 2 and X' 3 None of X' may be modified with a stable isotope, or even in this case, the one or more X' 2 and X' 3 At least one of the following is a stable isotope: D, 13 C. 18 O, or 15 It may be modified by N.

[0089] In formula (200), X' 21 , X' 22 , X' 23 , and X' 24 are, independently of each other, CHY', CDY', 13 CHY', 13 C.D.Y.', C.O. 13 CO, C 18 O, and 13 C 18 In a preferred embodiment, X' represents any one selected from the group consisting of 1 and 2. 21 , X' 22 , X' 23 , and X' 24 At least one of X' is CHY' or CDY', for example, 21 , X' 22 , X' 23 , and X' 24 Any of the above may be CHY or CDY. When the vitamin D compound of formula (200) is prepared using the alkyne compound of formula (100), X of formula (100) 21 , X 22 , X 23 , and X 24 The carbon atom of formula (200) is X' 21 , X' 22 , X' 23 , and X' 24 This corresponds to carbon.

[0090] The X' 21 , X' 22 , X' 23 , and X' 24 At least one of the following is a stable isotope: D, 13 C. 18 O, or 15 When modified by N, the X' 1 and X' 3 None of X' may be modified with a stable isotope, or even in this case, 1 and X' 3 Either or both of the above may be modified with a stable isotope. The X' 21 , X' 22 , X' 23 , and X' 24 Both are stable isotopes D, 13 C. 18 O, or 15 When not modified by N, the X' 1 and X' 3 Either or both may be modified with a stable isotope.

[0091] In formula (200), Y' and R' contained in Y' may be as described above in formula (III). That is, Y' is H, D, NH 2 , 15 NH 2 , O.H., 18 OH, SH, OR', O(CO)R', OSO 3 H, O.S.O. 3Na, and a sugar substituent, and R' represents an alkyl group, an alkenyl group, or an aryl group.

[0092] In one embodiment, the X' 21 , X' 22 , X' 23 , and X' 24 At least one of the following is CHY', CDY', 13 CHY', and 13 For example, X' 21 , X' 22 , X' 23 , and X' 24 Both are CHY', CDY', 13 CHY', and 13 CDY'. In this embodiment, preferably, Y' may represent any of H, D, OH, and OR', for example, any of H, D, OH, and OR'. For example, when two or more Y' are included in the vitamin D compound, the two or more Y' may be independently selected from H, D, OH, and OR'.

[0093] In a particularly preferred embodiment, the one or more X' 2 At least one of the X' is CHY' or CDY', for example, 2 Any of the may be CHY' or CDY'. In this embodiment, preferably, Y' may represent any of H, D, and OH, for example, any of H, D, and OH. For example, when two or more Y' are included in the alkyne compound, the two or more Y' may be independently selected from H, D, and OH. In this embodiment, preferably, the one or more X' 2 contains at least one CDD and at least one CDOH.

[0094] In formula (200), X' 3 is C or13 C. When the vitamin D compound of formula (200) is prepared using the alkyne compound of formula (100), X of formula (100) is 3 Carbon of formula (200) is X' 3 This corresponds to carbon.

[0095] As shown in equation (200), X' 1 and X' 3 may be bonded, i.e., the X' 1 , the X' 21 , X' 22 , X' 23 , and X' 24 and the X' 3 The ring structure may be formed by: This ring structure corresponds to the A ring in vitamin D compounds.

[0096] In the formula (200), the X' 1 , the X' 21 , X' 22 , X' 23 , and X' 24 and the X' 3 At least one of the following is a stable isotope: D, 13 C. 18 O, or 15 That is, the vitamin D compound of formula (200) is a compound having at least one stable isotope, and the at least one stable isotope is modified by the X' 1 , the X' 21 , X' 22 , X' 23 , and X' 24 and the X' 3 may be included in one or more of the following:

[0097] P contained in formula (200) is as explained above in formula (III), and the explanation also applies to formula (200).

[0098] The vitamin D compound of the present invention is 25-hydroxyvitamin D 3 (25OHD 3), 3-epi-25-hydroxyvitamin D 3 (3-epi-25OHD 3 ), 25-hydroxyvitamin D 2 (25OHD 2 ), 24R,25-dihydroxyvitamin D 3 (24R,25(OH) 2 D 3 ), 23S,25-dihydroxyvitamin D 3 (23S,25(OH) 2 D 3 ), 1α,25-dihydroxyvitamin D 3 (1α,25(OH) 2 D 3 ), 25-hydroxyvitamin D 3 -26R,23S-lactone (25OHD 3 -26R,23S-lactone), 1α,25-dihydroxyvitamin D 3 -26R,23S-Lactone (1α,25(OH) 2 D 3 -26R,23S-lactone), 1α,25-dihydroxyvitamin D 2 (1α,25(OH) 2 D 2 ), 1α, 24R, 25-trihydroxyvitamin D 3 (1α,24R,25(OH) 3 D 3 ), or 4β,25-dihydroxyvitamin D 3 (4β,25(OH) 2 D 3) may be a stable isotope-labeled compound. The chemical structures of these compounds are shown in FIG. 10. That is, the vitamin D compound of the present invention may be a vitamin D compound in which the A-ring portion of each of the chemical structures of the compounds shown in the figure is labeled with a stable isotope according to the present invention. In one embodiment, the vitamin D compound of the present invention may be a vitamin D compound in which at least one of the hydrogen atoms bonded to the carbon atoms forming the A-ring portion of each of the chemical structures of the compounds shown in the figure is deuterium. The number of deuterium atoms bonded to the carbon atoms forming the A-ring portion may be, for example, 2 or more, particularly 3 or more. The number of deuterium atoms bonded to the carbon atoms forming the A-ring portion may be, for example, 7 or less, particularly 6 or less, more particularly 5 or less.

[0099] 3-3. How to Use Vitamin D Compounds

[0100] The vitamin D compounds of the present invention may be used in various analytical methods, particularly in mass spectrometry, for example in isotope dilution mass spectrometry.The mass spectrometry may more particularly be LC-MS / MS or LC-MS.By carrying out the analytical method using the vitamin D compounds of the present invention, vitamin D compounds (e.g. vitamin D metabolites in biological samples) that have the same chemical structure but are not isotope-labeled can be detected or quantified.

[0101] The vitamin D compounds of the present invention may for example be utilized as standards in the above analytical methods, and in particular may be used as internal standards.

[0102] In the mass spectrometry method, the vitamin D compound of the present invention may be derivatized. The derivatization can improve detection accuracy. For the derivatization, a derivatization reagent known in the art may be used.

[0103] In the mass spectrometry method, the vitamin D compounds of the invention or the derivatized vitamin D compounds may be ionized, for example by electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), or matrix-assisted laser desorption ionization (MALDI) for more efficient ionization, but may also be ionized by other ionization methods, allowing for more accurate detection or quantification.

[0104] The mass spectrometry method may be performed, for example, by a liquid chromatography tandem mass spectrometer (LC-MS / MS) or a matrix-assisted laser desorption / ionization mass spectrometer (MALDI-MS), but the apparatus for performing the mass spectrometry step is not limited to these. Specific procedures in the mass spectrometry step can be appropriately set by those skilled in the art depending on, for example, the ionization method employed and the apparatus used.

[0105] 3-4. Manufacturing method of vitamin D compounds

[0106] The vitamin D compound of the present invention may be produced by reacting the alkyne compound of the present invention with a compound for forming a CD ring of the target vitamin D compound. The compound for forming a CD ring may be an olefin compound having a CD ring. As the olefin compound, a compound known in the art may be used. The olefin compound may be, for example, a compound having a structure represented by the following formula (II). As the compound, a compound known in the art may be used. [ka] In the compound of formula (II), C and D represent the moieties which form the C-ring and D-ring, respectively, when the vitamin D compound is synthesized. P in the compound of formula (II) may be, for example, the same as P described above in formula (III), but the hydroxy group contained in P may be protected by the alcohol protecting group described above in the compound of formula (II). The alcohol protecting group may be, for example, TES. Z in the compound of formula (II) may be a halogen or pseudohalogen group, for example, F, Cl, Br, I, or OTf. In one embodiment, Z is Br, i.e., the compound of formula (II) may be a bromoolefin compound.

[0107] That is, the method for producing a vitamin D compound of the present invention may include reacting an alkyne compound of formula (I) with a compound of formula (II). More specifically, the reaction may include a reaction between an alkynyl group in the alkyne compound of formula (I) and Z in the compound of formula (II). The reaction may result in a carbon bonded to Z in the compound of formula (II) and a carbon bonded to X, one of the two carbons constituting the alkynyl group in the alkyne compound of formula (I), being bonded to Z in the compound of formula (II). 3 A bond is formed between the carbon atom that is not a carbon atom and the carbon atom that is not a carbon atom, forming a seco-B ring. 1 and X 3 Thus, the A' ring and the seco-B ring are formed, forming the vitamin D compound of the present invention.

[0108] 4. Third embodiment (analysis method using vitamin D compound)

[0109] 4-1. Examples of analysis methods

[0110] The present invention also provides an analytical method using the vitamin D compound according to the present invention. The analytical method may include analyzing a sample by mass spectrometry as described in 3-3 above. The sample may be a sample that contains, or may contain (i.e., may not contain), a vitamin D compound having the same chemical structure as the vitamin D compound according to the present invention, but not isotopically labeled, as a target for detection or quantification, for example. The sample may be, for example, a biological sample, in particular a body fluid or a sample derived from a body fluid, and more specifically, any of serum, plasma, blood, urine, cerebrospinal fluid, and saliva.

[0111] According to one embodiment, the vitamin D compound of the present invention may be used as an internal standard in the analysis method. The method of using the vitamin D compound of the present invention as an internal standard may be a method known in the art. For example, in the analysis method, the amount or concentration of the compound to be analyzed (e.g., vitamin D metabolite) may be determined based on the relationship between the peak area ratio and the concentration ratio of the vitamin D compound of the present invention.

[0112] 4-2. Modifications of analysis method

[0113] In a modified version of the analytical method described in 4-1 above, vitamin D compounds not having a hydroxy group at position 1 of the A ring may be analyzed separately from vitamin D compounds having a hydroxy group at position 1 of the A ring. More specifically, the vitamin D compounds not having a hydroxy group at position 1 of the A ring may include at least one of 2-hydroxyvitamin D compounds and 4-hydroxyvitamin D compounds. Even more specifically, the vitamin D compounds not having a hydroxy group at position 1 of the A ring may include at least one of 4β,25-dihydroxyvitamin D3 compounds and 4-hydroxyvitamin D compounds. This modified version will be described in more detail below.

[0114] As mentioned above, there is a great demand for detection or quantification of vitamin D metabolites in biological samples in clinical and food nutritional aspects. Among various vitamin D metabolites, one of the most important metabolites in terms of physiological activity is 1α,25-dihydroxyvitamin D, also known as active vitamin D. 3 Therefore, selective extraction is extremely important. LC-MS / MS has high separation power by combining chromatographic separation and separation by m / z (mass-to-charge ratio) of fragment ions, and the sensitivity can be further increased by using derivatization reagents.

[0115] However, there are cases where two or more vitamin D metabolites with different chemical structures cannot be individually detected / quantified by LC-MS / MS alone. For example, there are cases where two or more positional isomers that differ only in the position of the hydroxyl group cannot be individually detected / quantified by LC-MS / MS alone. An example of such a case will be described with reference to FIG. 6. As shown in the figure, the sample to be analyzed contained 1α,25-dihydroxyvitamin D 3 (1α,25(OH) 2 D 3 ), 24R,25-dihydroxyvitamin D 3 (24R,25(OH) 2 D 3 ), and 4β,25-dihydroxyvitamin D 3 (4β,25(OH) 2 D 3 ) are included in the study. When the sample is derivatized using the derivatization reagent DAP-PA shown in the figure, three derivatized compounds are generated as shown on the right side of the figure. These three derivatives are cleaved at the positions indicated by the arrows in the figure. As can be seen from the products after the cleavage, 24R,25-dihydroxyvitamin D 3 1α,25-dihydroxyvitamin D 3 Fragment ion separation based on m / z is possible from 4β,25-dihydroxyvitamin D 3In addition, 1α,25-dihydroxyvitamin D 3 and 4β,25-dihydroxyvitamin D 3 It is also difficult to separate by chromatography.

[0116] By carrying out a predetermined separation process and then carrying out an analysis process using a vitamin D compound according to the present invention on a sample obtained by the separation process, two or more vitamin D compounds that are difficult to separate as described above can be individually detected or quantified. The present invention also provides an analysis method for selectively detecting or quantifying two or more vitamin D compounds that are difficult to separate by LC-MS / MS alone. That is, the analysis method of the present invention also provides an analysis method for individually detecting or quantifying two or more positional isomers of a vitamin D compound. The two or more positional isomers may be positional isomers in which the positions of the hydroxyl groups bonded to the A ring are different from each other, as described above. The two or more positional isomers may have the same molecular weight.

