JPH045287A - Reagent for determining vitamin ds and production thereof - Google Patents

Abstract

NEW MATERIAL:A compound shown by formula I (X is fluorescent coloring group or potentially chemical luminous group; Y is spacer). USE:For detecting and determining vitamin D, vitamin A and a metabolite thereof. PREPARATION:A compound shown by formula II or formula III (Q is hydroxymethyl or amino) is coupled with a compound shown by the formula X-COC1.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a dienephilic reagent having a b-functional group having a vitroscopy coloring or chemiluminescent ability, and a method for producing the same. The present invention relates to the detection of CPS-diene compounds, particularly vitamin D, vitamin A, and their metabolites, using such reagents, and to stationary analysis methods. -X (wherein, It is now well known that steroid hormones have many functions, not only in metabolism but also in the proliferation and differentiation of various target cells. In order to fully understand the biological role of vitamin D, it is essential to accurately know the concentrations of major metabolites in human plasma in various clinical settings. Since the commonly used competitive binding assays lack specificity, the target species must be separated prior to the two assays. Also, this analytical method lacks accuracy. The gas chromatography-tiger lid analysis method has its drawbacks, such as the complicated operation. Therefore, an accurate, specific, and highly sensitive method for analyzing vitamin 9 condensates is required. In each formula, X is a firefly, a chromophore or a chemiluminescent group, and Y is a spacer. Novel photo- or chemiluminescent reagents (I) that react rapidly, specifically and quantitatively with conjugated triene moieties of metabolites.
and (II) were synthesized. The compounds of the present invention are represented by - Shipura (I) and (a) below. Examples include those represented by -X22\l+L4 (in the formula, Z represents a conjugate base of an acid; R and R' may be the same or different and represent a hydrogen atom or an alkyl group). As a preferable f (c) of X, the following formula is particularly preferable. On the other hand, as an example of Y, °C is the following formula % formula %) (In the formula, -A- represents -0- or -N)I-. -B- is -NH--COO--80□N)i- [in the formula Z
has the same meaning as above (2)]. Indicates the selected member of the formula, t indicates an integer from 1 to 5, and 1
m represents an integer from 0 to 5, and n represents either 0 or 1]. However, the left end of each formula of Y is the general stock il+ or (with the phoneme atom of the salt of Ill. The right end is bonded to X. Preferred examples of Y are the following formula % formula %) (However, n has the same meaning as above) (shown) can be opened. Formula A)) Method by coupling of lyazolidine synth/ with chromophore synthon. It is particularly preferable if Preferred examples of the compounds of the present invention include: Formula % Formula % () A method for final ring closure of the semicarbazide compound. The following can be mentioned. The compounds represented by σ)-Funazo (I) and till of the present invention are synthesized in the following steps. (In the formula. (V button X and Y have the same meaning as above. Q represents a hydroxymethyl group or an amino group. W represents a C□-03 alkyl group.) A) As a method, general formula (V) and From (νlI)

From the reaction to obtain [1] or general formula (v1] and CVD) (it, l
What is the reaction to that? Acid chloride Cvu)'r:s water Dissolve in a solvent such as benzene, CV) t cis <Vl) 1st water D
MF #Hw processing, , carried out by flowing II. B] The reaction from general formula (vl) to (ll) is as follows. This is done in the presence of anhydrous potassium carbonate, etc. (by suspending ■ in a solvent such as anhydrous or ethanol, and flowing it through 1. In addition, if V:, the drug Il+ is unstable, use K11ll as shown in the reaction formula below. -X -X ([1)(I) The reaction to obtain 111 from the general formula till is Pb(OAc
),. This is done by oxidizing with a mild oxidizing agent such as PhI(OAc)2. Action Compounds represented by the general formulas (I) and (II) of the present invention, vitamin D, vitamin A, and their substitutes.
It is a strongly fluorescent dienephilic reagent that specifically detects the is-diene moiety. 4- selected as the wanted group! l conversion -1゜2,
4-) Riazoline-3,5-dione is highly reactive;
Reacts in situ with small amounts of substrate under mild conditions. Also, because it is symmetrical, it has the advantage that only one geometric isomer can be formed. The groups represented by X selected as fluorescent chromophores or potential chemiluminescent groups have high fluorescent efficiency and are also stable under oxidation conditions of the doriasilidine ring. The present invention will be explained in more detail below with reference to production examples and examples, but the present invention is not limited by these production examples and examples. The melting point was measured using a micro melting point meter (manufactured by Yanagimoto Seisakusho).
Uncorrected, IR spectrum is 1 JASCO FT/IR-7Q
Q [ ] type ℃泗” fixed. MS spectrum. JEOL JMS-D300 model uses electronic giI attack method (EI
) or o'H-NMFI spectrum measured by SIMS method on Hitachi M-2000 model 1 JEOL GX-27
Specified by type 0, tetramethylsll
Chemical shifts are shown as δ values using ane k as a standard. If + constant solvent was written for each compound. Naori 2
For measurements in 0, 5-(trimethylsily
l)-propionicacid-d4sodi
um 5alt 9 was used as an internal standard. The description of NMR is as follows σ) Abbreviation Knee C. s==sin
glet:d=cloublet;t=
triplet; q = quartet; m
= multiplet; arom = arom
atic; br = broad; Big = s
ignal. TLC (Thin Layer Off Togelahui) is
K18Sell gel GF254 (manufactured by MerCk) was used. For column chromatography, unless otherwise specified, silica gel was Kiesel gel manufactured by Merck.
l 60 (35-70mesh) k used. Reactions were routinely performed under argon flow. Ether 4-incyanatobenzoate (5,05'. 0.026 mot) anhydrous benzene l'g (260
In suJ, carbazic acid ether (3,1'
. After adding 0.033 mol, the reaction mixture was heated under reflux for 2 hours. After returning to room temperature, 1t'r of the crystals were sucked out.
Separated, crude semicarbazi) 1(7,25), yield 93.9
%) was obtained. Crystallized from EtO)i on the main island. Colorless scaly crystals (6,80P) fl were obtained. Compound 1: mp 194-195°C MS m/zN: 295 (M”, 6), 250 (7)
, 204151°1676CC=D) 'H-NMR (CDC63+DMSO-d6) δ: 1.
28 (3H, t, J=7.3Hz, C:H2CH3
)1.38 (3H, t, J=7.3Hz, CH2C
H3) 4.19 (2M, Q, J=7.3H
z, 0H20H3) 4.33 (28, q, J=7.
