JPH08245615A - 1,2-dioxetane derivative - Google Patents

Abstract

PURPOSE: To obtain the subject new compound excellent in chemical stability and the durability of light emission, giving stable data and useful for immunoassay, etc., being used as a chemically emitting reagent having high reproducibility. CONSTITUTION: This is a compound of formula I [R<1> to R<6> an each H, an alkyl, etc., and all of R<1> to R<6> can not be simultaneously H; R<7> is an alkyl; R<8> is an alkoxy, a group of the formula: OSi(R<9> R<10> R<11> ) (R<9> to R<11> are each R<7> ) or a phosphate group; X, X' are each H, an alkoxy, (a halogen an alkyl or an alkoxy substituted) phenyl, a halogen, R<7> , etc., and X and X' cannot be simultaneously H]] For example, 3-[3-(t-butyldimethylsiloxy)-5-methoxyphenyl]-4,4- diisopropyl-3-methoxy-1,2-dioxetane. A compound of formula I is obtained by reacting a compound of formula II with a compound of formula III to obtain α-alkoxystyrene derivative, treating the product by dealkylation to obtain a compound of formula IV, subsequently treating this compound with a silylating agent and a phosphoric-esterification agent followed by the reaction with a singlet oxygen.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel 1,2-dioxetane derivative. The 1,2-dioxetane derivative of the present invention can be used as a chemiluminescent reagent for immunoassay and the like.

[0002]

2. Description of the Related Art Conventionally, various 1,2-dioxetane derivatives have been synthesized, and it is known that a compound having a spiroadamantyl group bonded at the 3-position is particularly useful as a chemiluminescent substrate (for example, a special Japanese Patent Publication No. 5-21918 and Japanese Patent Publication No. 45590/1993).

[0003]

However, conventional compounds cannot be said to have sufficient effects on stability or luminescence sustainability, and improvements thereof have been desired.

That is, an object of the present invention is to provide a novel 1,2-dioxetane derivative having excellent stability and long-lasting luminescence.

[0005]

Means for Solving the Problems As a result of intensive studies to overcome the drawbacks of conventional compounds, the inventors of the present invention found a 1,2-dioxetane derivative having excellent stability and long-lasting luminescence. completed.

That is, the present invention provides a 1,2-dioxetane derivative represented by the following general formula [I].

[0007]

Embedded image (However, in the formula, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R
⁶ are each independently a hydrogen atom or an alkyl group (provided that R is
All of ^{1 to} R ⁶ may not be hydrogen atoms at the same time, and R ¹ and R ² and R ⁴ and R ⁵ may be integrated together to form a cyclic alkyl group), R ⁷ is an alkyl group, R ⁸ alkoxyl ^{group, -OSi (R 9 R 10 R} 11) ( wherein, R
⁹ , R ¹⁰ and R ¹¹ each independently represent an alkyl group) or a phosphate group, and X and X ′ are each independently substituted by a hydrogen atom, an alkoxyl group, a phenyl group (a halogen, an alkyl group or an alkoxyl group). ), A halogen or an alkyl group. (However, X and X'may not be hydrogen atoms at the same time, and X and X'may together form a ring.))

The present invention will be described in detail below.

In describing the present invention in detail below, the "alkyl group" in the present invention means a linear or branched alkyl group having 1 to 20 carbon atoms which may have a substituent. The alkyl group is a straight chain of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icodecyl. A group and a group in which the above alkyl group is appropriately branched to each other. The optionally substituted group is, for example, a hydroxyl group,
Examples thereof include an alkoxyl group, an aryl group and a heterocyclic group. Examples of the alkoxyl group include methoxy, ethoxy,
Propoxy, butoxy, pentyloxy, hexyloxy, methoxyethoxy, methoxypropoxy, ethoxyethoxy, ethoxypropoxy, methoxyethoxyethoxy groups and the like, and the aryl group thereof is, for example, phenyl, naphthyl group, etc. Examples of the group include furyl, thienyl and pyridyl groups.

As described above, the 1,2-dioxetane derivative of the present invention is represented by the above general formula [I].
In the formula [I], R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are each independently a hydrogen atom or an alkyl group, and R ^{1 to} R
Not all ⁶ can be hydrogen atoms at the same time. Further, R ¹ and R ² and R ⁴ and R ⁵ may be integrated with each other to form a cyclic alkyl group. As a preferred example, R
¹ , R ² , R ⁴ and R ⁵ are alkyl groups (especially methyl groups)
And R ³ and R ⁶ are hydrogen atoms. R ⁷ is an alkyl group. R ⁸ is an alkoxyl group, —OSi (R ⁹ R ¹⁰ R ¹¹ ) (provided that R 8
⁹ , R ¹⁰ and R ¹¹ each independently represent an alkyl group) or a phosphate group, and among these, —OSi (R ⁹ R
¹⁰ R ¹¹ ) is preferred. Among them, -OSi (Me) ₂ t
-Bu is particularly preferred.