[0117] In one embodiment, the analytical method of the present invention comprises: fractionating the sample into at least one first fraction in which vitamin D compounds having a hydroxy group at position 1 of the A ring are recovered and at least one second fraction in which vitamin D compounds having a hydroxy group at position 1 of the A ring are not recovered; analyzing either or both of said at least one first fraction and said at least one second fraction using a vitamin D compound of the present invention. may include: In this embodiment, the second fraction may include vitamin D compounds that do not have a hydroxy group at position 1 of the A ring. The sample does not necessarily contain a vitamin D compound having a hydroxy group at position 1 of the A ring, i.e., the first fraction may be a fraction obtained by performing an operation for recovering a vitamin D compound having a hydroxy group at position 1 of the A ring, and the fraction may not contain the vitamin D compound. The fractionation step and the analysis step will be described below.

[0118] 4-2-1.Fraction process The sample to be subjected to the fractionation step may be the sample described in 4-1 above.

[0119] The vitamin D compound having a hydroxy group at position 1 of the A ring may be, for example, a vitamin D compound having the structure shown on the left of FIG. 7, i.e., a 1-hydroxyvitamin D compound. The 1-hydroxyvitamin D compound has a hydroxy group at position 1 of the A ring as shown in the figure. Furthermore, the 1-hydroxyvitamin D compound may have a hydroxy group at position 3 of the A ring. P contained in the 1-hydroxyvitamin D compound may be as described above with respect to formula (III).

[0120] The vitamin D compound not having a hydroxy group at position 1 of the A ring may be a vitamin D compound having the structure shown on the right of Figure 7, i.e., a 2-hydroxyvitamin D compound or a 4-hydroxyvitamin D compound. The 2-hydroxyvitamin D compound or the 4-hydroxyvitamin D compound has a hydroxy group at position 2 or 4 of the A ring as shown in the figure. Furthermore, the 2-hydroxyvitamin D compound or the 4-hydroxyvitamin D compound may have a hydroxy group at position 3 of the A ring. The P contained in the 2-hydroxyvitamin D compound or the 4-hydroxyvitamin D compound may be as described above with respect to formula (III).

[0121] In particular, the 1-hydroxyvitamin D compound and the 2-hydroxyvitamin D compound or the 4-hydroxyvitamin D compound may be the same except for the difference in the position of the hydroxy group attached to the A ring. The 1-hydroxyvitamin D compound is, for example, 1α,25-dihydroxyvitamin D, as shown on the left in FIG. 3 The 4-hydroxyvitamin D compound may be 4β,25-dihydroxyvitamin D, as shown on the right side of the figure. 3 It may be. That is, the sample to be subjected to the above-mentioned analytical method contains 1α,25-dihydroxyvitamin D 3 and / or 4β,25-dihydroxyvitamin D 3 The sample may include:

[0122] It should be noted that the sample subjected to the above-mentioned analytical method does not necessarily need to contain the vitamin D compound to be analyzed; that is, the analysis may reveal that the sample does not contain the vitamin D compound to be analyzed.

[0123] The fractionation step may be carried out, for example, using a capture substance that selectively captures (e.g., binds or adsorbs) only vitamin D compounds having a hydroxy group at position 1 of the A ring. Examples of the capture substance include, but are not limited to, antibodies and aptamers. Reagents known in the art may be used as the capture substance, and those skilled in the art can appropriately select the capture substance depending on the vitamin D compound to be captured.

[0124] In one embodiment, in the fractionation step, the sample may be applied to a column having an antibody that selectively captures only vitamin D compounds having a hydroxyl group at position 1 of the A ring. An example of the column is an antibody column, for example, 1,25-(OH) 2 -Vitamin D 3 / D 2 -ImmuTube can be mentioned. By adding the sample to the column, the antibody in the column captures vitamin D compounds having a hydroxy group at position 1 of the A ring. Next, the vitamin D compounds that do not have a hydroxy group at position 1 of the A ring that have not been captured are recovered from the column to obtain the second fraction. Next, the captured vitamin D compounds having a hydroxy group at position 1 of the A ring are recovered from the column by a specified elution process to obtain the first fraction.

[0125] 4-2-2.Analysis process

[0126] In the analyzing step, either one or both of the at least one first fraction and the at least one second fraction are analyzed using the vitamin D compound of the present invention. The analysis may be performed by mass spectrometry as described in 4-1 above.

[0127] For example, the second fraction may be subjected to mass spectrometry using, as a standard, a vitamin D compound of the present invention (a compound having a chemical structure in which the A ring of the vitamin D compound is labeled with a stable isotope) corresponding to the target vitamin D compound expected to be contained in the second fraction. 3 If yes, then 4β,25-dihydroxyvitamin D 3 Alternatively, LC-MS / MS may be performed using a compound having a chemical structure in which the A ring of the compound is labeled with a stable isotope as an internal standard substance.

[0128] Furthermore, the first fraction may be subjected to mass spectrometry using, as a standard substance, a vitamin D compound of the present invention corresponding to a target vitamin D compound expected to be contained in the first fraction (a compound having a chemical structure in which the A ring of the target vitamin D compound is labeled with a stable isotope). 3 If yes, 1α,25-dihydroxyvitamin D 3 Alternatively, LC-MS / MS may be performed using a compound having a chemical structure in which the A ring of the compound is labeled with a stable isotope as an internal standard substance.

[0129] The analytical method described above makes it possible to selectively detect or quantify two or more vitamin D compounds that are difficult to separate by LC-MS / MS alone.

[0130] For example, the analytical method can selectively recover 2(or 4)-hydroxyvitamin D compounds, which can then be analyzed by LC-MS / MS (liquid chromatography-tandem mass spectrometry), regardless of the mass spectrometer manufacturer. Among them, 4β,25-dihydroxyvitamin D 3 is called the active form of vitamin D and is physiologically the most important 1α,25-dihydroxyvitamin D. 3 They are positional isomers of 4β,25-dihydroxyvitamin D, and have identical molecular weights, MS / MS fragmentation patterns, and almost identical HPLC chromatographic behavior, making them extremely difficult to separate. 3 1α,25-dihydroxyvitamin D 3 Its physiological role was unknown. 3 The phenomenon that was thought to be the activation of 4β,25-dihydroxyvitamin D 3 It is possible that this is due to the presence of 4β,25-dihydroxyvitamin D. 3 This allows the determination of 4β,25-dihydroxyvitamin D 3 and further, 1α,25-dihydroxyvitamin D 3 The analytical method of the present invention is also useful for more accurately evaluating the function or activity of vitamin D compounds. Thus, the analytical method of the present invention is believed to greatly contribute to elucidating the mechanism of action of vitamin D compounds.

[0131] 5. Fourth embodiment (method of producing vitamin D compound)

[0132] The present invention also provides a method for producing the vitamin D compound of the present invention. The method may be carried out as described in 3-4 above. That is, in the method for producing the present invention, the alkyne compound represented by formula (I) may be used as a material for forming the A ring of the vitamin D compound. An example of a chemical reaction formula in this case is shown in FIG. 9A. As shown in the figure, in the method for producing the present invention, the alkyne compound represented by formula (I) may be reacted with a compound having a structure represented by formula (II) to produce the vitamin D compound represented by formula (III).

[0133] In a preferred embodiment, in the production method of the present invention, the alkyne compound represented by the formula (100) may be used as a material for forming the A ring of a vitamin D compound. An example of a chemical reaction formula in this case is shown in FIG. 9B. As shown in the figure, in the production method of the present invention, the vitamin D compound represented by the formula (200) may be produced by reacting the alkyne compound represented by the formula (100) with a compound having a structure represented by the formula (II). EXAMPLES

[0134] 6. Working Example The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0135] 6-1. Example 1: Preparation of alkyne compounds and vitamin D compounds

[0136] (1) Synthesis of alkyne compound 10

[0137] Alkyne compound 10 was prepared by the synthetic route shown below, with each reaction in the synthetic route being explained below. [ka]

[0138] (1-1)d3 Synthesis of -diol 2 [ka] Under a hydrogen atmosphere, diol 1 (4.0 g, 21.0 mmol) was dissolved in deuterium oxide (80 mL), ruthenium carbon (4.0 g) was added at room temperature, and the mixture was stirred at 80° C. for 24 hours. After stirring, the reaction solution was filtered through Celite (ethyl acetate / methanol=8 / 1) and the solvent was distilled off. After the solvent was distilled off, the residue was purified by silica gel column chromatography (hexane / ethyl acetate=1 / 1) and deuterium oxide was obtained. 3 The -diol 2 (3.84 g, 96% yield) was obtained as a colorless liquid. 1 H NMR (400 MHz, CDCl 3 ) δ 4.34-4.42 (m, 1H), 4.12-4.17 (m, 1H), 1.68-1.81 (m, 2H), 1.19 (s, 9H). HRMS (ESI, M+Na) calcd. for C 9 H 15 D 3 O 4 Na 216.1291, found 216.1268.

[0139] (1-2)d 3 -Synthesis of tosylate 3 [ka] Under argon atmosphere, d 3 -Diol 2 (7.8 g, 40.4 mmol) was dissolved in pyridine (80.8 mL), tosyl chloride (8.08 mL, 42.4 mmol) was added, and the mixture was stirred at room temperature for 15 hours. After stirring, distilled water (80 mL) was added to the reaction solution, which was then extracted with methylene chloride. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane / ethyl acetate=3 / 1) and d 3 -Tosylate 3 (7.05 g, 50% yield) was obtained as a colorless liquid. 1H NMR (300 MHz, CDCl 3 ) δ 7.80 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 8.7 Hz, 2H), 4.23-4.29 (m, 1H), 4.09-4.16 (m, 1H), 2.45 (s, 3H), 1.76-1.83 (m, 1H), 1.67-1.73 (m, 1H), 1.18 (s, 9H). HRMS (ESI, M+Na) calcd. for C 16 H 21 D 3 O 6 SNa 370.1380, found 370.1413.

[0140] (1-3)d 3 -Synthesis of epoxide 4 [ka] Under argon atmosphere, d 3 -Tosylate 3 (10.8 g, 31.1 mmol) was dissolved in dehydrated dimethylformamide (156 mL), sodium hydride (1.49 g, 37.3 mmol) was added under ice cooling, and the mixture was stirred at room temperature for 0.5 hours. After stirring, distilled water (100 mL) was added to the reaction solution, which was extracted with methylene chloride. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After distilling off the solvent, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 8 / 1) and d 3 -Epoxide 4 (3.96 g, 73% yield) was obtained as a colorless liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 4.22 (t, J = 6.0 Hz, 2H), 1.80-1.94 (m, 2H), 1.20 (s, 9H). HRMS (ESI, M+Na) calcd. for C 9 H 13 D 3 O 3 Na 198.1185, found 198.1213.

[0141] (1-4)d 3 -Synthesis of alkyne 5 [ka] Under an argon atmosphere, trimethylsilylacetylene (6.4 mL, 45.6 mmol) was dissolved in anhydrous tetrahydrofuran solution (24.8 mL), and n-butyllithium (1.6 M hexane solution) (13.7 mL, 35.6 mmol) was added dropwise at -78°C to prepare a reaction mixture. The reaction mixture was stirred at the same temperature for 1 hour, and then cooled to 5°C. 3 A solution of epoxide 4 (3.47 g, 19.8 mmol) in tetrahydrofuran and boron trifluoride diethyl ether complex (3.23 mL, 25.7 mmol) were added dropwise in sequence. The reaction mixture was stirred at the same temperature for another 30 minutes. A saturated aqueous solution of ammonium chloride (20 mL) was added to the reaction mixture, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane / ethyl acetate=10 / 1) to give d 3 -Alkyne 5 (3.33 g, 61% yield) was obtained as a yellow liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 4.27-4.33 (m, 1H), 4.15-4.21 (m, 1H), 1.88-1.95 (m, 1H), 1.75-1.81 (m, 1H), 1.18 (s, 9H), 0.15 (s, 9H). HRMS (ESI, M+Na) calcd. for C 14 H 23 D 3 O 3 SiNa 296.1737, found 296.1732.

[0142] (1-5)d 3 -Synthesis of alkyne 6 [ka] Under argon atmosphere, d 3 -Alkyne 5 (3.2 g, 11.7 mmol) was dissolved in dehydrated dimethylformamide (23.4 mL), and imidazole (2.15 g, 31.6 mmol), tert-butyldimethylsilyl chloride (3.50 g, 23.4 mmol), and 4-dimethylaminopyridine (143 mg, 1.17 mmol) were added and stirred at room temperature for 3 hours. After stirring, distilled water (23 mL) was added to the reaction solution, which was extracted with a mixed solvent of hexane / ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After distilling off the solvent, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 10 / 1) and d 3 -Alkyne 6 (4.61 g, 100% yield) was obtained as a yellow liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 4.19-4.25 (m, 1H), 4.05-4.13 (m, 1H), 1.93-2.00 (m, 1H), 1.73-1.79 (m, 1H), 1.22 (s, 9H), 0.88 (s, 9H), 0.14 (s, 9H), 0.08 (d, J = 10.5 Hz, 6H). HRMS (ESI, M+Na) calcd. for C 20 H 37 D 3 O 3 S 2 Na 410.2602, found 410.2578.