3) 1z, CH20H3) 7.52 and 7.92 (4H, AA'BB', J:8.6Hz, aromH)
7.70 (IH, br, sig, NH) 7.80
(IH, br, sig, NH)8.57 (I
H, br, sig, NH) original ticket analysis two calculated values (C; 13H17N, 0.): C, 52, 87;
H, 5, sO; N, 14.23 Measured value: C,
52,71; H, 5,72; N, 14.14 (Production Example 2 Nosemicarbazide 1 (I, 181P, 4 mmoz)
After bath-dissolving and pulverizing anhydrous vertical mill potassium (556 mm, 4 mmj) t) was added to lItm water EtO)i (5 Qa/), the reaction solution was heated under reflux for 5 hours. EtO under reduced pressure
)iftm left. Add the remaining 11EK water-cooled water, and then add dilute hydrochloric acid to the water layer at 1°C.
ki! 12 annotations. The aqueous layer is ACOEt (3X
60 soap) and the Acogt layer was washed with saturated edible water. It was dried over anhydrous magnesium sulfate, and the solvent was distilled off. Crystal a residue ffi (901ag) was purified by silica gel column chromatography (45)) and purified with 10% MeO
H-containing Ac0Et #Triazolidine 2 (8
084', yield 811%] was obtained in crystalline form. Nasal crystals were obtained from Ac OEt and colorless needle crystals were obtained. Compound 2 IJ) 206-207°C MS m/Z (%): 249 (M”, 35), 221
(31). 2 (74 (I00), 191 (I3). IH-NMR (CDC; t, +DMSO-d,) δ:
1.40 (3H, t, J = 7.3Hz, 0H20
H,)4.37 (2H,Q,J=7.3)iz,C
H20H3) 7.71 and 8.10 (4H, AA'
BB'. J = 8.9 Hz, aroma H) 10.29
(2M, br, s, 2xNH) Elemental analysis: 11 calculation value (C1lH1□N304): C, 53, 0; H, 4,
45; N, 16.86 measurement m: C, 53,00
: H, 4, 51; N, 16.95 (Production Example 3) Method 1> Esthetics A'#2C555m9.145mmot) f) Anhydrous T) IF (8,5j17) solution dry ice and tetrachloride R* bath The cooled and stirred solution was cooled to -20°C using a water volumized diimpdel aluminum (642 m/;
1 mot/l) was added to the solution (1) over a period of about 20 minutes. The reaction mixture was further stirred at -20°C for 4 hours, and then diluted with dilute hydrochloric acid (2su, 19.3mmO
6) was added to make it acidic. Vacuum lower tower t
& is adsorbed to the cruel 21 silica gel obtained by distilling off,
It was subjected to silica gel column chromatography (40Sf) and subjected to Me. The column was cHat3 containing 10% MeOH.
After taking a bath, the samurai, MeOHK! MJ&Made. Pure benzyl alcohol 3 (240 μ, yield 8'L)
4%] as colorless needle crystals. Method 1) - Using one ester group 2 (t5761t5.32mmot1), which is suitable for large-scale reactions, the reaction and post-treatment were carried out in the same manner as in method 1. ~. Passed through a cotton plug to remove insoluble matter. Solution F was Ambly filled with water.
erlite The column was washed with water (I500a/tc). MeOH
When adsorbed at ℃ and eluted 6 at ℃, a solid substance fi(
-117 cl) was despised and recrystallized from MeOHz, and 3 (794 ~, yield 60
．． 6%]. Compound 6: mp > aoo°C MS m/Z (%): 207CM”, 82),
178 (I DO). 148 (22), t32N4), 120 (29), 10
7(47), 58(55). Br IRνmaX cs-”: 3344 (OH),
1682(C=0)')1-NMFI(DMSO-d,
) δ: 4.54 (2H, S, 0H2Ph) 7.40
(4H, S, aromH) 105B <28. S
, 2xNH) Elemental analysis two calculated values (G, H, N, Oj): C, 52, 17; H, 4,
38;N, 20.28#j constant value: C, 51,9
9; H, 444; N, 20.25 incyanep-nitroaznyl (5,0?. 30.5 mmot), carbazine rice ether (3,
91-. 3 Q, 5 mm0t) and anhydrous penze: /< 13
The mixed solution was heated for 15 hours and heated for 11 hours. The precipitated pale yellow crude crystals were collected with suction and thoroughly washed with a bay bottle. Semicarbazide 4 (7.85N, yield 97.6%) was obtained. P+ crystals were obtained from Honjima y MeOH to obtain colorless scale crystals. Compound 4: J) 216-217°C (Lit, mp21 B-
0-218.5°C) MS m/Z (%): 26B (M
”, 0.5), 222(3). 1553 and 1338(NO2)'H-NMR(DMSO-(I,)δ:t21 (3
H, t, J ~6.9hz, CH2C; H3) 4.08
(2H,Q,J==6.9Hz, CH20H3)7
．． 73 (2H, br, sig, aromH)8.1
5 (2H, aromH) 8.36.9-02 and 948 C! IH, br, s, 3XN) l) (literature) F,
Arnat, L., Loewe, and A., Tarla.
n-AkOn. Rev, Fac, SCi, Forest Univ, l
5 tanbul. Back, 127 (I948); chem, Abstr, 42.8190 (I94B)
(Production Example 5) Pulverized anhydrous potassium carbonate (2,5C1,15,2mm
ot) and anhydrous, EtOH (200at) 9)s~
g't Heat R flow for 5 hours. The reaction mixture turned from yellow to bright red. Satoshi, who removed the EtOH under pressure, was cruelly given ice-cold water (500d)? Add r, 2N HGt vc
-c&note. This aqueous layer is Ac0Kt, (3xl 5
0), dried with Glauber's salt, ACOEt Ik
When you leave it. Crude crystalline acid produced @(6,7CJ1f) was obtained, and when this was P+ crystallized from Geer 7 tons, colorless needle crystals of 5(2,73CJ1f) were obtained.
t, yield 80.7%) was obtained. Compound 5: mp267-269°C (Lit, mp270.0-27
15℃) Semicarbazide 4 (4.08?, 15.2mm
ot). 1528 and 1348 (No□) 'H-NMR (DMSO-d6) δ Near, 90 and 8.34 (4H, AA' BEy, J
=9.2H2゜arom H) 10.78 (2H1br, s, 2xl) (literature)
G., Zinner and W., Daucker, Ar.
ch, Pharm. 294.370 (I961) (low snow fl16) Compound 6: mp > soo℃ MS m/Z (%): 192 (M”, 99), 154
(I00). NO□ NH2 Nitro 15 (787'%', &54 mmoz
) cbEtDH solution (60y) i PtO□ (80y
mg), catalytic reduction was carried out in a hydrogen stream at normal pressure and in a stream of hydrogen. Approximately 40 to 50 minutes after the start of water t8, crystals began to precipitate in the first reaction vessel. After 2 hours, the theoretical hydrogen consumption (2
40IE/) Ki! The reaction was terminated. Precipitated crystals. fk was dissolved using ACOEt and gtoH and suction filtered through Celite. The solvent of liquid F was distilled off under reduced pressure to obtain crude amino compound 6 (720 μm, quantitative yield) in the form of crystals. The main island is EtOH-ACOEt (I: 1; V
/V) to form colorless needle crystals.