In the formula [I], X and X'independently of each other are a hydrogen atom, an alkoxyl group, a phenyl group (which may be substituted with a halogen, an alkyl group or an alkoxyl group),
It is a halogen or alkyl group, and X and X'cannot be hydrogen atoms at the same time. In addition, X and X ′ may together form a ring. Of these, preferred are those in which X is an alkoxyl group (particularly a methoxy group) and X'is a hydrogen atom, X is a phenyl group or a substituted phenyl group and X'is a hydrogen atom, and X and X'are represented by the following formulas: [I
As shown in [I], the case where they are integrally formed into a ring can be mentioned.

[0012]

Embedded image (However, in the formula [II], R ¹⁴ and R ¹⁵ each independently represent hydrogen or a lower alkyl group.)

The 1,2-dioxetane derivative represented by the formula [I] of the present invention can be produced according to the following reaction formula. In the following chemical formulas, reaction formulas, and explanations, the same substituents as the above-mentioned substituents are denoted by the same reference numerals, and the meaning thereof is as described above. Further, R ⁸¹ is an alkoxyl group or —O of R ^8.
Si (R ⁹ R ¹⁰ R ¹¹ ) is shown, and R ⁸² is —O of R ^8.
Indicates Si (R ⁹ R ¹⁰ R ¹¹ ) or a phosphate group.

[0014]

Embedded image

Each step of the above reaction formula will be described below.

First Step In this step, an aromatic carboxylic acid ester represented by the general formula [III] is reacted with a ketone represented by the general formula [IV],
It is for producing an α-alkoxystyrene derivative represented by the general formula [VI].

The reaction is preferably carried out in the presence of titanium. Usually, titanium is preferably used in the reaction by forming a reduced state of titanium halide such as titanium chloride using a reducing agent such as lithium aluminum hydride and a base such as triethylamine.

The reaction is preferably carried out in ether such as tetrahydrofuran (THF). Reaction is 0-100
Although it proceeds at 0 ° C., it is preferable to carry out under reflux of a solvent such as THF from the viewpoint of operation and reactivity. The reaction time is usually about 30 minutes to 5 hours.

The compound of the general formula [VI] is prepared by subjecting the compound of the general formula [V] in which the second step R ⁸ is lower alkyloxy (alkoxy, preferably methoxy) to a dealkylation reaction.

The reaction is carried out by heating the compound of the general formula [VI] with an alkali metal salt of lower alkyl mercaptan (for example, sodium or potassium salt of methanethiol or ethanethiol) in an aprotic polar solvent such as dimethylformamide and heating (50 to 150). ° C). The reaction time is usually 1 to 10 hours.

Third Step This step can be achieved by reacting [V] with a silylating agent or by a phosphoric acid esterification reaction according to a conventional method (see Reference Example below).

Fourth Step In this step, the α-alkoxystyrene derivative represented by the general formula [VI] is reacted with singlet oxygen to produce the 1,2-dioxetane derivative represented by the general formula [I]. is there.

The reaction with singlet oxygen is carried out by the reaction of the above general formula [VI]
The α-alkoxystyrene derivative represented by is dissolved in a solvent such as dichloromethane, dichloroethane, carbon tetrachloride or alcohol, and is visible in the presence of a photosensitizer such as methylene blue, rose bengal or tetraphenylporphine under an oxygen atmosphere. It is achieved by performing light irradiation. The reaction can be carried out at -80 ° C to 0 ° C, and the reaction time is usually 30.
It is about 5 minutes to 5 hours. By directly subjecting the compound represented by the general formula [V] to this fourth step, R ^{8 in the} compound represented by the general formula [I] of the present invention is an alkoxyl group or —OSi (R ⁹ R ¹⁰ R ¹¹ ) can be obtained.

The 1,2-dioxetane derivative of the present invention is
An enzyme such as tetrabutylammonium fluoride, potassium fluoride or alkaline phosphatase cleaves the O—Si bond, O—P bond or O—C bond in R ⁸ and emits light at this time. Therefore, if this luminescence is measured, it can be used as a substrate in enzyme immunoassay.

[0025]

EXAMPLES The present invention will be described more specifically below based on examples and reference examples. However, the present invention is not limited to the following examples.

Reference Example 1

[Chemical 4]

Titanium trichloride 4.5 at 0 ° C. under nitrogen atmosphere
g (29 mmol) and 0.57 g (15 mmol) of lithium aluminum hydride were sequentially added to 75 ml of anhydrous THF and stirred. 2.1m of triethylamine in this solution
1 (15 mmol) was added at room temperature, followed by heating under reflux for 15 minutes. Methyl 3,5-dimethoxybenzoate (compound [1]) dissolved in 25 ml of anhydrous THF was added to this solution.
A solution of 1.76 g (8.98 mmol) and diisopropyl ketone 1.8 ml (12.7 mmol) was added dropwise over 20 minutes, and the mixture was heated under reflux for 2 hours. After adding 100 ml of water to the reaction mixture at 0 ° C., the mixture was extracted with ethyl acetate. The extract layer was washed with water, dried over magnesium sulfate, and concentrated. When the concentrate was applied to a silica gel column and poured out with a mixed solvent of hexane and dichloromethane of 3: 1,
1,1-diisopropyl-2-methoxy-2- (3,5
-Dimethoxyphenyl) ethene (compound [2]) is 45
0 mg, yield 18.0%, was obtained as a colorless oil.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.93 (d, J = 6.8 Hz, 6
H), 1.24 (d, J = 6.8Hz, 6H), 2.30 (sept, J = 6.8Hz, 1H), 2.50
(sept, J = 6.8Hz, 1H), 3.21 (s, 3H), 3.80 (s, 6H), 6.41 (s, 3
H) ppm IR (liquid film): 2955, 1590cm ^-1 Mass (m / z,%): 278 (M ⁺ , 34), 264 (100), 231 (41), 205 (2
9), 189 (18), 165 (19), 128 (16), 115 (39)