[0143] (1-6)d 3 -Synthesis of alkyne 7 [ka] Under argon atmosphere, d 3-Alkyne 6 (4.51 g, 11.6 mmol) was dissolved in methanol (107 mL), and sodium methoxide (5.0 M methanol solution) (9.28 mL, 46.4 mmol) was added dropwise under ice cooling, and the mixture was stirred at room temperature for 24 hours. After the stirring, a saturated aqueous ammonium chloride solution (20 mL) was added to the reaction solution, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After the solvent was distilled off, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 30 / 1) and d 3 -Alkyne 7 (2.32 g, 86% yield) was obtained as a yellow liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 3.79-3.85 (m, 1H), 3.71-3.77 (m, 1H), 1.99 (s, 1H), 1.91-1.97 (m, 1H), 1.77-1.84 (m, 1H), 0.89 (s, 9H), 0.11 (s, 6H). HRMS (ESI, M+Na) calcd. for C 12 H 21 D 3 O 2 SiNa 254.1632, found 254.1631.

[0144] (1-7)d 3 -Synthesis of alkyne 8 [ka] Under argon atmosphere, d 3 -Alkyne 7 (1.0 g, 4.32 mmol) was dissolved in methylene chloride (14 mL), triethylamine (2.1 mL, 15.1 mmol), tosyl chloride (1.65 g, 8.64 mmol), and 4-dimethylaminopyridine (52 mg, 0.43 mmol) were added, and the mixture was stirred at room temperature for 2 hours. Distilled water (20 mL) was added to the reaction solution, which was then extracted with methylene chloride. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. The residue was purified by silica gel column chromatography (hexane / ethyl acetate = 20:1) and d3 -Alkyne 8 (1.67 g, 99% yield) was obtained as a colorless liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 7.79 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.2 Hz, 2H), 4.13 (dd, J = 7.3, 5.5 Hz, 2H), 2.45 (s, 3H), 2.06-1.94 (m, 2H), 1.82 (dt, J = 14.2, 5.5 Hz, 1H), 0.82 (s, 9H), 0.02 (d, J = 22.9 Hz, 6H). HRMS (ESI, M+Na) calcd. for C 19 H 27 D 3 O 4 SSiNa 408.1720, found 408.1677.

[0145] (1-8)d 3 -Synthesis of alkyne 9 [ka] Under argon atmosphere, d 3 -Alkyne 8 (1.67 g, 4.27 mmol) was dissolved in dimethyl sulfoxide (14 mL), sodium cyanide (314 mg, 6.41 mmol) was added at room temperature, and the mixture was stirred at 90°C for 30 minutes. After stirring, distilled water (20 mL) was added to the reaction solution, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 20:1) to obtain d 3 -Alkyne 9 (1.0 g, 100% yield) was obtained as a colorless liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 2.47-2.37 (m, 2H), 2.08-1.95 (2H), 1.94-1.80 (m, 1H), 0.89 (s, 9H), 0.11 (d, J = 1.4 Hz, 6H). HRMS (ESI, M+Na) calcd. for C 13 H 20 D 3 NOSiNa 263.1635, found 263.1632.

[0146] (1-9)d 3 -Synthesis of alkyne 10 [ka] Under argon atmosphere, d 3 -Alkyne 9 (506 mg, 2.09 mmol) was dissolved in dehydrated methylene chloride (11 mL), and diisobutylaluminum hydride (1.0 M hexane solution) (2.5 mL, 2.50 mmol) was added dropwise under ice cooling, followed by stirring for 1 hour. After stirring, isopropanol (1.75 mL), silica gel, and distilled water were added to the reaction solution, which was stirred for 30 minutes, filtered, and the solvent was removed by distillation. After removing the solvent, the residue (d 3 -aldehyde) was used in the next step without further purification. Under an argon atmosphere, methyltriphenylphosphonium iodide (2.79 g, 6.90 mmol) was dissolved in dehydrated tetrahydrofuran solution (15 mL), and sodium hexamethyldisilazide (1.9 M tetrahydrofuran solution) (3.2 mL, 6.15 mmol) was added dropwise under ice cooling. The reaction mixture was stirred at the same temperature for 30 minutes, and then d 3 A solution of aldehyde (599 mg, 2.46 mmol) in tetrahydrofuran was added dropwise. The reaction mixture was stirred at the same temperature for another hour. After stirring, distilled water (20 mL) was added to the reaction mixture, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. The residue was purified by silica gel column chromatography (hexane) to obtain d 3 -Alkyne 10 (461 mg, 61% yield over two steps) was obtained as a colorless liquid. 3 -Alkyne 10 is also referred to herein as "alkyne compound 10." 1 H NMR (300 MHz, CDCl 3) δ 5.89-5.76 (m, 1H), 5.03 (dd, J = 16.9, 1.8 Hz, 1H), 4.96 (d, J = 10.1 Hz, 1H), 2.21-2.00 (m, 2H), 1.97 (s, 1H), 1.79-1.66 (m, 1H), 1.66-1.58 (m, 1H), 0.89 (s, 9H), 0.07 (d, J = 4.6 Hz, 6H).

[0147] (2) Synthesis of alkyne compound 17

[0148] Alkyne compound 17 was produced by the synthetic route shown below. As shown in the synthetic route, alkyne compound 17 was produced by the reaction of d 3 -alkyne 7. Each reaction in the synthesis pathway is described below. [ka]

[0149] (2-1)d 3 -Synthesis of alkyne 11 [ka] Under argon atmosphere, d 3 -Alkyne 7 (0.80 g, 3.46 mmol) was dissolved in dimethyl sulfoxide (34.6 mL), 2-iodoxybenzoic acid (1.93 g, 6.91 mmol) was added, and the mixture was stirred at 50°C for 1.5 hours. After the stirring, a saturated aqueous solution of sodium thiosulfate (34.6 mL) was added to the reaction solution, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After the solvent was distilled off, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 50 / 1) to give d 3 -Alkyne 11 (0.57 g, 72% yield) was obtained as a colorless liquid. 1 H NMR (300 MHz, CDCl 3) δ 9.82 (t, J = 1.8 Hz, 1H), 2.77-2.64 (m, 2H), 2.02 (s, 1H), 0.86 (d, J = 2.7 Hz, 9H), 0.09 (d, J = 13.3 Hz, 6H). HRMS (ESI, M+Na) calcd. for C 12 H 19 D 3 O 2 SiNa 252.1475, found 252.1503.

[0150] (2-2)d 3 -Synthesis of alkyne 12 [ka] Under argon atmosphere, d 3 -Alkyne 11 (0.55 g, 2.41 mmol) was dissolved in dehydrated methylene chloride (24.1 mL), and (ethoxycarbonylmethylene)triphenylphosphorane (1.68 g, 4.83 mmol) was added under ice cooling and stirred for 1 hour. After stirring, distilled water (24.1 mL) was added to the reaction solution, extracted with ethyl acetate, the organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After distilling off the solvent, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 100 / 1) and d 3 -Alkyne 12 (0.72 g, 99% yield) was obtained as a colorless liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 6.99-6.91 (m, 1H), 5.88 (dd, J = 15.6, 1.4 Hz, 1H), 4.18 (q, J = 7.2 Hz, 2H), 2.54 (q, J = 7.2 Hz, 1H), 2.42 (q, J = 7.3 Hz, 1H), 2.00 (s, 1H), 1.28 (t, J = 7.1 Hz, 3H), 0.88 (s, 9H), 0.07 (d, J = 6.4 Hz, 6H). HRMS (ESI, M+Na) calcd. for C 16 H 25 D 3 O 3 SiNa 322.1894, found 322.1939.

[0151] (2-3)d 3 -Synthesis of alkyne 13 [ka] Under argon atmosphere, d 3 -Alkyne 13 (0.72 g, 2.41 mmol) was dissolved in dehydrated toluene (24.1 mL), and diisobutylaluminum hydride (1.0 M hexane solution) (7.22 mL, 7.22 mmol) was added dropwise under ice cooling, and the mixture was stirred for 0.5 hours. After the stirring, isopropanol (5.05 mL), silica gel, and distilled water (24.1 mL) were added to the reaction solution, and the mixture was stirred and filtered through Celite, and the solvent was removed by distillation. After the solvent was removed by distillation, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 10 / 1) and d 3 -Alkyne 13 (0.60 g, 98% yield) was obtained as a colorless liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 4.11 (d, J = 4.1 Hz, 2H), 2.41-2.37 (m, 1H), 2.28 (dd, J = 12.6, 4.4 Hz, 1H), 1.98 (s, 1H), 0.89 (s, 9H), 0.07 (d, J = 6.9 Hz, 6H). HRMS (ESI, M+Na) calcd. for C 14 H 23 D 3 O 2 SiNa 280.1788, found 280.1791.

[0152] (2-4)d 3 -Synthesis of alkyne 14 [ka] Under an argon atmosphere, methylene chloride (18 mL) was added to MS4A (1.18 g) and incubated at -20°C for 1 h. i P) 4 (0.21 mL, 0.70 mmol), L-(+)-DET(0.15 mL, 0.85 mmol), d 3 -Alkyne 13 (0.60 g, 2.35 mmol) in methylene chloride (6 mL) was added, and TBHP (3.5 M in CH 2 Cl 2 , 1.34 mL, 4.7 mmol) was added dropwise and stirred at the same temperature for 1 hour. After this stirring, dimethyl sulfide (2.5 mL) and saturated aqueous sodium fluoride solution (70 mL) were added to the reaction solution and stirred for 12 hours. After this stirring, the reaction solution was filtered through Celite (methylene chloride) and the solvent was distilled off. The residue was extracted with methylene chloride, and the organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. The residue was purified by silica gel column chromatography (hexane / ethyl acetate = 20 / 1) and distilled off. 3 -Alkyne 14 (0.58 g, 91% yield) was obtained as a colorless liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 3.89 (dd, J = 12.8, 2.3 Hz, 1H), 3.57 (dd, J = 12.6, 4.4 Hz, 1H), 3.08-3.05 (m, 1H), 2.96-2.94 (m, 1H), 1.97 (s, 1H), 1.83-1.80 (m, 1H), 1.74 (q, J = 7.2 Hz, 1H), 0.87 (s, 9H), 0.07 (s, 6H). HRMS (ESI, M+Na) calcd. for C 14 H 23 D 3 O 3 SiNa 296.1737, found 296.1736.

[0153] (2-5)d 3 -Synthesis of alkyne 15 [ka] Under argon atmosphere, d 3 -Alkyne 14 (0.51 g, 1.85 mmol) was dissolved in anhydrous tetrahydrofuran solution (9.25 mL), and triphenylphosphine (1.46 g, 5.55 mmol), imidazole (756 mg, 11.1 mmol), and iodine (1.41 g, 5.55 mmol) were added in sequence at -20°C, and the mixture was stirred at the same temperature for 0.5 hours, and then stirred at room temperature for 0.5 hours. After the stirring, a saturated aqueous solution of sodium thiosulfate (9.25 mL) was added to the reaction solution, which was extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After the solvent was distilled off, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 80 / 1) and d 3 -Alkyne 15 (0.62 g, 88% yield) was obtained as a colorless liquid. 1 H NMR (400 MHz, CDCl 3 ) δ 3.24-3.19 (m, 1H), 3.13-3.00 (m, 2H), 2.99-2.95 (m, 1H), 1.98 (s, 1H), 1.90-1.82 (m, 1H), 1.74-1.66 (m, 1H), 0.89 (s, 9H), 0.09 (s, 6H). HRMS (ESI, M+Na) calcd. for C 14 H 22 D 3 IO 2 SiNa 406.0755, found 406.0762.

[0154] (2-6)d 3 -Synthesis of alkyne 16 [ka] d 3-Alkyne 15 (0.58 g, 1.51 mmol) was dissolved in methanol (14.5 mL), acetic acid (0.58 mL), and zinc metal treated with hydrochloric acid (346 mg, 5.29 mmol) were added, stirred for 1 hour, filtered through Celite, and the solvent was distilled off. After the solvent was distilled off, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 80 / 1) and d 3 -Alkyne 16 (0.41 g, 99% yield) was obtained as a colorless liquid. 1 H NMR (400 MHz, CDCl 3 ) δ 5.92-5.82 (m, 1H), 5.30-5.24 (m, 1H), 5.10 (qt, J = 5.0, 1.4 Hz, 1H), 4.42-4.38 (m, 1H), 1.99 (s, 1H), 1.93-1.73 (m, 2H), 0.90 (s, 9H), 0.11 (s, 6H). HRMS (ESI, M+Na) calcd. for C 14 H 23 D 3 O 2 SiNa 280.1788, found 280.1804.

[0155] (2-7)d 3 -Resolution of alkyne 16 [ka] Under argon atmosphere, d 3 -Alkyne 16 (333 mg, 1.29 mmol) was dissolved in dehydrated toluene (26 mL) and heated at -60 °C to give (2S,3R)-HyperBTM (40.1 mg, 0.13 mmol, 10 mol%) and iPr 2 NEt (68 μL), (CO i P) 2O (65 μL) was added and the mixture was stirred at −60° C. for 8 hours. After the stirring, distilled water (20 mL) was added to the reaction solution, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After the solvent was distilled off, the residue was purified by silica gel column chromatography (hexane / ethyl acetate=100 / 1) to obtain d 3 The -alkyne (-)-16 (270 mg, 82% yield) was obtained as a colorless liquid.