【Gave. 'H-NMR (DMSO-d6)+5:5.21 (
2H1br, s, NH2) 6.60 and 6.97 (
4H1AA'BB'. J = 8.6 Hz, aroma H) 10.14
(2H, br, s, 2xNH) (I!! Example 7) C, tAAN=Q=Q −+01AANH−n HNH
■OEt3-chloropropyl isocyanate (5,C
1°4't, smmoz) of anhydrous benzene solution (I20
a/) to ethyl carbazate (5,2SF, 5α2 m
The mixture was heated under reflux for 1 hour. When the solvent was distilled off under reduced pressure, a crude product (9,25?) was obtained in crystal form, which was recrystallized from a single piece of AcOEt-gt,o.
Pure semicarbazide 7C1>59t)'l was obtained. lh
Crystal mother solution (2,24Pl' silica gel column chromatography 4- (Wacogel G-200, I0Off
) at! 1liL. Further Z(5
17av) was obtained. The total yield was 76.0%. Compound 7: mp 107.0-108.5°C MS m/Z (% Ri: 225 (M”+2.0.08)
, 223 (M”0.2), 178 (2), 180 (0,
7). 1721 and 1649 (C = 0) 'H-NMR (CDCl2) δ: +29 (3H, t, J = 6.9Hz, OH, 0H3
)2.00 (2H, m, 0H20H20H,)
&39 (2H,m, -OH,NH-)3.5
9 (2H, t, J=5,8Hz, CtCH2-)4
．． 21 (2H, q, J==6.9Hz, OH, OH
,) 5.68 (IH, m, NH) 6.76 and 6.81 (respectively I H, br, s. 2XN) l) (I!! Preparation Example 8) cz/XAsH-c-NHsHcoogt + semicarbazide 7 (5,256F, 23.5mmoz)
Anhydrous DMF# solution (55Nt) KNaN3 (I830P
. 28.2 mmoz) k was added and heated at 100°C for 6.5 hours. The mixture was heated and stirred. DMF%: Distilled off under reduced pressure, and water (5%) was added to the residue.
0a/) and extracted with AcOEt (5x70m/)K. The combined Ac0Et # is washed with water, dried with sodium chloride, and L! By solvent distillation, the crude azide group 8 (5,131 yield 9
468%] was obtained in crystalline form. Noble crystals were obtained from the main ether, and colorless crystals (&6C1) were obtained. Compound 8: J) 81-82°C MS m/Z (%): nom-185(2), 141
(3). 116 (2), 104 (I00) 2102 (N3) 1764 and 1671 C0 = 0) 〆) 1-NMR (GDC; t3) δ: t29 C3H0t, J = 7.5Hz, 0H20H8
)t80 (2H0m, 0H20H2C) (
2) 3.35 (2)1. II+, -0H
28H-)3.37 (2H, t, J=6.6Hz,
N30H2-)4.21 (2H, q, J=7.3H
z, CH20H3) 5.64 (IH, m, NH) and 66 and 6.73 (each tr IH, br, s. 2xNH) (Production Example 9) and anhydrous EtOH (I40d) were heated to reflux 6 I did it for an hour. EtOH was distilled off under reduced pressure, and water (50%
wLl)'Ik was added and made # acidic with 2NHC; t. 0) @Majesty's water layer should be adjusted with water in advance.
I passed it. After washing Arum with water (750117). Formed 'm11K: was desorbed and eluted from the resin with MeOH. The combined MeOH eluate * mountain part was distilled off to obtain a crude crystal product, which was recrystallized from 9I'AOOEt to give pure 9
(2,58P, yield 898%) was obtained as colorless flakes. Compound 9 m9140-141°C MS m/z (%): noM", 141 (20
), 129(39). Azide body 8 (5,6c1.15.6 mmot), crushed anhydrous potassium carbonate (2,60?, 18.7 mm
ot)1667(C=O)')(-NMR(DMSO-d6)δ:1.78 (
2H,quirltet,J=6.6Hz. -CH2C20H2-) 3.38 (2H, t, J=6.6Hz) 3.43
(2H1t, J=6.6H2)10.06 (2)
1. br, s, 2xNH) (Production Example 10) Azide tK9 (t7(In, 9.23mmoz)
vEtOH<191Lt) and H,O (7,5m/)
The mixture was dissolved in K and subjected to atmospheric hydrogen catalytic reduction in the presence of 10% pa-C:(I77ag). Ni! 1 reaction solution after carrying out the reaction for 96 hours. It was thoroughly removed through Celite. When liquid F is removed under m pressure, a crystalline residue (I, 6291) is obtained, and H, O
- When recrystallized from MeOH, free amino acid 1,0 (tO
O7F, yield 690%] was obtained as colorless needle crystals. Compound 10 J) 227-229°C 129 (47), 101 (95), 56 (94). 'H-NMR (D20) δ: 2.00 (2H, m, -CH2 to H20H2-)3
．． 03 (2H, t, J=Z4Hz, 0H2NH2)
3.60 (2H, t, J = 6.6H2, -C
H2-N) Salt of Compound 10 #I1. Salt azide form 9 (I40Iv, 0.76 mmot)'
kEtO)i (20m) and -dissolved in hydrochloric acid (0,4d) in the presence of 10% pd-C(305i&), 3.0
Medium pressure catalytic reduction was carried out at 2 h/on hydrogen pressure. After 4 hours at Tomitsu. When the reaction is complete and treated as above, 10 ml of HCl
The salt is obtained in crystalline form, MeOH-Et20! r)
Recrystallized pure 1 [IHCA salt (93, Omg),
Yield: 63.0%] was obtained as colorless needle crystals. MS m/z<9! o): 158 (M”, 100), 1
41 (50). Dinitroveratrol 11 (2,28F, 10m
mot) vm was dissolved in warm EtOH (,100a/). Catalytic reduction was carried out in the presence of -2Pω2(0,23)) at room temperature and in a hydrogen stream at normal pressure. After 2365 hours of stirring, the consumption of hydrogen completely stopped (theoretical hydrogen consumption: 1.3 t), so the reaction was terminated. The reduced product was quickly passed through a celite catalyst under a stream of phonemes, and the catalyst was removed, and the F solution was directly introduced into a reaction vessel in which dietherketomalone (I741, 1() mmot) had been previously placed. The reaction mixture solution was immediately heated under reflux for 1 hour and then left at room temperature overnight. Collect the precipitated crystals by suction. Crude crystalline product 12 (2,11?, yield 75.8%) was obtained. Recrystallization was performed from EtOH-OH20t2 to obtain yellow-green needle crystals (I98P). On the other hand, crystal F liquid (0.80t
] is silica gel column chromatography (4Fl)
From the CHC63 eluate containing 1% MeOH,
Furthermore, 12(0,33)) was obtained, with a total yield of 840%.
Met. Compound 12 mp250-251°C (Lit, 255°C) MS m
/Z (%): 278℃M, 100), 233(7)
. 'H-NMR (CDO13+CD30D) δ: 1.48
(3H, t, J=ZO)12. C1H2C)i3)
3.99 and 4.06 (each5H,s,2xO
CH3) 4.56 (2H, q, J=ZOHz, CH
2CH3) 6.94 and 138 (IH, s, a respectively
romH) (literature) z, 5uaesinsky a
nd A, Valenta. Co11ection Czechoslov, Che
m.Commun. 36.2527 (I971) (Production Example 12) NaH(0,26), 1197mm0t) anhydrous DM
12 (2,035', 7.31 mmot) of anhydrous D.
The MF (60at) solution was added over a period of about 20 minutes. After confirming that the generation of hydrogen gas had ended and the color of the solution had changed from yellow to tan (about 20-30 minutes), methyl iodide (0,68id, 1αq7mmot) was added dropwise over about 5 minutes. The reaction mixture solution was further stirred for 15 hours under water cooling, and then poured into ice-cold water (I50IL1).
15QMl). GHGt, the extracted layer was washed with saturated sodium chloride at °C and dried with mirabilite. The solvent was evaporated to give a yellow residue ac2.74t)k. Unproducts were analyzed by silica gel column chromatography (I30)
), GHGt3: Benzene (I
:1; v/v) From the elution part, 0-methyl compound 14 (0,1
0? , yield 4.7%] as pale yellow crystals. In addition, N-methyl form 1 (I, 7
2F. It was obtained as yellow crystals (yield: 80.4%). N-methyl compounds 1 to 6 were recrystallized from EtOH-Et, O to obtain yellow needle crystals, and 1 was gt, on-Hexane J
: It was recrystallized and pale yellow needle-like crystals were obtained. Compound 16 m9166-167°C MS m/Z (%): 292 (M, 100), 27
8 (I1). 277 (I2), 247 (I6), 220 (45),
205(6), 192(22). 'f(-NMR(CDC63)δ: t44 (3H, t, J=7.0Hz, 0H2(J,
) 3.73 (3H,s,N-0H3) 3.94 and 4. C5 (each 3B, 6°2xoca,) 4.51 (2H, Q, J=7.0Hz, OH, 0H
3) 6.71 and z56 (each IH, s, a
rom) i) Compound 14 mp123-125°C MS m/Z (%I): 292 (M”, 100), 2
47 (I6). 220 (45), 205 (I8), 190'H-NMR
(C;DGt,)-2t46 (3H,t, J=8.QHz, 0H20H,
) 4.00, 4.06 and 4.16 (each
5H. S, 3xOOH,) 4.52 (2H, q, J=8.0) 1z, 0H20
H8) 7.10 and 7.41 (6aChIH,S,
&rOmH) Elemental analysis: Calculated value measurement WL: Solution (51) was added and stirred for 10 minutes at room temperature.The reaction solution was converted to weak fIR using INHCz, and then cHat. The combined CHC layer was washed with water, dried with mirabilite, and the solvent was distilled off to obtain crude yellow crystals of carbon al15 (Q, 31°, yield 90.7%). The product was recrystallized from CHCL3 to obtain yellow needle-shaped crystals. Compound 15 mp241-243℃ (Lit, m9222℃) MS
m/Z (%): 264 (M”-27)-220 (I00
) - (Reference) T, Iwata, M, Yamagu
chi, S., Hara. M, Nakamura, and Y, Ohkura. J, Gchromatogr., 362.209 (I98
6) (Production N14) υ and N-methyl form 13 (D, 48t, t63mmo4
) 1,4-dioxane (5m) solution 11CIN
NaOH aqueous p-rubonic acid 15 (470m9.178m
A mixed solution of moz ) and 5OCz, (I0m ) 1
The mixture was heated to reflux for an hour. Under reduced pressure, excess 5OCz 2%: @i
The reddish brown crystals obtained were recrystallized from anhydrous benzene-n-hexane to give acid chloride 16 (43
5Iv. Yield: 86.6%]. Compound 16 mp 272-275°C (decomposition) [Lit, mp26
1℃] MS m/Z (% Ri: 284 (M”+2.21)
, 282(M”, 62). (Reference) T, Iwata, M, Yamaguch
i, S, Hara and M, Nakamura, J.
Ghrormtogr, 362. (Example 1) Acid chloride 16C113jlS', 0.4mm0L)
Dissolved in fk anhydrous benzene (8WLt), stirred and refluxed the solution, and added benzyl alcohol group 3 (I24~, 0.
6 m mot) of anhydrous DMF' (Q, 511+17)
The solution was added all at once and the reflux was continued for 15 hours. A yellow precipitate began to form upon addition of 6. The reaction solution was cooled to tO°C, and the precipitated crystals were collected by suction and washed with benzene. The yellow crude crystals (I84~) were swollen with chloroform and subjected to Sephadex LH-20 column chromatography.
CHCt3 containing 5% MeOH
[13 [10311 g, yield 56.8%] was obtained from the eluate. This product was recrystallized from MeOH-CHC; t3 to obtain yellow crystals. Compound iIA mp 279-280°C (decomposition) MS (+SIMS) m/z: 454 (M"+1)'H-
NMR (CDC63+DMSO-d,) J:'5.75
(6H,s,N-1c)I3) 3.92 and 4.0
5 (3H, s, 2xOCH, respectively) 5.43 (
2H, s, OH2ph) 691 and Z36 (41 each
H, s, arom H) 7.54 and 7.58
(4H, AAJ3B'. J=8.6Hz, aromH. 10.56 (2H, br, s, 2XNH) Elemental analysis: Total 'lI1. value (C2□H1gN50.) OH30H: C, 54, 43
; fi, 4.78; N, 14.45 measured value
: C, 54,42; H, 4,35; N, 14.8
3 (Example 2) Liquid %: After returning to room temperature, the precipitated crystals were collected by suction, and a jk color crude crystal product [IB (I1,3, yield 55.1%)] was obtained. The main island is extremely Jk11i!