[0156] (2-8)d 3 -Synthesis of alkyne 17 [ka] Under argon atmosphere, d 3 -Alkyne (-)-16 (259.1 mg, 1.01 mmol) was dissolved in dimethylformaldehyde (2.0 mL), and imidazole (116.4 mg, 1.71 mmol), tert-butyldimethylsilyl chloride (167 mg, 1.11 mmol), and 4-dimethylaminopyridine (6.8 mg, 0.056 mmol) were added and stirred at room temperature for 1 hour. After stirring, distilled water (10 mL) was added to the reaction solution, which was extracted with methylene chloride. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After distilling off the solvent, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 100 / 1) and d 3 -Alkyne 17 (308.9 mg, 82% yield) was obtained as a colorless liquid. 1 H NMR (400 MHz, CDCl 3 ) δ 5.87-5.76 (m, 1H), 5.20-5.02 (m, 2H), 4.23-4.19 (m, 1H), 1.97 (s, 1H), 1.91-1.78 (m, 1H), 1.65 (dd, J = 13.8, 4.8 Hz, 1H), 0.89 (s, 18H), 0.09-0.04 (m, 12H). HRMS (ESI, M+Na) calcd. for C 20 H 37 D 3 O3 S 2 Na 394.2653, found 394.2677.

[0157] (3) Synthesis of alkyne compound 30

[0158] Alkyne compound 30 was prepared by the synthetic route shown below, with each reaction in the synthetic route being explained below. [ka]

[0159] (3-1)d 5 Synthesis of -triol 19 [ka] Under a hydrogen atmosphere, triol 18 (1.62 g, 15.3 mmol) was dissolved in deuterium oxide (30 mL), ruthenium carbon (5%, 1.62 g, 16.0 mmol) was added at room temperature, and the mixture was stirred at 80° C. for 72 hours. After stirring, the reaction solution was filtered through Celite (ethyl acetate / methanol=8 / 1) and the solvent was distilled off. After the solvent was distilled off, the residue was purified by silica gel column chromatography (ethyl acetate / methanol=8 / 1) and deuterium oxide was obtained. 5 The -triol 19 (1.62 g, 100% yield) was obtained as a colorless liquid. 1 H NMR (300 MHz, D 2 O) δ 3.13 (s, 1H), 1.45 (dd, J = 31.0, 14.1 Hz, 2H). HRMS (ESI, M+Na) calcd. for C 4 H 5 D 5 O 3 Na 134.0842, found 134.0823.

[0160] (3-2)d 5 -Synthesis of alcohol 20 [ka] Under argon atmosphere, d 3 -Triol 19 (500 mg, 4.50 mmol) was dissolved in acetone (7 mL), p-toluenesulfonic acid (55.6 mg, 0.292 mmol) was added, and the mixture was stirred at room temperature for 12 hours. After stirring, triethylamine (0.233 mL) was added to the reaction solution, and the solvent was distilled off. After the solvent was distilled off, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 1 / 1) and d 5 The alcohol 20 (538 mg, 79% yield) was obtained as a colorless liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 2.10 (s, 1H), 1.81 (s, 2H), 1.43 (s, 3H), 1.36 (s, 3H). HRMS (ESI, M+Na) calcd. for C 7 H 9 D 5 O 5 Na 174.1155, found 174.1156.

[0161] (3-3)d 5 Synthesis of ester 21 [ka] Under argon atmosphere, d 5 Alcohol 20 (538 mg, 3.56 mmol) was dissolved in methylene chloride (7.1 mL), triethylamine (1.49 mL, 10.7 mmol) and benzoyl chloride (0.615 mL, 5.34 mmol) were added, and the mixture was stirred at room temperature for 5 hours. After stirring, distilled water (7 mL) was added to the reaction solution, which was then extracted with methylene chloride. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane / ethyl acetate=4 / 1) and d 5 The resulting ester 21 (909 mg, 100% yield) was obtained as a colorless liquid. 1 H NMR (300 MHz, CDCl3 ) δ 8.04-8.01 (m, 2H), 7.58-7.53 (m, 1H), 7.45-7.41 (m, 2H), 2.08-1.96 (m, 2H), 1.42 (s, 3H), 1.36 (s, 3H). HRMS (ESI, M+Na) calcd. for C 14 H 13 D 5 O 4 Na 278.1417, found 278.1400.

[0162] (3-4)d 5 Synthesis of -diol 22 [ka] Ester 21 (909 mg, 3.56 mmol) was dissolved in distilled water (1.34 mL), acetic acid (8.04 mL) was added at room temperature, and the mixture was stirred for 12 hours, after which the solvent was distilled off. After the solvent was distilled off, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 1 / 1) to give d 5 The diol 22 (784 mg, 88% yield) was obtained as a colorless liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 8.03-8.00 (m, 2H), 7.58-7.52 (m, 1H), 7.45-7.40 (m, 2H), 2.73 (s, 2H), 1.85 (dd, J = 23.4, 14.1 Hz, 2H). HRMS (ESI, M+Na) calcd. for C 11 H 9 D 5 O 4 Na 238.1104, found 238.1125.

[0163] (3-5)d 5 Synthesis of -bissilyl ether 23 [ka] Under argon atmosphere, d5 -Triol 22 (784 mg, 3.64 mmol) was dissolved in methylene chloride (18 mL), and 2,6-lutidine (1.12 mL, 9.47 mmol) and tert-butyldimethylsilyl triflate (2.09 ml, 9.11 mmol) were added under ice cooling, and the mixture was stirred at room temperature for 30 minutes. After stirring, distilled water (18 mL) was added to the reaction solution, which was extracted with methylene chloride. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After distilling off the solvent, the residue was purified by silica gel column chromatography (hexane / ethyl acetate=20 / 1) and d 5 The bis-silyl ether 23 (1.77 g, 100% yield) was obtained as a colorless liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 8.06-8.03 (m, 2H), 7.58-7.53 (m, 1H), 7.44 (t, J = 7.6 Hz, 2H), 2.07 (d, J = 14.1 Hz, 1H), 1.82-1.75 (m, 1H), 0.89 (s, 18H), 0.06 (t, J = 2.4 Hz, 12H). HRMS (ESI, M+Na) calcd. for C 23 H 37 D 5 O 4 S 2 Na 466.2833, found 466.2862.

[0164] (3-6)d 5 -Synthesis of alcohol 24 [ka] Under argon atmosphere, d 5-Bissilyl ether 23 (195 mg, 0.438 mmol) was dissolved in methanol (2 mL) and dichloromethane (2 mL), camphorsulfonic acid (5.1 mg, 0.0219 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After stirring, saturated aqueous sodium bicarbonate solution (4 mL) was added to the reaction solution, and the solvent was distilled off. After distillation of the solvent, the residue was purified by silica gel column chromatography (hexane / ethyl acetate) and d 5 -Alcohol 24 (77 mg, 58% yield) was obtained as a colorless liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 8.04-8.01 (m, 2H), 7.59-7.54 (m, 1H), 7.44 (t, J = 7.7 Hz, 2H), 2.02-1.76 (m, 2H), 0.94-0.89 (m, 9H), 0.12-0.07 (m, 6H). HRMS (ESI, M+Na) calcd. for C 17 H 23 D 5 O 4 SiNa 352.1968, found 352.2010.

[0165] (3-7)d 4 -Synthesis of alkyne 25 [ka] Under an argon atmosphere, oxalyl chloride (0.24 mL, 2.8 mmol) was dissolved in dehydrated methylene chloride (7 mL), and dimethyl sulfoxide (0.497 mL, 7.00 mmol) was added dropwise at -78°C. The reaction mixture was stirred at the same temperature for 10 minutes, and then cooled to 5°C. 5 A methylene chloride solution (7 mL) of alcohol 24 (461 mg, 1.40 mmol) and triethylamine (1.94 mL, 14.0 mmol) were added dropwise in that order. The reaction mixture was stirred at the same temperature for another hour. After stirring, distilled water (14 mL) was added to the reaction mixture, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After the solvent was removed by distillation, the residue (d4 -aldehyde) was used in the next step without further purification. Under an argon atmosphere, trimethylsilylacetylene (0.996 mL, 7.2 mmol) was dissolved in anhydrous tetrahydrofuran solution (12 mL), and n-butyllithium (2.6 M hexane solution) (2.3 mL, 6.0 mmol) was added dropwise at -78°C to prepare a reaction mixture. The reaction mixture was stirred at the same temperature for 30 minutes, and then d 4 A solution (12 mL) of -aldehyde (772 mg, 2.40 mmol) in tetrahydrofuran was added dropwise. After the addition, the reaction mixture was stirred at the same temperature for another 15 minutes. After the stirring, a saturated aqueous ammonium chloride solution (24 mL) was added to the reaction mixture, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After the solvent was removed by distillation, the residue was purified by silica gel column chromatography (hexane / ethyl acetate=30 / 1) to give d 4 -Alkyne 25 (910 mg, 90% yield) was obtained as a colorless liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 8.06-8.02 (m, 2H), 7.59-7.54 (m, 1H), 7.45 (t, J = 7.6 Hz, 2H), 2.38-1.89 (m, 2H), 0.93-0.90 (m, 9H), 0.16-0.06 (m, 15H). HRMS (ESI, M+Na) calcd. for C 22 H 32 D 4 O 4 S 2 Na 447.2301, found 447.2289.

[0166] (3-8)d 4 -Synthesis of alkyne 26 [ka] Under argon atmosphere, d 4-Alkyne 25 (297 mg, 0.700 mmol) was dissolved in methylene chloride (7 mL), and 2,6-lutidine (0.199 mL, 1.68 mmol) and tert-butyldimethylsilyl triflate (0.193 ml, 0.840 mmol) were added under ice cooling, and the mixture was stirred at room temperature for 1 hour. After stirring, distilled water (7 mL) was added to the reaction solution, which was extracted with methylene chloride. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After distilling off the solvent, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 40 / 1) and d 4 The bis-silyl ether 26 (381 mg, 100% yield) was obtained as a colorless liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 8.06-8.03 (m, 2H), 7.55 (t, J = 7.4 Hz, 1H), 7.44 (t, J = 7.6 Hz, 2H), 2.19-1.90 (m, 2H), 0.93-0.87 (m, 18H), 0.24-0.03 (m, 21H). HRMS (ESI, M+Na) calcd. for C 28 H 46 D 4 O 4 S 3 Na 561.3166, found 561.3141.

[0167] (3-9)d 4 -Synthesis of alkyne 27 [ka] D 4 -Alkyne 26 (314 mg, 0.587 mmol) was dissolved in methanol (2 mL), and potassium carbonate (324 mg, 2.35 mmol) was added under ice cooling, and the mixture was stirred at room temperature for 1 hour. After stirring, distilled water (2 mL) was added to the reaction solution, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane / ethyl acetate=30 / 1) and d 4-Alcohol 27 (152 mg, 76% yield) was obtained as a colorless liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 2.41 (s, 1H), 1.99-1.59 (m, 2H), 0.90 (d, J = 1.7 Hz, 18H), 0.16-0.09 (m, 12H). HRMS (ESI, M+Na) calcd. for C 18 H 34 D 4 O 3 S 2 Na 385.2508, found 385.2549.

[0168] (3-10)d 4 -Synthesis of alkyne 28 [ka] Under argon atmosphere, d 4 Alcohol 27 (136 mg, 0.375 mmol) was dissolved in anhydrous tetrahydrofuran solution (1.6 mL), and triphenylphosphine (509 mg, 2.40 mmol), imidazole (76.6 mg, 1.13 mmol), and iodine (306 mg, 1.20 mmol) were added in that order at -20°C, and the mixture was stirred for 30 minutes, and then stirred at room temperature for 20 minutes. After stirring, a saturated sodium thiosulfate solution (1.6 mL) was added to the reaction solution, which was extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After the solvent was distilled off, the residue was purified by silica gel column chromatography (hexane) and d 4 The -iodide 28 (174 mg, 98% yield) was obtained as a colorless liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 2.39 (s, 1H), 2.20-2.06 (m, 2H), 0.90 (d, J = 2.1 Hz, 18H), 0.14-0.12 (m, 12H). HRMS (ESI, M+Na) calcd. for C18 H 33 D 4 IO 2 S 2 Na 495.1526, found 495.1497.

[0169] (3-11)d 4 Synthesis of nitrile 29 [ka] Under argon atmosphere, d 4 -Iodide 28 (174 mg, 0.369 mmol) was dissolved in dimethylformamide (0.9 mL), sodium cyanide (27.1 mg, 0.554 mmol) was added at room temperature, and the mixture was stirred at 90°C for 20 minutes. After stirring, saturated aqueous sodium bicarbonate solution (1 mL) was added to the reaction mixture, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 80 / 1) to give d 4 The -nitrile 29 (130 mg, 94% yield) was obtained as a colorless liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 2.42 (s, 1H), 1.98 (dd, J = 24.1, 13.8 Hz, 2H), 0.90 (s, 18H), 0.14-0.11 (m, 12H). HRMS (ESI, M+Na) calcd. for C 19 H 33 D 4 NO 2 S 2 Na 394.2540, found 394.2512.