As a note, it is used without purification in the following reactions. Compound 11B'H-NMFI (DMSO-d6)-2 3.78
(3H,s,N-OH,)6.89 and 4.03
(3H, s, 2xOcH3 respectively) Zlo and Z47
(respectively I H, S, MOm H) 7.45 and Hif,
85 (4H, AA'BB'. J = 9.0f (z, arom H) 10.42 (2H, 8, 2XNH) (Production Example 15) Acid chloride 16 (20jllf, Q, 07mmoz)
To a refluxing solution of anhydrous benzene (2,5id), 6 (
91111F, 0.047 mmot) anhydrous DMF
(0,1d) solution was added all at once and the reaction mixture was heated for 1 hour. Reacted dinitroberatrol 11
(0,91?, 4 mmot) in EtOH (80
d), catalytic reduction was carried out in the presence of Pto□ (89#) at room temperature in a hydrogen stream at normal pressure. The reaction continued for about 17 hours until hydrogen consumption (theoretical consumption 154,017) completely stopped. The resulting pale yellow-green bath liquid was passed through celite in a kiln gas stream to remove the catalyst, and the tF liquid was saturated with 2-ketoglutaric acid (
The mixture was collected in a reaction vessel containing 0.58y, 4mmoz) and immediately heated to reflux. 1 reaction after 15 hours? :P, done. When the reaction solution was cooled, orange-yellow crystals were precipitated. The precipitated crystals were suctioned and quinoxalinone 17 (0,8
0), yield 71.9%], which was fluorolysed from EtO) to give yellow needle crystals (0,62) 1-. Compound 17 mp250-252°M3 m/z (%ri: 278 (M”, 36)
, 260 (I00). 232 (88n, 217(32), 189'H-NMR
<CDC; t3) a: 2.80 (2H, t, J
=7.5Hz, -0H2Co-)3,ts (2H,t
, J=7. sHz, 'cH-) 3.92 and 3.93 (
3H, s, 2xOCH3) 6.78 and 118 respectively
(1H, S, 2LrOmH, respectively) 11.92 (I
H,br,sig,-C;0OH)18 El2G0
0CH8 20h=cooH Method I> Ice-cold NaH (I76W, 7.3mmoz) o>
Add compound 17 (6) to a suspension in anhydrous DMF (3a()) with stirring.
06 Satoshi, 2.2 mm06) f) Anhydrous DMF (20
m) Leave the solution for about 20 minutes. FLK
Stirring was started at 0.514 °C.Next, the reaction volume 1i
FK methyl iodide (455pi, 7.3mmot)
K was added dropwise over about 10 minutes, and the entire reaction solution was then heated to 0.
Stir for 15 hours at <0>C and pour into ice-cold water (50m). Mixed bath liquid. The organic layer was washed with saturated brine, dried with sodium sulfate, and the solvent was distilled off. A crude product (570 my) was obtained. This is the main structure. Silica gel column chromatography (Wacogel
Purified with G-200, 24)), n-Hexane
:Ac0Et (4: 1-2: 1; v/v
) ty> N-methyl form 18 (492 mg
, yield 73 o 8%) was obtained and recrystallized from MeOH-cHct3 to obtain pale yellow needle crystals. Only a trace amount of 0-methyl compound 19 was obtained (see Method I). On the other hand, the aqueous layer after CHO6 extraction was made acidic with Q,5NHC6, and then re-extracted with GHC13 containing 5% MeOH, and the organic layer was washed with saturated brine, dried with mirabilite, and the solvent was distilled off. Carboxylic acid 20 (86 cm. 13.5%) was obtained in crystalline form. Main island fMeOH-c
Recrystallization from Hct3 gave yellow needles. Method l> Carvone [17 (t00?, 3.60 mmoz)
%: OH, OL, -MeOH (I: 1: V/V
, 100WLt) K& was made cloudy, and diazomethane ether bath solution V was added to the solution under stirring at room temperature until 17 of the raw material V disappeared. Excess diazomethane is distilled off using a rapid accumulative air stream, and then 1 kftj of the medium is removed under reduced pressure.
N) was obtained. This product was purified by silica gel column chromatography 4-(Wacogel C;-2[]0,5
Qf) Made of K shaft, n-Hexane: Ac0J? :
O-methyl compound 1 from the t(6: 1; V/V) elution part
9 C48011jf, yield 43.6%) and n-f
(exane: Ac0ji: t (I: 1 ; v/v
) N-methyl compound 18 (5281Iy, yield 48.0%) was obtained from the eluate. The 0-methyl body 19 is NaOH-CHOP3! The crystals were recrystallized to obtain colorless prism crystals. Compound 18 mp174-175°C MS m/z (%): 306 CM”, 100), 2
74 (I5). 258 (I4), 247 (88), 231 (24)
, 203(20) Br IRymaX5++-1:1740 and 1651 (C
=0)') i-NMR (CDCj3) δ: 2-86 (2)1. t-J=ZIH2-CHzGO
)3.25 (28,t, J=ZIHz, )-a)C
2-]6.69 and 3.70 (respectively 3H, S, N-
CH3゜C100CH3) 6.96 and 4.01 (3H,s, 2xOCH respectively
3) 6.68 and 125 (IH, s, aromaH, respectively)
) Compound Th19 mp 137-169°C MS m/z (%): 306 (M”, 51), 27
5 (I0). Compound 20 ml) 239-241°C MS l/Z (%): 292 (M”, 533.2
74(99). 246 (I00), 231 (53), 203 (41),
175 (27) Br IRymaXa*-”: 1764 and 1628 (0=
0)') 1-NMR (DMSO-d6) δ: 2.70
(2H, t, J=6.9Hz, 0H2CO) &00
(2H, t, J=49Hz, 0H2082Go)6-
65 (3H-s -N -G Hs ) 584 and 5.94 (6H, s, 2xOCH, respectively) 6.9
8 and 122 (IH, SlrOmH, respectively)')l −
NMR (C; DC, C3) δ: 2.89 (2H, m
) 3.24 C2H,m) 3.71 (3H0,s,C00CR,)4.00 and 402 and 4.07 (3H.,s,5xOC;H,) 7.17 and 7.27 (eachll( ,s,aro
mH) 18 2O N-methyl form 18 (tl 513.75 mmoj
) tl, 4-shi:ir*ty (I8su) and IN NaOH7jC solution (IDia/)K dissolved,
The reaction was stirred and reacted for 10 minutes under N temperature. The reaction mixture contains dilute salt t
After it is acidic. Extraction was repeated with cHct containing 10% MeOH until the bottom of the aqueous layer disappeared. The combined organic layer is The crude crystalline carboxylic acid 20 (I, OFl, yield 95.9%) was obtained by washing with saturated saline, drying with sodium sulfate, and distilling off the solvent. Motojima was recrystallized from MeOH-Of (Cz3). Pure product 20 (852~) was obtained, which matched the compound !1120 previously attended. (Production Example 18) Carboxylic acid 20 (500 Iv, 171 mmot)
t' Dissolve one bottle of anhydrous DMF (I2jl/)K and add triethylamine (0.36wr!, 2.57 m) to the stirred solution.
moz), phosphoric azide (0,55
ml, 2.57 mmol) was then added dropwise. After 1 hour of reaction, diphenylphosphorus e17)” (0,54
mmot) k, stirred at room temperature for 15 hours, #! Yellow-yellow-green liquid liquid. The bath was immediately removed under reduced pressure and the residue was dried under high vacuum. Anhydrous pensene (2011J)' was added to the dried product, and the mixed solution was heated under reflux for 1 hour. After the reaction solution was returned to temperature, carbazic acid ether (I78m&t711 [1m0t) was added. The reaction solution has M of 0.5
After refluxing IWl for a while, stir for 0.5 h under humid M. #
an-distilled. Obtained pale yellow residue aC1,976t)
teeth. Purification was performed by silica gel column chromatography (91:l), and semicarbazide 21 (230m9) was obtained from the eluate of CHC43 containing 4 to 5% M80H.
P) In addition, 21 (I64 mg) was obtained with a total yield of 58.6%. Compound 21 MS m/Z (%): 393 (M”, 5), 347 (0
, 8). Ro 30 (I), 289 (92), 246 (47). 'H-NMR (DMSO-d,) δ: 1.18 (3H,t, J = 6.9Hz, 0H2C
H3) 2.95 (2H, t, J=5.9Hz, CH
20H2NH-)3.52 (2H,m, CH,
NH) 167 (5H,s, N-CH,) 3.89 and 3.97 (3H,s, 2xOCH, respectively
3) 4.02 (2H, Q, J=6.9Hz, (H2
0H3)6.40 (IH,br,sig,NH)6
．． 91 and z31 (IH, S, lLromH, respectively) 7
．． 70 (IH,br,s,NH)8.65 (I
H, br, sig, NH) semicarbazide 21 (272
~, 0.69 mmot). Anhydrous potassium carbonate with stopper frame (I91~, i8mmot)
The mixture was heated under reflux for 6 hours in a bath of abs EtOH (20 ysi). The solvent was distilled off under reduced pressure, and the remaining aK water (60
In addition to Muri', it was made acidic with 2NHC6K. water 1164
! , 10%MeO)1 *OHC; t3(3x50
au) Extracted with K, the organic layer was washed with saturated saline,
After drying and evaporating the solvent, pale yellow crystals (4 g of 222I) were obtained.
) was obtained = MeOf(-Crystalized from CHCL3,
90(I30%') 9 was obtained as yellow prism crystals. The crystal mother liquor (70 mg) was purified by silica gel column chromatography (I (I), and purified with 5% MeOH-containing G
HGt3#a[SC') further t/Cr1OC64119)
The total yield was 80.8%. Compound-C ml) 250-255°C MS Vz (%): 547 (M”, 5), 289 (
2). 246 (I00), 231 (35), 205I
RymaXa*-1:1694 and 1640 (C=0)
'H-NMR (DMSO-d6) δ: 3.02 (2)1. t, J = 6.9Hz. 3.65 (3H,s,N-0H3)3.80 (
2H,t,J=6.9Hz. 3.84 and 3.95 (3H, 8, 2XOCH respectively
3) 6.96 and Z26 (lH, s, aromaH, respectively
)9.95 (2H,S,2XNft). (Example 5) Triazo') Schiff IC (IQ, Qap, 0.
029mmot) in anhydrous DMF (Im) solution was added PhI (
OAc), (I12~, 0.035 mmot) 9 was added, and the mixture was stirred at room temperature for 1 hour. Removal of DMFlk under reduced pressure gave a red solid residue. Main island v cH2at2-
When benzene was crystallized, red prismatic crystals of the desired IC were obtained, although the amount was very small. the current,
Methods (sublimation method, etc.) to obtain pure products in large quantities are currently being considered, including amino acid (98 mm width, 0.05 mmot) and coal plate potassium (I5.2 mm, 0.11 mmot). 7-chloro-4-nitropenzofurazane (8,34', α041 mrnol) in aqueous solution (0,25 m)
The tOH(I-) solution was added over about 5 minutes. The resulting dark purple solution was stirred at room temperature for 10 minutes and then heated to 60-70°C.
The mixture was heated and stirred for 5 minutes. Dark purple reaction suspension solution. When acidified with 2NHO1K, a dark red solution was obtained. The solvent was distilled off under reduced pressure, and the residue fi (44,5~) was obtained. It was produced using ODS column chromatography (WaterS Co., ODS packing material for dispersion 5?). The column is 2
Elute with water containing 0% MθOH, triazolidine tl
DC9.5~, yield 70.9%] was obtained as a yellow-red sticky substance. Compound [ID Br IRνmax cm-”: 1688 (C=0)
1586 and 1301 (NO,) MS l/Z (%): noM'''H-NMR (DMSO-d,) a: t99 (2H, m) 54-&5 (4H, m) 6.42 (I) 1.d, J=9.2Hz, aroma
H) 851 (IH, d, J=9.2Hz, aroma
H) 9.42 (If(0m, Nf() IQ, 10 (2H,s, 2xNH) (Actual #ilI Example 6) fiE O N-methylacridinium (22~, 0.038m m
ot) 17) Anhydrous DMF (0.5 mg) il:7
t/JinU C7,6Iv, 0.048mm06l- was added, and the resulting reaction suspension was heated and stirred at an external temperature of 50-60°C for 6 hours. DMF was distilled off under reduced pressure, leaving a dark yellow residue (43,
An aqueous hydrochloric acid solution (an amount corresponding to 0,057 mmoz of hydrogen chloride) and MeO) were added to the remaining solution. The solvent was distilled off again, and the residual Sephadex LH-2 [
1 column chromatography (551) and moxibustion. Fill the column with 1 ml of EtO. Elution was performed with EtOH. Elution volume 67-46d yellow fraction thlt collected compound rJEc22.4■, yield 94
．． 8%). Compound lIE 'H-NMR (DMSO-d6) δ: 167 (2H,m, -HNCH,CH2O) (2-
)2.46 (2H,t,J =7.3Hz,-()
12G-)2.94 (2H0t, J=7.6Hz,
Ph0H2-)3.06 (2H,m-
HNCH2-)5.36 (2H,t,J=6.6f
(z, -0H2N)4.97 (3H,s,
10N-CH5) 746 and 7.65 (
4H, AA'BB'. J:8.6H2, aromH) 7.92 (IHom, NH) 8.19 (2)1, m, aromH)
8.57 (2H, m, aroma H)8.6
2 (2H, d, J=8.6Hz, aromJ8.9
6 (2)1. d, J=9.2Hz, aromaH)1
0.10 (2H, br, s, 2XNH) Below, an example of the reaction between the compound of the present invention and vitamin Ds will be explained. (Example 7) am R1=R2=Rg=H b= R1=R3=HR2=OH C=R□:OHR2,F13=f(d:R,=R2=O)I R3=He=R1
=R3=OHR! =H Method 1) Oxidation of one area with Pb(OAc)4,
Continuation of bow, A, reaction Vl tamtn D3 22 a (91W
, 0.0 2 4 mm01)L[I A (I
2.9W, 0.028mmot), anhydrous DMF (0
, 2d), anhydrous THF (0,3id) and g4. A.C.O.