[0170] (3-12)d 4 -Synthesis of alkyne 30 [ka] Under argon atmosphere, d 4Nitrile 29 (108 mg, 0.291 mmol) was dissolved in dehydrated dichloromethane (1.5 mL), and diisobutylaluminum hydride (1.0 M hexane solution) (0.35 mL, 0.35 mmol) was added dropwise under ice cooling, and the mixture was stirred for 20 minutes. After the stirring, isopropanol (0.245 mL), silica gel (700 mg), and distilled water (1 mL) were added to the reaction solution, and the mixture was stirred for 30 minutes. After filtration through Celite, the solvent was distilled off. After the solvent was distilled off, the residue (d 4 -aldehyde) was used in the next step without further purification. Under an argon atmosphere, methyltriphenylphosphonium iodide (623 mg, 1.54 mmol) was dissolved in dehydrated tetrahydrofuran solution (1.5 mL), and sodium hexamethyldisilazide (1.9 M tetrahydrofuran solution) (0.766 mL, 1.46 mmol) was added dropwise under ice cooling. The reaction mixture was stirred at the same temperature for 30 minutes, and then d 4 A solution (1.5 mL) of -aldehyde (109 mg, 0.291 mmol) in tetrahydrofuran was added dropwise to prepare a reaction mixture. The reaction mixture was stirred at the same temperature for another 30 minutes. After stirring, a saturated aqueous ammonium chloride solution (3 mL) was added to the reaction mixture, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. The residue was purified by silica gel column chromatography (hexane / ethyl acetate=100 / 0) to give d 4 -Alkyne 30 (55.9 mg, 52% yield) was obtained as a colorless liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 5.88-5.77 (m, 1H), 5.05-4.94 (m, 2H), 2.35 (s, 1H), 1.78-1.56 (m, 2H), 0.91 (s, 18H), 0.12 (dd, J = 13.8, 7.9 Hz, 12H). HRMS (ESI, M+Na) calcd. for C 20 H 36 D 4 O 2 S 2Na 395.2716, found 395.2699.

[0171] (4) Synthesis of non-isotopically labeled alkyne compound 42

[0172] In the experiments described below, non-isotopically labeled vitamin D compounds were used. To produce the vitamin D compounds, non-isotopically labeled alkyne compound 42 was prepared by the synthetic route shown below. Each reaction in the synthetic route is described below. [ka]

[0173] (4-1) Synthesis of Alcohol 32 [ka] Under an argon atmosphere, triol 31 (4.0 g, 37.3 mmol) was dissolved in acetone (60 mL), p-toluenesulfonic acid (461 mg, 2.4 mmol) was added, and the mixture was stirred at room temperature for 12 hours. Triethylamine (1.9 mL) was added to the reaction solution, and the solvent was distilled off. After the solvent was distilled off, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 1 / 1) to obtain alcohol 32 (2.8 g, 52% yield) as a colorless liquid.

[0174] (4-2) Synthesis of ester 33 [ka] Under an argon atmosphere, alcohol 32 (2.8 g, 3.6 mmol) was dissolved in methylene chloride (40 mL), triethylamine (8 mL, 58.0 mmol) and benzoyl chloride (3.3 mL, 29.0 mmol) were added, and the mixture was stirred at room temperature for 5 hours. After stirring, distilled water (40 mL) was added to the reaction solution, which was then extracted with methylene chloride. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 4 / 1) to obtain ester 33 (4.8 g, 100% yield) as a colorless liquid.

[0175] (4-3) Synthesis of diol 34 [ka] Ester 33 (5.6 g, 22.6 mmol) was dissolved in distilled water (6.5 mL), and acetic acid (40 mL) was added at room temperature. After stirring for 12 hours, the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 1 / 1) to obtain diol 34 (4.4 g, 88% yield) as a colorless liquid.

[0176] (4-4) Synthesis of bis-silyl ether 35 [ka] Under an argon atmosphere, diol 34 (300 mg, 1.4 mmol) was dissolved in methylene chloride (7.0 mL), and 2,6-lutidine (0.44 mL, 3.7 mmol) and tert-butyldimethylsilyl triflate (0.82 ml, 3.6 mmol) were added under ice cooling, and the mixture was stirred at room temperature for 30 minutes. After stirring, distilled water (7.0 mL) was added to the reaction solution, which was extracted with methylene chloride. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After distilling off the solvent, the residue was purified by silica gel column chromatography (hexane / ethyl acetate=20 / 1) to obtain bissilyl ether 35 (627 mg, 100% yield) as a colorless liquid.

[0177] (4-5) Synthesis of alcohol 36 [ka] Under an argon atmosphere, the bissilyl ether 35 (627 mg, 1.4 mmol) was dissolved in methanol (8.5 mL) and dichloromethane (8.5 mL), camphorsulfonic acid (40 mg, 0.17 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After stirring, a saturated aqueous solution of sodium bicarbonate (17 mL) was added to the reaction solution, and the solvent was distilled off. After distilling off the solvent, the residue was purified by silica gel column chromatography (hexane / ethyl acetate=10 / 1) to obtain alcohol 36 (77 mg, 54% yield) as a colorless liquid.

[0178] (4-6) Synthesis of alkyne 37 [ka] Under an argon atmosphere, oxalyl chloride (0.48 mL, 5.6 mmol) was dissolved in dehydrated methylene chloride (7.0 mL), and dimethyl sulfoxide (1.0 mL, 14.0 mmol) was added dropwise at -78°C. The reaction mixture was stirred at the same temperature for 10 minutes, and then a methylene chloride solution (7.0 mL) of alcohol 36 (902 mg, 2.78 mmol) and triethylamine (3.9 mL, 27.8 mmol) were added dropwise in that order. The reaction mixture was stirred at the same temperature for another hour. After the stirring, distilled water (14 mL) was added to the reaction solution, which was extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After the solvent was distilled off, the residue was filtered using silica gel column chromatography (hexane / ethyl acetate = 1 / 1) to obtain the aldehyde. This was used in the next step without further purification. Under an argon atmosphere, trimethylsilylacetylene (1.0 mL, 7.2 mmol) was dissolved in anhydrous tetrahydrofuran solution (12 mL), and n-butyllithium (2.6 M hexane solution) (2.3 mL, 6.0 mmol) was added dropwise at -78°C. The reaction mixture was stirred at the same temperature for 30 minutes, and then the tetrahydrofuran solution of the aldehyde (12 mL) was added dropwise in sequence. The reaction mixture was stirred at the same temperature for another 15 minutes. After the stirring, a saturated aqueous ammonium chloride solution (24 mL) was added to the reaction solution, which was extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After the solvent was distilled off, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 30 / 1) to obtain alkyne 37 (910 mg, yield 90%) as a colorless liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 8.05-8.03 (m, 2H), 7.59-7.53 (m, 1H), 7.44 (t, J = 7.6 Hz, 2H), 4.56-4.26 (m, 3H), 4.04-3.99 (m, 1H), 2.33-1.87 (m, 2H), 0.92-0.88 (m, 9H), 0.16-0.08 (m, 15H). HRMS (ESI, M+Na) calcd. for C 22 H 36 O 4 S 2 Na 443.2050, found 443.2017.

[0179] (4-7) Synthesis of alkyne 38 [ka] Under an argon atmosphere, alkyne 37 (264 mg, 0.63 mmol) was dissolved in methylene chloride (6.0 mL), and 2,6-lutidine (0.22 mL, 1.9 mmol) and tert-butyldimethylsilyl triflate (0.22 ml, 0.94 mmol) were added under ice cooling, and the mixture was stirred at room temperature for 1 hour. After stirring, distilled water (6.0 mL) was added to the reaction solution, which was extracted with methylene chloride. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After distilling off the solvent, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 40 / 1) to obtain alkyne 38 (334 mg, 100% yield) as a colorless liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 8.06-8.03 (m, 2H), 7.58-7.53 (m, 1H), 7.44 (t, J = 7.4 Hz, 2H), 4.54-4.36 (m, 2H), 4.28 (d, J = 4.5 Hz, 1H), 3.94-3.89 (m, 1H), 2.19-1.92 (m, 2H), 0.91-0.87 (m, 18H), 0.15-0.02 (m, 21H). HRMS (ESI, M+Na) calcd. for C 28 H 50 O 4 S 3 Na 557.2915, found 557.2872.

[0180] (4-8) Synthesis of alkyne 39 [ka] Alkyne 38 (206 mg, 0.39 mmol) was dissolved in methanol (1.3 mL), and potassium carbonate (213 mg, 1.5 mmol) was added under ice cooling, followed by stirring at room temperature for 1 hour. After stirring, distilled water (1.3 mL) was added to the reaction solution, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane / ethyl acetate=10 / 1) to obtain alkyne 39 (104 mg, 76% yield) as a colorless liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 4.36 (q, J = 2.2 Hz, 1H), 3.94 (q, J = 4.8 Hz, 1H), 3.80-3.75 (m, 2H), 2.41 (d, J = 2.1 Hz, 1H), 2.15-1.82 (m, 3H), 0.90 (d, J = 1.7 Hz, 18H), 0.16-0.09 (m, 12H). HRMS (ESI, M+Na) calcd. for C 18 H 38 O 3 S 2 Na 381.2257, found 381.2263.

[0181] (4-9) Synthesis of iodide 40 [ka] Under an argon atmosphere, alkyne 39 (136 mg, 0.38 mmol) was dissolved in anhydrous tetrahydrofuran solution (1.6 mL), and triphenylphosphine (193 mg, 0.91 mmol), imidazole (76.6 mg, 1.1 mmol), and iodine (306 mg, 1.2 mmol) were added in sequence at -20°C, and the mixture was stirred for 30 minutes, and then stirred at room temperature for 20 minutes. After the stirring, a saturated sodium thiosulfate solution (1.6 mL) was added to the reaction solution, which was extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After the solvent was distilled off, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 100 / 0) to obtain iodide 40 (149 mg, 84% yield) as a colorless liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 4.25 (q, J = 2.1 Hz, 1H), 3.79-3.68 (m, 1H), 3.41-3.18 (m, 2H), 2.39 (d, J = 2.4 Hz, 1H), 2.19-2.12 (m, 2H), 0.90 (d, J = 2.1 Hz, 18H), 0.14-0.12 (m, 12H). HRMS (ESI, M+Na) calcd. for C 18 H 37 IO 2 S 2 Na 491.1274, found 491.1243.

[0182] (4-10) Synthesis of nitrile 41 [ka] Under an argon atmosphere, iodide 40 (133 mg, 0.28 mmol) was dissolved in dimethylformamide (0.7 mL), sodium cyanide (21.0 mg, 0.43 mmol) was added at room temperature, and the mixture was stirred at 90°C for 20 minutes. After stirring, saturated aqueous sodium bicarbonate solution (1.0 mL) was added to the reaction solution, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 80 / 1) to obtain nitrile 41 (105 mg, 94% yield) as a colorless liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 4.27 (q, J = 2.1 Hz, 1H), 3.85-3.80 (m, 1H), 2.51-2.36 (m, 3H), 2.12-1.89 (m, 2H), 0.90 (s, 18H), 0.15-0.11 (m, 12H). HRMS (ESI, M+Na) calcd. for C 19 H 37 NO 2 S 2 Na 390.2261, found 390.2249.

[0183] (4-11) Synthesis of alkyne 42 [ka] Under an argon atmosphere, nitrile 41 (32.5 mg, 0.089 mmol) was dissolved in dehydrated dichloromethane (0.4 mL), and diisobutylaluminum hydride (1.0 M hexane solution) (0.11 mL, 0.11 mmol) was added dropwise under ice cooling, and the mixture was stirred for 20 minutes. After the stirring, isopropanol (0.074 mL), silica gel (200 mg), and distilled water (1 mL) were added to the reaction solution, and the mixture was stirred for 30 minutes. After filtration through Celite, the solvent was distilled off. After the solvent was distilled off, the residue was filtered using silica gel column chromatography (hexane / ethyl acetate = 10 / 1) to obtain the aldehyde. This was used in the next step without further purification. Under an argon atmosphere, methyltriphenylphosphonium iodide (623 mg, 1.5 mmol) was dissolved in anhydrous tetrahydrofuran solution (0.75 mL), and sodium hexamethyldisilazide (1.9 M tetrahydrofuran solution) (0.77 mL, 1.5 mmol) was added dropwise under ice cooling. The reaction mixture was stirred at the same temperature for 30 minutes, and then a tetrahydrofuran solution (0.75 mL) of the aldehyde (109 mg, 0.29 mmol) was added dropwise. The reaction mixture was stirred at the same temperature for another 30 minutes. After the stirring, a saturated aqueous ammonium chloride solution (3 mL) was added to the reaction solution, which was extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was distilled off. After the solvent was distilled off, the residue was purified by silica gel column chromatography (hexane / ethyl acetate=100 / 0) to obtain alkyne 42 (55.9 mg, yield 62%) as a colorless liquid. 1 H NMR (300 MHz, CDCl 3 ) δ 5.89-5.76 (m, 1H), 5.05-4.93 (m, 2H), 4.23 (q, J = 2.3 Hz, 1H), 3.73 (dd, J = 10.8, 4.6 Hz, 1H), 2.35 (d, J = 2.4 Hz, 1H), 2.20-2.07 (m, 2H), 1.79-1.59 (m, 2H), 0.91 (s, 18H), 0.11 (dd, J = 13.4, 7.6 Hz, 12H). HRMS (ESI, M+Na) calcd. for C 20 H 40 O 2 S 2 Na 391.2465, found 391.2442.