A suspension of H (5 μt) was cooled to 75°C and stirred with one tube of Pb(OAc), (I8.9 mg, 0.043 m
mO/-) was added. The reaction suspension was PbC0Ac)4
Five minutes after the addition, the mixture turned red. The reaction solution was heated for 1 hour-7
After stirring at 5°C, the mixture was returned to room temperature and the reaction was continued for an additional hour. Pour the reaction mixture into ice-cold water and add cHct3 (3X1
5awj)K and extracted. The cHat 3 layer was washed with saturated saline, dried with Glauber's salt, and the solvent was distilled off. Obtained yellow sticky substance (22, [3W)
veTLO (Merck company lit, 5ilica
gel 6OF254゜20 tx (W) X I
Q cm (H) X O,5am (T) !C6-αH] (G, -βH: 1.6#) 'i' was obtained. The total yield was 555%. and apricot crystallization from MeOH-C; H2O12 to obtain yellow needle-like crystals. Compound-a (G6-αH) mp 145-145°C UVλIJQHnm (log g): 217 (4
, 72). 24 (Shoulder), 322 (3,80). 4ON4.07). Elemental analysis two calculated values (048H61N5011-Hρ): C, 67,50
:H, 7, 45: N, 8.20° 11J constant 11
:C,67,08:)1.7.18;N,8.12
゜<Method 2> PhI (OAc) 2 and 11A4)
Oxidation, iJ followed by reaction with vitamin D (Iid) FItK, PhI (OAC), (7,
5av, 102!1mm0A)t' was added, and stirring was continued at room temperature for about 0.5 to 1 hour until the first reaction solution developed a sufficiently red color. This red solution D, 24.25 (OH), D, 22e (
5, Oav. 0.012 mmOL) was added thereto, and the mixture was further stirred for 5 hours until the red color of the solution disappeared. The solvent was distilled off under reduced pressure. A member-colored adhesive substance (24,1~) was obtained and prepared.
ativeTLCC Merck H5ilica ge
l 60 F2. . 20cm (W) X 10m (H) X O, 5aa*
(T)
L2) was purified at °C and a mixture of adducts c6-αH anti-βh, 10.4 μm. It was obtained as a non-crystalline a'R colored adhesive powder (yield: fixed). Table 1 shows the differential production ratio and yield of the adduct of vitamin D and the reagent of the present invention obtained in this way. Compounds 1c, ld and Ie are mixed
The r-no-liquid was analyzed by HPLC (high performance liquid chromatography) under the following conditions. HPLC separation conditions A: Li chromospher 10CIRP
15lel (5,czm)4 aa X 250
m #* Agent: Water: Methanol = 27: 73 (v/v
) Flow rate: 11/min Detection 2 EX395nm, Em500nm6nP))
/10 μt ethanol results are shown in FIG. In the figure, lc, l (I, let etc.N) l\no l-1/ is the main production ml Ic', tumor d'0zene' is l1ll raw WV it l ~ no r\//-++/ represents. The two isomers K (C; 6-αH and βH) of compound pHa were separated by preparative TLC, and then 'H-NMRkaJ was determined. The results are shown in Table 4. Mixture of compounds rvb, rvc and l'Vd which are adducts of vitamin D3 and the reagent of the present invention (compound 1111G)/++//2-no--1-iPLc separation using i, 1-iPLc It was conducted under the following conditions. HPLC separation conditions column: Li chromospher 10CIRP-
18f81 (5μm) 4gm8250g+ #Removal: [@By linear gradient bathing] 0 minutes Water: Methanol = 40:60'50 minutes Water: Methanol = 20:80 After 40 minutes Water: Methanol =
20:80 flow rate = 11E//min detection: EX, 37Qnm, Em, 44Qnm. IVd(-15nfP): 1/10 μtz/-L The results are shown in FIG. In the figure, IVb, IVC, and IVd are represented by 1/1N. Examples of the reaction between the compound of the present invention and vitamin A will be explained below. Reaction of fluorescent water-injected diene reagent with vitamin Ap injection (Execution 91J8-10) 1) = Y = -0H201-12 11A (22!, 50 μm06) in DMF
+/) to PhI(OAC)2 (I61+19.50μ
moz) Y and stirred at room temperature for 30 minutes, then added IA? Prepare. This solution Yrettnoic acid (I[]al
, 33μmot) 17)C)12Gt2 (20
aj) Add the dissolved NKO dropwise under an Ar stream at °C. After immersing at Q℃ for 60 minutes, the solvent was distilled off and the silica gel TLC was 10% M.
e 1aH'T247 using OH/G HCZ 3
．． 218hm, IR (KBr) 1770. 1719, 1655clR-1'H to MRCCDGL3) 61.05 and 1.14 (5H, &, Me-1, respectively), 1.75 (3H,s, M
e-5). 1.81(3)1. s, Me-9), 2.21 (3f
(, s. Me-13), 3.73 (6H, s, NM6), 3.
94 and 4.04 (3H, a, OMe, respectively),
4.85 (Ih. t,, H7), 4.85 (IH, d, J=8.6.H-
10), 5.47 (IH, a,, H-8), 5.83 (
IH, t, H-14), 5.45 (2H, s. 0-OH), 6.021) 1. dd, J=15 and 8.
6. H-11), 6.47 (IH, d, J = 15°H-
12), 6.67 and 7.37 (each I H,
s. Ki/1r4jsu7), 7.55 and 7.58 (eac
h2 H. AA'BB', J=9, Ar) (9
0fNf'J9) and lmyA acid (E6tinOi
PhI(OAc)2 (7,8 mW, 24.g
mot) and stirred at room temperature for 30 minutes, and the pale red solution of 1c f
f! L-manufactured. Fletinoic acid (
3-5III, 12 pUaOL) of CH2Cl2
(I0m/) Add Ar at 0°C to the bath solution with the above ratio.
Stir for 60 minutes under a stream of air, remove the solvent, and purify the residue with 10% MθOH/C:H2Gt2 using silica gel TL and G to obtain the adduct Qb (5!.66%). Vb UV (95% EtOH) 368,244.213
nmr-IFl (KBr) 1771.1715.1649z-1
M8 m/8646CM”-1), 601,399.
355/HNMR (CDCl2) δ0.88 and 0.9
5 (3H, s, Me-1 respectively), 'L59 (3H,
s, Me-5). t77 (3H, @, Me-9), 2.19 (3H, r,
. Me-13),! 1.67 (3H, @, NMe), 3
．． 93 and 4.01 (respectively "ki, s, OMB"),
4.61 (IH, @, H-7), 4.74 (IH, d,
J=8.6. H-10), 5.37 (IH,s, H-8
). 5.86 (IH, i, H-14), 5.96 (IH. dd, J=15 and 8.6, 8-11), 6.3
DMF solution f retinol (24)
(I Qll, 35 pmot)ノOH,C;t
2 (2051/) solution at -78°C and stirred for 60 minutes. Solvent distillation flk residue is silicage A/17) Column A OOE t / G HCZ a / E t O
f(5Q:50:1 solvent) to purify the adduct v
1 (I49, 55%) is obtained. Compound V+: IR(KBr)3440.1771.1711.165
5ffi-”/HNMR (GDC, t3) 0.992 and 0.98
7 (6H, s, Me-1 respectively), t66 (5H, s,
Me-5). t99c5H, s, Me-9), 1.91 (3H
,s. Me-13), 3.74 (3f(,s,NMe), 19
5 and 4.05 (3 each)1. a, OMe), 5.5
6 (IH, dd. J=8 and 4, OH, D, 0ffi7FO disappearance),
4.24 and 3.87 (IH, rn, H-15 respectively),
4.44-(IH. (I, J=6.8-14), 5.22(I8, d, J=
Kai H-10), 5.31 (I H, m, H-11)
, 5.70 (IH, m, H-12), 5.99 (I)
1. d, J=16.5. H-8), 6.17 (IH, d
, J=16.5°302.220 DETAILED DESCRIPTION OF THE INVENTION As described in detail, the fluorescent or chemiluminescent reagents synthesized according to the present invention react rapidly, specifically and routinely with compounds containing C15-dienes. These reagents are therefore cps-
It can be said that it is an excellent reagent suitable for the detection and quantitative analysis of diene-containing compounds, especially vitamins D and A, and their metabolites.

[Brief explanation of the drawing]

FIG. 1 is a chromatogram showing the peaks of adducts of vitamin D, adducts of various adducts, and the reagent IA of the present invention detected by separation at °C using vHpLc. FIG. 2 is a chromatogram showing the peaks of each adduct of vitamin D3 derivatives and the reagent IC of the present invention detected after separation by t-1pLc. Patent applicant Agent of Biosensor Institute Co., Ltd.
Patent attorney Kyozo Yuasa: ', -,' Oni I (4 others)

Claims (1)

[Claims] 1. General formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) (In the formula, X represents a fluorescent chromophore or a potential chemiluminescent group, and Y represents a spacer.) Compound represented. 2. General formula (II) ▲Mathematical formulas, chemical formulas, tables, etc. are available▼(II) (In the formula, X represents a fluorescent chromophore or potential chemiluminescent group, and Y represents a spacer.) A compound represented by the following. 3. A group where X is a group represented by the following formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ may be the same or different and represent a hydrogen atom or an alkyl group] Y is a member selected from the following formula - (CH_2)_l- ▲ There are mathematical formulas, chemical formulas, tables, etc.
A- represents -O- or -NH-; -B- represents -NH
-, -COO-, -SO_2NH-, ▲mathematical formula, chemical formula,
Indicates a member selected from the group consisting of tables, etc.▼ and ▲Mathematical formulas, chemical formulas, tables, etc.▼, l indicates an integer from 1 to 5, m indicates an integer from 0 to 5, n represents either 0 or 1) (However, the left end of each formula of Y is bonded to the nitrogen atom of the ring, and the right end is bonded to X). The compound according to 1 or 2. 4. X is a member selected from the group consisting of groups represented by the following formula ▲ There are mathematical formulas, chemical formulas, tables, etc. (in the formula, Z represents the conjugate base of the acid), and Y is the following formula ▲ There are chemical formulas, tables, etc.▼,▲mathematical formulas, chemical formulas,
There are tables, etc. ▼ -(CH_2)_n- (in the formula, n indicates either 0 or 1) is a member selected from the group consisting of the groups represented by (However, the left end of each formula of Y is The nitrogen atom of the ring and the right end is X
). A compound according to claim 3. 5. A compound according to claim 3. 6. General formula (V) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (V) or (VI) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (VI) (In the formula, Y represents a spacer, and Q represents hydroxy Coupling a compound represented by the general formula (VII) X-COCl (VII) (in which X represents a fluorescent chromophore or a potential chemiluminescent group) General formula (I) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (I) (in the formula, X and Y have the above meanings) or general formula (II) ▲ Numerical formulas, chemical formulas, tables, etc. etc. ▼(II) A method for producing a compound represented by (wherein, X and Y have the above meanings). 7. General formula (VIII) ▲Mathematical formulas, chemical formulas, tables, etc.▼(VIII) General formula II characterized by ring-closing a semicarbazide represented by A method for producing a compound. 8. General formula (I) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (I) (In the formula, X represents a fluorescent chromophore or potential chemiluminescent group, and Y represents a spacer) or general formula (II) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (II) (In the formula, X and Y have the above meanings) The compound is used as a fluorescent or chemiluminescent reagent, and contains CiS in the chemical structure. - Detection and quantitative analysis method for compounds with diene structure. 9. General formula (I) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (I) (In the formula, X represents a fluorescent chromophore or potential chemiluminescent group, and Y represents a spacer) or general formula (II) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (II) Vitamin D, vitamin A characterized by using the compound represented by (in the formula, X and Y have the above meanings) as a fluorescent or chemiluminescent reagent. or detection and quantitative analysis methods for these metabolites.