[0184] (6) Vitamin D Compound 44 (25OH D 3 -3,4,4'-d 3 "25OHD 3 -d 3 " is also called.) Synthesis of

[0185] As shown in the following reaction scheme, the alkyne compound 10 (d 3 The vitamin D compound 44 was prepared by reacting -alkyne 10) with bromoolefin 43. [ka]

[0186] Under argon atmosphere, d 3 -Alkyne 10 (10 mg, 0.04 mmol) and bromoolefin 60 (29.3 mg, 0.06 mmol) were dissolved in dehydrated toluene (1.0 mL) and triethylamine (1.0 mL), triphenylphosphine tetrakispalladium was added, and the mixture was stirred at 90°C for 1 hour. After stirring, the reaction solution was filtered through Celite (ethyl acetate) and the solvent was distilled off. After the solvent was distilled off, the residue (vitamin D3-3, 25-bissilyl ether-d 3 (V.D. 3 -3,25-bissilyl ether-d 3 ) was filtered using silica gel column chromatography (hexane / ethyl acetate = 50 / 1) and used in the next step without further purification.

[0187] Under argon atmosphere, VD 3 -3,25-bissilyl ether-d 3The compound was dissolved in methanol (0.5 mL), and methanesulfonic acid (10 μL) was added dropwise under ice cooling, followed by stirring for 30 minutes. After stirring, a saturated aqueous solution of sodium bicarbonate was added to the reaction solution, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane / ethyl acetate=1:1) and reverse phase HPLC to obtain vitamin D compound 44 (25OHD 3 -d 3 The compound was obtained as a colorless solid (0.29 mg, 1.8% yield, 2 steps). The physical properties of the produced vitamin D compound 44 were as follows, and its NMR spectrum data is shown in FIG. 1A. 1 H NMR (400 MHz, CDCl 3 ) δ 6.23 (d, J = 11.5 Hz, 1H), 6.03 (d, J = 11.5 Hz, 1H), 5.05 (s, 1H), 4.82 (s, 1H), 2.82 (d, J = 12.6 Hz, 1H), 2.37-2.42 (m, 1H), 2.15-2.20 (m, 1H), 1.96-2.04 (m, 2H), 1.84-1.94 (m, 2H), 1.25-1.70 (m, 16H), 1.21 (s, 6H), 0.95 (d, J = 5.2 Hz, 3H), 0.54 (s, 3H). HRMS (ESI, M+Na) calcd. for C 27 H 41 D 3 O 2 Na 426.3427, found 426.3454.

[0188] (7) Vitamin D Compound 46(25OHD 3 -3,4,4'-d 3 -26R,23S-lactone, 25OHD 3 -26R,23S-lactone-d 3 " (also called ")

[0189] Vitamin D compound 46 was synthesized by carrying out the same synthesis as in (6) above, except that the following bromoolefin 45 was used instead of bromoolefin 60. [ka] The physical property data measured for the produced vitamin D compound 46 are as follows, and the NMR spectrum data thereof is shown in FIG. 1B. 1 H NMR (400 MHz, CDCl 3 ) δ 6.23 (d, J = 11.4 Hz, 1H), 6.03 (d, J = 10.5 Hz, 1H), 5.05 (s, 1H), 4.81 (s, 1H), 4.54-4.36 (m, 1H), 3.75-3.56 (m, 2H), 2.83 (d, J = 12.8 Hz, 1H), 2.51-2.31 (m, 2H), 2.26-2.10 (m, 1H), 2.11-1.80 (m, 6H), 1.81-1.43 (m, 8H), 1.42-1.15 (m, 6H), 1.02 (d, J = 6.4 Hz, 3H), 0.55 (s, 3H). HRMS (ESI, M+Na) calcd. for C 27 H 37 D 3 O 4 Na 454.3013, found 454.3009.

[0190] (8) Vitamin D compound 47(1α,25(OH) 2 D 3 -3,4,4'-d 3 , "1α,25(OH) 2 D 3 -d 3 " (also called ")

[0191] Vitamin D compound 47 was synthesized by carrying out the same synthesis as in (6) above, except that alkyne compound 17 was used instead of alkyne compound 10. [ka] The physical properties of the produced vitamin D compound 47 were as follows, and its NMR spectrum data is shown in FIG. 1C. 1 H NMR (300 MHz, CDCl 3 ) δ 6.38 (d, J = 11.0 Hz, 1H), 6.01 (d, J = 11.7 Hz, 1H), 5.33 (s, 1H), 5.00 (s, 1H), 4.42-4.42 (m, 1H), 2.82 (d, J = 12.4 Hz, 1H), 1.25-2.05 (m, 20H), 1.21 (s, 6H), 0.93 (d, J = 6.2 Hz, 3H), 0.54 (s, 3H). HRMS (ESI, M+Na) calcd. for C 27 H 41 D 3 O 3 Na 442.3376, found 442.3336.

[0192] (9) Vitamin D Compound 48(1α,25(OH) 2 D 3 -3,4,4'-d 3 -26R,23S-lactone, “1α,25(OH) 2 D 3 -26R,23S-lactone-d 3 " (also called ")

[0193] The following vitamin D compound 48 was synthesized by carrying out the same synthesis as in (6) above, except that the above bromoolefin 45 was used instead of bromoolefin 43 and the alkyne compound 17 was used instead of alkyne compound 10. [ka] The physical properties of the produced vitamin D compound 48 were as follows, and its NMR spectrum data is shown in FIG. 1D. 1 H NMR (300 MHz, CDCl 3 ) δ 6.37 (d, J = 11.4 Hz, 1H), 6.01 (d, J = 10.7 Hz, 1H), 5.33 (s, 1H), 5.00 (s, 1H), 4.46-4.46 (m, 2H), 2.83 (d, J = 13.1 Hz, 1H), 2.39 (dd, J = 13.2, 5.0 Hz, 1H), 1.25-2.05 (m, 20H), 1.02 (d, J = 6.5 Hz, 3H), 0.55 (s, 3H). HRMS (ESI, M+Na) calcd. for C 27 H 37 D 3 O 5 Na 470.2962, found 470.2963.

[0194] (10) Vitamin D compound 49 (24R,25(OH) 2 D 3 -3,4,4'-d 3 , "24R,25(OH) 2 D 3 -d 3 " (also called ")

[0195] The following vitamin D compound 50 was synthesized by carrying out the same synthesis as in (6) above, except that the following bromoolefin 49 was used instead of bromoolefin 43. [ka] The physical properties of the produced vitamin D compound 67 were as follows, and its NMR spectrum data is shown in FIG. 1E. 1 H NMR (400 MHz, CDCl 3) δ 6.23 (d, J = 11.0 Hz, 1H), 6.03 (d, J = 11.4 Hz, 1H), 5.05 (s, 1H), 4.82 (s, 1H), 3.34 (t, J = 6.0 Hz, 1H), 2.83 (d, J = 12.8 Hz, 1H), 2.35-2.42 (m, 1H), 2.18-2.20 (m, 1H), 1.16-2.01 (m, 24H), 0.94 (d, J = 6.4 Hz, 3H), 0.55 (s, 3H). HRMS (ESI, M+Na) calcd. for C 27 H 41 D 3 O 3 Na 442.3376, found 442.3402.

[0196] (11) Vitamin D compound 51 (1α, 24R, 25(OH) 3 -3,4,4'-d 3 D 3 , "1α,24R,25(OH) 3 D 3 -d 3 " (also called ")

[0197] The following vitamin D compound 51 was synthesized by carrying out the same synthesis as in (6) above, except that the above-mentioned bromoolefin 49 was used instead of bromoolefin 43 and the alkyne compound 17 was used instead of alkyne compound 10. [ka] The physical properties of the produced vitamin D compound 51 were as follows, and its NMR spectrum data is shown in FIG. 1F. 1 H NMR (500 MHz, CD 3OD) δ 6.36 (d, J = 11.5 Hz, 1H), 6.12 (d, J = 11.5 Hz, 1H), 5.33 (s, 1H), 4.90 (s, 1H), 4.38 (t, J = 6.0 Hz, 1H), 3.24 (d, J = 9.7 Hz, 1H), 2.87-2.92 (m, 1H), 1.24-2.09 (m, 18H), 1.20 (s, 3H), 1.16 (s, 3H), 1.00 (d, J = 6.3 Hz, 3H), 0.62 (s, 3H). HRMS (ESI, M+Na) calcd. for C 27 H 41 D 3 O 4 Na 458.3326, found 458.3334.

[0198] (12) Vitamin D compound 52(4β,25(OH) 2 D 3 -1,1',3,4-d 3 , "4β,25(OH) 2 D 3 -d 4 " (also called ")

[0199] Vitamin D compound 52 was synthesized by carrying out the same synthesis as in (6) above, except that alkyne compound 30 was used instead of alkyne compound 10. [ka]

[0200] Under an argon atmosphere, the alkyne compound 30 (d 4 -alkyne, 6.5 mg, 0.027 mmol) and bromoolefin 43 (9.8 mg, 0.032 mmol) were dissolved in dehydrated toluene (0.3 mL) and triethylamine (0.3 mL), triphenylphosphinetetrakispalladium was added, and the mixture was stirred at 90°C for 1 hour. After stirring, the reaction solution was filtered through Celite (ethyl acetate) and the solvent was distilled off. After distillation of the solvent, the residue (vitamin D 3-3,4,25-trisilyl ether -d 4 (V.D. 3 -3,4,25-trisilyl ether-d 4 ) was filtered using silica gel column chromatography (hexane / ethyl acetate = 50 / 1) and used in the next step without further purification.

[0201] Under argon atmosphere, VD 3 -3,4,25-trisilyl ether-d 4 The residue was dissolved in tetrahydrofuran solution (0.4 mL), and tetra-n-butylammonium fluoride (1 M tetrahydrofuran solution) (0.075 mL, 0.075 mmol) was added dropwise under ice cooling, followed by stirring for 12 hours. After stirring, a saturated aqueous solution of ammonium chloride was added to the reaction solution, which was then extracted with ethyl acetate. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by distillation. After removing the solvent, the residue was purified by silica gel column chromatography (hexane / ethyl acetate=1:1) and reverse phase HPLC to obtain vitamin D compound 52 (d4-4β,25(OH) 2 Vitamin D 3 The compound was obtained as a colorless solid (1.8 mg, 27% yield, 2 steps). The physical properties of the produced vitamin D compound 52 were as follows, and its NMR spectrum data is shown in FIG. 1G. 1 H NMR (400 MHz, CDCl 3 ) δ 6.51 (d, J = 11.4 Hz, 1H), 6.04 (d, J = 11.4 Hz, 1H), 5.17 (d, J = 2.3 Hz, 1H), 4.92 (d, J = 2.3 Hz, 1H), 2.86 (d, J = 13.7 Hz, 1H), 2.00-1.98 (m, 2H), 1.90-1.80 (m, 2H), 1.53-1.34 (m, 5H), 1.32-1.25 (m, 1H), 1.22 (s, 6H), 1.09-1.04 (m, 1H), 0.94 (d, J = 6.4 Hz, 3H), 0.54 (s, 3H). HRMS (ESI, M+Na) calcd. for C 27 H 40 D 4 O 3 Na 443.3439, found 443.3459.

[0202] (13) Non-isotopically labeled vitamin D compound 54(4β,25(OH) 2 D 3 Synthesis of

[0203] Vitamin D compound 54 shown below was synthesized by carrying out the same synthesis as above (12), except that non-isotopically labeled alkyne compound 53 was used instead of alkyne compound 30. [ka] The physical property data of the obtained vitamin D compound 54 are as follows, and the NMR spectrum data thereof is shown in FIG. 1H. 1 H NMR (500 MHz, CDCl 3 ) δ 6.51 (d, J = 10.9 Hz, 1H), 6.04 (d, J = 11.5 Hz, 1H), 5.17 (s, 1H), 4.92 (s, 1H), 4.22 (s, 1H), 3.86 (s, 1H), 2.86 (d, J = 13.2 Hz, 1H), 2.38-2.35 (m, 1H), 2.18-2.13 (m, 1H), 2.01 (d, J = 10.9 Hz, 2H), 1.85 (d, J = 6.3 Hz, 3H), 1.71-1.33 (m, 17H), 1.21 (s, 6H), 1.05 (q, J = 9.9 Hz, 1H), 0.93 (d, J = 6.3 Hz, 3H), 0.54 (s, 3H). HRMS (ESI, M+Na) calcd. for C 27 H 40 D 4 O 3Na 439.3188, found 439.3181.

[0204] 6-2. Example 2: 25OHD in pooled serum 3 -26R,23S-lactone and 1α,25(OH) 2 D 3 LC-MS / MS analysis of -26R,23S-lactone

[0205] The above two vitamin D compounds contained in pooled serum were quantitatively analyzed by LC-MS / MS using the stable isotope-labeled vitamin D compounds synthesized in Example 1. The procedure for the quantitative analysis is as follows.

[0206] (1) Preparation of calibrator 25OHD 3 -26R,23S-lactone standard and 1α,25(OH) 2 D 3 A 30% acetonitrile solution of -26R,23S-lactone standard was prepared at the concentration shown in Table 2 below.

[0207] [Table 2]

[0208] (2) Preparation of IS (internal standard) solution Vitamin D Compound 46 (25OHD 3 -26R,23S-lactone-d 3 ) and vitamin D compound 48 (1α,25(OH) 2 D 3 -26R,23S-lactone-d 3 A 30% acetonitrile solution of the standard was prepared at the concentration shown in Table 3 below.

[0209] [Table 3]

[0210] (3) Sample pretreatment The sample was pretreated according to the following procedure. (3-1) SLE (solid-liquid) extraction 1) 100 μL of serum was mixed with 200 μL of IS solution. 2) 300 μL of serum / IS mixture was loaded onto the SLE column. 3) 600 μL of hexane / ethyl acetate (50 / 50, v / v) was added for elution. This was repeated three times. (3-2) Drying: Nitrogen spray or centrifugal concentration for about 20 minutes (3-3) Derivatization 1) Preheat the derivatization reagent DAP-PA (80°C, 15 minutes). (The structure of DAP-PA and its derivatization reaction are shown in Figure 2G.) 2) 100 μL of DAP-PA (pre-warmed) was added to the dried sample. 3) Leave to stand at room temperature (15 minutes). 4) Drying: Centrifugal concentration for about 15 minutes 5) Dissolve in 50 μL of reconstitution solution (50% acetonitrile).

[0211] (4)LC analysis conditions The LC analysis conditions were as follows. Equipment: Waters ACQUITY UPLC I-Class Analytical column: Osaka Soda Co., Ltd. CAPCELL CORE C18 2.1 mmI.D. x 75 mm Elution conditions: Flow rate 0.4 mL / min Solvent A: 0.1% formic acid-water B: 0.1% formic acid-acetonitrile The detailed elution conditions are shown in Table 4 below.

[0212] [Table 4]

[0213] (5)MS / MS analysis conditions The MS / MS analysis conditions were as follows: Equipment: Waters Xevo TQ-XS triple quadrupole mass spectrometer Ionization conditions: ESI positive ion mode The SRM parameters were as shown in Table 5 below.

[0214] [Table 5]

[0215] (6) Analysis results of the sample The results of the analysis of the preparations are shown in Figures 2A to 2D. Figure 2A shows the 25OHD 3 -26R,23S-lactone-d 3 This is an SRM chromatogram of a standard sample. Figure 2B shows 1α,25(OH) 2 D 3 -26R,23S-lactone-d 3 This is an SRM chromatogram of a standard sample. Figure 2C shows 25OHD 3 This is a calibration curve for the -26R,23S-lactone standard. FIG. 2D shows 1α,25(OH) 2 D 3 This is a calibration curve for the -26R,23S-lactone standard.

[0216] (7) 25OHD in pooled serum 3 -26R,23S-lactone and 1α,25(OH) 2 D 3 -26R,23S-lactone quantitative value Based on the above analysis results, 25OHD in pooled serum 3 -26R,23S-lactone and 1α,25(OH) 2 D 3 -26R,23S-lactone was quantified as shown in Table 6. Thus, by using the vitamin D compound of the present invention as a standard substance (particularly an internal standard substance) in mass spectrometry, vitamin D metabolites in a sample (particularly a biological sample) can be quantified.

[0217] Figure 2E shows the 25OHD in pooled serum.3 This is an SRM chromatogram of -26R,23S-lactone. Figure 2F shows the 1α,25(OH) 2 D 3 This is an SRM chromatogram of -26R,23S-lactone.

[0218] [Table 6]

[0219] 6-3. Example 3: 25OHD in pooled serum 3 , 24R,25(OH) 2 D 3 LC-MS / MS analysis of

[0220] The above two vitamin D compounds contained in pooled serum were quantitatively analyzed by LC-MS / MS using the stable isotope-labeled vitamin D compounds synthesized in Example 1. The procedure for the quantitative analysis was as follows.

[0221] (1) Calibrator As a calibrator, JeoQuant Kit for LC / MS / MS Analysis of Vitamin D Metabolites (JEOL Ltd.) was used. The concentrations were as shown in Table 7 below.

[0222] [Table 7]

[0223] (2)IS solution Vitamin D Compound 44(25OHD 3 -d 3 ) and vitamin D compound 50 (24R,25(OH) 2 D 3 -d 3 A 30% acetonitrile solution of the standard was prepared at the concentration shown in Table 8 below.

[0224] [Table 8]

[0225] (3) Sample pretreatment The sample was pretreated according to the following procedure. (3-1) SLE (solid-liquid) extraction 1) 50 μL of serum and IS solution (25OHD 3 -d 3 20 μL, 24R,25(OH) 2 D 3 -d 3 2 μL) and dilute with 230 μL of 30% acetonitrile solution. 2) 302 μL of the serum / IS mixture was loaded onto the SLE column. 3) 600 μL of hexane / ethyl acetate (50 / 50, v / v) was added for elution. This was repeated twice. (3-2) Centrifugal concentration for about 20 minutes (3-3) Derivatization 1) Add 100 μL of the derivatization reagent DAP-PA and heat at 80°C for 15 minutes (see Figure 2H). 2) Drying: Centrifugal concentration for about 15 minutes 3) Dissolve in 100 μL of reconstitution solution (50% acetonitrile).

[0226] (4)LC analysis conditions Equipment: Shimadzu Corporation UFLC LC-20AD Analytical column: Osaka Soda Co., Ltd. CAPCELL CORE C18 2.1 mmI.D. x 100 mm Elution conditions: Flow rate 0.3 mL / min Solvent A: 0.1% formic acid-water B: 0.1% formic acid-acetonitrile Detailed elution conditions were as shown in Table 9 below.

[0227] [Table 9]

[0228] (5)MS / MS analysis conditions Equipment: Shimadzu Corporation LCMS8040 triple quadrupole mass spectrometer Ionization conditions: ESI positive ion mode The SRM parameters were as shown in Table 10 below.

[0229] [Table 10]

[0230] (6) Analysis results of the sample The results of the analysis of the preparations are shown in Figures 3A to 3D. Figure 3A shows the 25OHD 3 This is a calibration curve for the standard. FIG. 3B shows 24R,25(OH) 2 D 3 This is a calibration curve for the standard.

[0231] (7) 25OHD in pooled serum 3 and 24R,25(OH) 2 D 3 Quantitative value of Based on the above analysis results, 25OHD in pooled serum 3 and 24R,25(OH) 2 D 3 was quantified as shown in Table 11 below. Thus, by using the vitamin D compound of the present invention as a standard substance (particularly an internal standard substance) in mass spectrometry, vitamin D metabolites in a sample (particularly a biological sample) can be quantified.

[0232] Figure 3C shows the 25OHD in pooled serum. 3 4 is an SRM chromatogram of the above. Figure 3D shows the 24R,25(OH) 2 D 3 4 is an SRM chromatogram of the above.

[0233] [Table 11]

[0234] 6-4. Example 4: 4β,25-(OH) 2 D 3 Separation and measurement of

[0235] There are two or more vitamin D compounds that have different positions or types of groups bonded to the A ring, but the molecular weights of the substances generated by cleavage after derivatization for mass spectrometry are exactly the same. It has been difficult to detect or quantify each of the two or more vitamin D compounds. Each of said two or more vitamin D compounds can be detected and / or quantified by an analytical method using the vitamin D compounds of the present invention and carrying out a specific procedure. In the following, 4β,25-(OH) 2 D 3 An example of quantifying the above will be described.

[0236] (1) Material Standard material: 4β,25-(OH) 2 D 3 (Vitamin D Compound 54 prepared in Example 1 above) Stable isotope label: 4β,25-(OH) 2 D 3 -d 4 (Vitamin D Compound 52 prepared in Example 1 above) Vitamin D-free serum: MSG1000 (Golden West Biologicals) Antibody column: 1,25-(OH) 2 -Vitamin D 3 / D 2 Immuno Tube(TM) Extraction Kit (Immunodiagnostik) SLE column: Isolute SLE+ column (Biotage) Derivatization reagent (DAP-PA):JeoQuant Kit for LC-MS / MS Analysis of Vitamin D Metabolites (JEOL Ltd.)

[0237] (2) IS (internal standard) solution 4β,25-(OH) 2 D 3 -d 4 Aqueous solutions were prepared with the following concentrations:

[0238] [Table 12]

[0239] (3) Sample pretreatment (3-1) 4β,25-(OH) 2 D 3 200 μL of vitamin D-free serum adjusted to a concentration of 50 pg / mL and 30 μL of IS were added to a microtube and stirred to prepare a sample. (3-2) The sample was applied to the antibody column, the lid was closed, and the column was gently mixed. (3-3) The mixture was mixed and rotated at room temperature for 1 hour using a rotator to allow the antigen-antibody reaction. (3-4) The outlet of the antibody column was opened, and the column was centrifuged at 550 g for 2 minutes to elute the antibody, and the non-adsorbed fraction was collected. (3-5) 500 μL of WASHSOL included in the antibody column kit was added, and the mixture was centrifuged at 550 g for 2 minutes twice, and the eluate was discarded. (3-6) 250 μL of ELUREAG included in the antibody column kit was added, and the column was centrifuged at 550 g for 2 minutes for elution, and the adsorbed fraction was collected. (3-7) The non-adsorbed fraction was applied to an SLE column, left to stand for 10 minutes, and then eluted with 600 μL of ethyl acetate / hexane (50 / 50, v / v). This procedure was repeated three times, and the eluates were combined and concentrated using a centrifugal evaporator. (3-8) The adsorbed fraction was concentrated directly using a centrifugal evaporator.

[0240] (4) Derivatization The adsorbed fraction and the non-adsorbed fraction were each subjected to a derivatization treatment as follows. (4-1) The derivatization reagent was heated at 80°C for 15 minutes, and then 100 μL was added to the concentrated and dried sample (see Figure 2H). (4-2) After standing at room temperature for 15 minutes, the mixture was concentrated using a centrifugal evaporator. (4-3) Dissolve in 50 μL of reconstitution solution (50% acetonitrile aqueous solution).

[0241] (5)LC analysis conditions After the derivatization treatment, liquid chromatography treatment was carried out under the following conditions. Equipment: Agilent 1290 Infinity liquid chromatography system Analytical column: Osaka Soda Co., Ltd. CAPCELL CORE C18 2.1 mmI.D. x 75 mm Elution conditions: Flow rate 0.4 mL / min Solvent A: 0.1% formic acid-water B: 0.1% formic acid-acetonitrile Detailed elution conditions were as shown in Table 13 below.

[0242] [Table 13]

[0243] (6)MS / MS analysis conditions After the liquid chromatography treatment, mass spectrometry was carried out under the following conditions. Equipment: AB SCIEX QTRAP4500 triple quadrupole mass spectrometer Ionization conditions: ESI positive ion mode The SRM parameters were as shown in Table 14 below.

[0244] [Table 14]

[0245] (7)Analysis results FIG. 4A shows an SRM chromatogram of the antibody column non-adsorbed fraction. FIG. 4B shows the SRM chromatogram of the antibody column adsorption fraction. From these results, 4β,25-(OH) 2 D 3was confirmed to be present only in the fraction not adsorbed to the antibody column. Therefore, by performing mass spectrometry on the non-adsorbed fraction, 4β,25-(OH) 2 D 3 It has been found that it is possible to separate it from other components and detect and / or quantitate it.

[0246] 6-5. Example 5: 4β,25-(OH) in pooled serum 2 D 3 LC-MS / MS analysis of

[0247] (1) Material Standard material: 4β,25-(OH) 2 D 3 (Vitamin D compound 54 prepared in Example 1 above), 1α,25-(OH) 2 D 3 (SIGMA) Stable isotope label: 4β,25-(OH) 2 D 3 -d 4 , (vitamin D compound 52 prepared in Example 1 above), 1α,25-(OH) 2 D 3 - 13 C 3 (SIGMA) Vitamin D-free serum: MSG1000 (Golden West Biologicals) Antibody column: 1,25-(OH) 2 -Vitamin D 3 / D 2 Immuno Tube(TM) Extraction Kit (Immunodiagnostik) SLE column: Isolute SLE+ column (Biotage) Derivatization reagent (DAP-PA):JeoQuant Kit for LC-MS / MS Analysis of Vitamin D Metabolites (JEOL Ltd.)

[0248] (2) Calibrator Vitamin D-free serum with 4β,25-(OH) 2 D3 Calibrators were prepared by adding the following concentrations:

[0249] [Table 15]

[0250] (3) IS (internal standard) solution 4β,25-(OH) 2 D 3 -d 4 Aqueous solutions were prepared with the following concentrations:

[0251] [Table 16]

[0252] (4) Sample pretreatment (4-1) Calibrator or pooled serum: 200 μL, IS: 180 μL, and additive: 20 μL were added to a microtube and mixed to prepare a sample. The calibrator contained 1α,25-(OH) 2 D 3 Aqueous solution (1 ng / mL), 1α,25-(OH) 2 D 3 - 13 C 3 The pooled serum contained 1α,25-(OH) 2 D 3 - 13 C 3 10 μL each of aqueous solution (1 ng / mL) and water were added. (4-2) The sample was applied to the antibody column, the lid was closed, and the column was gently mixed. (4-3) The mixture was mixed and rotated at room temperature for 1 hour using a rotator to allow the antigen-antibody reaction. (4-4) The outlet of the antibody column was opened, and the column was centrifuged at 550 g for 2 minutes to elute the antibody, and the non-adsorbed fraction was collected. (4-5) 500 μL of WASHSOL included in the antibody column kit was added, and the mixture was centrifuged at 550 g for 2 minutes twice, and the eluate was discarded. (4-6) 250 μL of ELUREAG included in the antibody column kit was added, and the column was centrifuged at 550 g for 2 minutes for elution, and the adsorbed fraction was collected. (4-7) The non-adsorbed fraction was applied to an SLE column, left to stand for 10 min, and then eluted with 600 μL of ethyl acetate / hexane (50 / 50, v / v). This procedure was repeated three times, and the eluates were combined and concentrated using a centrifugal evaporator. (4-8) The adsorbed fraction was concentrated directly using a centrifugal evaporator.

[0253] (5) Derivatization (5-1) The derivatization reagent was heated at 80°C for 15 minutes, and then 100 μL was added to the concentrated and dried sample. (5-2) After standing at room temperature for 15 minutes, the mixture was concentrated using a centrifugal evaporator.

[0254] (6)LC analysis conditions Equipment: Agilent 1290 Infinity liquid chromatography system Analytical column: Osaka Soda Co., Ltd. CAPCELL CORE C18 2.1 mmI.D. x 100 mm Elution conditions: Flow rate 0.3 mL / min Solvent A: 0.1% formic acid-water B: 0.1% formic acid-acetonitrile

[0255] [Table 17]

[0256] (7)MS / MS analysis conditions Equipment: AB SCIEX QTRAP4500 triple quadrupole mass spectrometer Ionization conditions: ESI positive ion mode The SRM parameters were as shown in Table 18 below.

[0257] [Table 18]

[0258] (8)Analysis results FIG. 5A shows 4β,25-(OH) 2 D 3 The calibration curve for the standard is shown. FIG. 5B shows an SRM chromatogram of the antibody column non-adsorbed fraction of pooled serum. FIG. 5C shows an SRM chromatogram of the antibody column adsorption fraction of pooled serum. From these results, 4β,25-(OH) 2 D 3 and 1α,25(OH) 2 D 3 was isolated and 4β,25-(OH) 2 D 3 In the antibody column non-adsorbed fraction, 1α,25(OH) 2 D 3 It was confirmed that each of the above was recovered in the antibody column adsorption fraction.

[0259] (9) 4β,25-(OH) in pooled serum 2 D 3 Quantitative value of

[0260] [Table 19] Thus, 4β,25-(OH) 2 D 3 1α,25(OH) 2 D 3 Separated from 4β,25-(OH) 2 D 3 It has been found that only 100% of the 100% 15 ...

Claims

1. An alkyne compound represented by the following formula (100): 【Chemistry 1-1】 [In formula (100), A represents a linear carbon chain having an alkynyl group and a vinyl group at each of its two ends, X １ is CH or １３ C-H, X ２１ , X ２２ , X ２３ , and X ２４ are each independently CHY, CDY, １３ CHY, １３ C.D.Y., C.O. １３ C.O., C. １８ O, and １３ C １８ O, Y is H, D, NH ２ , １５ N.H. ２ , O.H., １８ OH, SH, OR, O(CO)R, OSO ３ H, OSO ３ Na, and a sugar substituent, and when two or more Y's are included in formula (100), the Y's may be the same or different from each other; R is an alkyl group, an alkenyl group, an aryl group, or an alcohol protecting group selected from t-butyldimethylsilyl, triisopropylsilyl, and triethylsilyl; X ３ is C or １３ C, X １ , X ２１ , X ２２ , X ２３ , X ２４ , and X ３ At least one of the above is a stable isotope D, １３ C. １８ O, or １５ is modified by N, The sugar substituent has the structure: 【Chemistry 1-2】 With respect to Wo in the sugar substituent represented by this structure, W represents a substituent of the ring carbon of the sugar, and o represents the number of the substituents and is an integer of 3 to 5. W is H, OH, CH ２ OH, COOH, NH ２ , and NHAc, which may be the same or different from each other. (However, the following alkyne compounds: 【Chemistry 1-3】 (Excluding.)

2. An alkyne compound represented by the following formula (100): 【Chemistry 2-1】 [In formula (100), A represents a linear carbon chain having an alkynyl group and a vinyl group at each of its two ends, X １ is CH or １３ C-H, X ２１ , X ２２ , X ２３ , and X ２４ are each independently CHY, CDY, １３ CHY, １３ C.D.Y., C.O. １３ C.O., C. １８ O, and １３ C １８ O, Y is H, D, NH ２ , １５ N.H. ２ , O.H., １８ OH, SH, OR, O(CO)R, OSO ３ H, OSO ３ Na, and a sugar substituent, and when two or more Y's are included in formula (100), the Y's may be the same or different from each other; Here, X ２１ , X ２２ , X ２３ , and X ２４ At least one of the following is CHY, CDY, １３ CHY, and １３ C, D, Y, or NH ２ , １５ N.H. ２ , O.H., １８ OH, SH, OR, O(CO)R, OSO ３ H, OSO ３ Na, and a sugar substituent; R is an alkyl group, an alkenyl group, an aryl group, or an alcohol protecting group selected from t-butyldimethylsilyl, triisopropylsilyl, and triethylsilyl; X ３ is C or １３ C, X １ , X ２１ , X ２２ , X ２３ , X ２４ , and X ３ At least one of the above is a stable isotope D, １３ C. １８ O, or １５ is modified by N, The sugar substituent has the structure: 【Chemistry 2-2】 With respect to Wo in the sugar substituent represented by this structure, W represents a substituent of the ring carbon of the sugar, and o represents the number of the substituents and is an integer of 3 to 5. W is H, OH, CH ２ OH, COOH, NH ２ , and NHAc, which may be the same or different from each other.

3. In formula (100), X ２１ , X ２２ , X ２３ , and X ２４ The alkyne compound according to claim 2, wherein at least one of is CHY or CDY.

4. A vitamin D compound represented by the following formula (200): 【Chemistry 3-1】 [In formula (200), A' represents a cyclic carbon chain corresponding to the A ring of a vitamin D compound; X' １ is C or １３ C, X' ２１ , X' ２２ , X' ２３ , and X′ ２４ are each independently CHY', CDY', １３ CHY', １３ C.D.Y.', C.O. １３ C.O., C. １８ O, and １３ C １８ O, Y' is H, D, NH ２ , １５ N.H. ２ , O.H., １８ OH, SH, OR', O(CO)R', OSO ３ H, OSO ３ Na, and a sugar substituent, and when two or more Y' are included in formula (200), Y' may be the same or different from each other, R' is an alkyl group, an alkenyl group, an aryl group, or an alcohol protecting group selected from t-butyldimethylsilyl, triisopropylsilyl, and triethylsilyl; X' ３ is C or １３ C, X' １ , X' ２１ , X' ２２ , X' ２３ , X' ２４ , and X′ ３ At least one of the above is a stable isotope D, １３ C. １８ O, or １５ is modified by N, P is a C substituted or unsubstituted with a hydroxy group. 1 ~C 10 C which is or is not substituted with an alkyl group or a hydroxy group 2 ~C 10 an alkenyl group, wherein the alkyl group or the alkenyl group may have a lactone group substituted with a hydroxy group; The sugar substituent has the structure: 【Chemistry 3-2】 With respect to Wo in the sugar substituent represented by this structure, W represents a substituent of the ring carbon of the sugar, and o represents the number of the substituents and is an integer of 3 to 5. W is H, OH, CH ２ OH, COOH, NH ２ , and NHAc, which may be the same or different from each other. (However, the following vitamin D compounds: 【Chemistry 3-3】 (Excluding.)

5. A vitamin D compound represented by the following formula (200): 【Chemistry 4-1】 [In formula (200), A' represents a cyclic carbon chain corresponding to the A ring of a vitamin D compound; X' １ is C or １３ C, X' ２１ , X' ２２ , X' ２３ , and X′ ２４ are each independently CHY', CDY', １３ CHY', １３ C.D.Y.', C.O. １３ C.O., C. １８ O, and １３ C １８ O, Y' is H, D, NH ２ , １５ N.H. ２ , O.H., １８ OH, SH, OR', O(CO)R', OSO ３ H, OSO ３ Na, and a sugar substituent, and when two or more Y' are included in formula (200), Y' may be the same or different from each other, R' is an alkyl group, an alkenyl group, an aryl group, or an alcohol protecting group selected from t-butyldimethylsilyl, triisopropylsilyl, and triethylsilyl; X' ３ is C or １３ C, X' １ , X' ２１ , X' ２２ , X' ２３ , X' ２４ , and X′ ３ At least one of the above is a stable isotope D, １３ C. １８ O, or １５ N, where X' ２１ , X' ２２ , X' ２３ , and X′ ２４ At least one of the isotopes is stable １３ C. １８ O, or １５ N or modified by X' ２１ , X' ２２ , X' ２３ and X' ２４ At least two of X' are modified with a stable isotope D, or ２１ , X' ２２ , X' ２３ , and X′ ２４ At least one of the above is CDD; P is a C substituted or unsubstituted with a hydroxy group. 1 ~C 10 C which is or is not substituted with an alkyl group or a hydroxy group 2 ~C 10 an alkenyl group, wherein the alkyl group or the alkenyl group may have a lactone group substituted with a hydroxy group; The sugar substituent has the structure: 【Chemistry 4-2】 With respect to Wo in the sugar substituent represented by this structure, W represents a substituent of the ring carbon of the sugar, and o represents the number of the substituents and is an integer of 3 to 5. W is H, OH, CH ２ OH, COOH, NH ２ , and NHAc, which may be the same or different from each other.

6. 6. The vitamin D compound of claim 5, wherein, in formula (200), P has any of the following structures: 【Chemistry 5】 [In the structure relating to P, Q, Q', Q'', Q''', and Q'''' are each independently H, CH ２ , C.H. ３ , C ２ H ５ , O, OH, and a sugar substituent.

7. 6. An analytical method using a vitamin D compound according to claim 4 or 5.

8. The method of analysis includes analyzing a sample by mass spectrometry; In the analysis, the vitamin D compound is used as an internal standard. The analytical method according to claim 7.

9. The method according to claim 8, wherein the mass spectrometry is LC-MS / MS or LC / MS.

10. 8. The method according to claim 7, wherein vitamin D compounds not having a hydroxy group at position 1 of the A ring are analyzed separately from vitamin D compounds having a hydroxy group at position 1 of the A ring.

11. The method according to claim 10, wherein the vitamin D compound not having a hydroxy group at position 1 of the A ring includes at least one of a 2-hydroxyvitamin D compound and a 4-hydroxyvitamin D compound.

12. The method according to claim 10, wherein the vitamin D compound not having a hydroxy group at position 1 of the A ring includes at least one of a 4β,25-dihydroxyvitamin D3 compound and a 4-hydroxyvitamin D compound.

13. The method for producing a vitamin D compound according to claim 4 or 5, wherein an alkyne compound represented by the following formula (100) is used as a material for forming an A ring of the vitamin D compound. 【Chemistry 6-1】 [In formula (100), A represents a linear carbon chain having an alkynyl group and a vinyl group at each of its two ends, X １ is CH or １３ C-H, X ２１ , X ２２ , X ２３ , and X ２４ are each independently CHY, CDY, １３ CHY, １３ C.D.Y., C.O. １３ C.O., C. １８ O, and １３ C １８ O, Y is H, D, NH ２ , １５ N.H. ２ , O.H., １８ OH, SH, OR, O(CO)R, OSO ３ H, OSO ３ Na, and a sugar substituent, and when two or more Y's are included in formula (100), the Y's may be the same or different from each other; Here, X ２１ , X ２２ , X ２３ , and X ２４ At least one of the following is CHY, CDY, １３ CHY, and １３ C, D, Y, or NH ２ , １５ N.H. ２ , O.H., １８ OH, SH, OR, O(CO)R, OSO ３ H, OSO ３ Na, and a sugar substituent; R is an alkyl group, an alkenyl group, an aryl group, or an alcohol protecting group selected from t-butyldimethylsilyl, triisopropylsilyl, and triethylsilyl; X ３ is C or １３ C, X １ , X ２１ , X ２２ , X ２３ , X ２４ , and X ３ At least one of the above is a stable isotope D, １３ C. １８ O, or １５ is modified by N, The sugar substituent has the structure: 【Chemistry 6-2】 With respect to Wo in the sugar substituent represented by this structure, W represents a substituent of the ring carbon of the sugar, and o represents the number of the substituents and is an integer of 3 to 5. W is H, OH, CH ２ OH, COOH, NH ２ , and NHAc, which may be the same or different from each other.

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