Reference Example 2

Embedded image

Sodium hydride (60%) and 80 mg (2.0 mmol) were added to anhydrous DMF at 0 ° C. under a nitrogen atmosphere.
Ethanethiol 0.15 was added to the solution which was suspended in 2 ml and stirred.
ml (2.0 mmol) was added dropwise. Anhydrous D in this solution
Compound [2] synthesized in Reference Example 1 dissolved in 2 ml of MF
A 200 mg (0.719 mmol) solution was added, followed by heating under reflux for 3 hours. The reaction mixture was poured into saturated saline and extracted with ethyl acetate. The extract layer was washed with saturated saline,
The extract was dried over magnesium acetate and concentrated. The concentrate was applied to a silica gel column and poured out with a 5: 1 mixed solvent of hexane and ethyl acetate to give 1- (3-hydroxy-5-methoxyphenyl) -2,2-diisopropyl-1-methoxyethene (compound [3] ) Is 150 mg, and the yield is 79.0.
% As a pale yellow oil.

¹ H NMR (400 MHz, CDCl ₃ ): δ0.92 (d, J = 6.8 Hz, 6
H), 1.23 (d, J = 6.8Hz, 6H), 2.31 (sept, J = 6.8Hz, 1H), 2.49
(sept, J = 6.8Hz, 1H), 3.21 (s, 3H), 3.79 (s, 3H), 4.83 (broa
ds, 1H), 6.33 (d, J = 1.0Hz, 1H), 6.36 (t, J = 2.4Hz, 1H), 6.3
9 (d, J = 1.0Hz, 1H) ppm IR (liquid film): 3400, 2960, 1605cm ^-1 Mass (m / z,%): 264 (M ⁺ , 62), 249 (100), 221 (88 ), 207 (1
4), 189 (50), 175 (26), 151 (24), 123 (16), 108 (12)

Reference Example 3

[Chemical 6]

83 mg (0.314 mmol) of the compound [3] synthesized in Reference Example 2 was added to anhydrous D at 0 ° C. under a nitrogen atmosphere.
It was dissolved in 2 ml of MF and stirred. To this solution were sequentially added 1.0 ml (7.2 mmol) of triethylamine and 0.10 g (0.66 mmol) of t-butyldimethylchlorosilane, and subsequently stirred at room temperature overnight. The reaction mixture was poured into water and extracted with ethyl acetate. The extract layer was washed with saturated brine, dried over magnesium sulfate, and concentrated. The concentrate was applied to a silica gel column and was poured out with a mixed solvent of hexane and ethyl acetate at a ratio of 5: 1 to give 1- [3- (t-butyldimethylsiloxy) -5-methoxyphenyl] -2,2.
-Diisopropyl-1-methoxyethene compound [4] was obtained as a colorless oily substance with a yield of 53 mg and a yield of 44.6%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.20 (s, 6H), 0.92
(d, J = 6.8Hz, 6H), 0.98 (s, 9H), 1.23 (d, J = 6.8Hz, 6H), 2.30
(sept, J = 6.8Hz, 1H), 2.48 (sept, J = 6.8Hz, 1H), 3.20 (s, 3
H), 3.78 (s, 3H), 6.35 (d, J = 2.0Hz, 2H), 6.43 (s, 1H) ppm IR (liquid film): 2950, 1590, 1360, 1150cm ^-1

Example 1

[Chemical 7]

43 mg of the compound [4] synthesized in Reference Example 3
(0.114 mmol) and 5 mg of TPP were dissolved in 10 ml of dichloromethane, and the mixture was stirred at -78 ° C under an oxygen atmosphere. This solution was irradiated with a Na lamp (940W) for 2 hours. The reaction mixture was concentrated, the concentrate was applied to a silica gel column, and was poured out with a 10: 1 mixed solvent of hexane and ethyl acetate. 3- [3- (t-butyldimethylsiloxy) -5-methoxyphenyl] -4,4 -Diisopropyl-3-methoxy-1,2-dioxetane (Compound [5]) was obtained as a pale yellow oil in 35 mg, yield 75.0%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.20 (s, 6H), 0.52
(d, J = 6.8Hz, 3H), 0.92 (d, J = 6.8Hz, 3H), 0.98 (s, 9H), 1.16
(d, J = 6.8Hz, 3H), 1.29 (d, J = 6.8Hz, 3H), 2.45 (sept, J = 6.8
Hz, 1H), 2.63 (sept, J = 6.8Hz, 1H), 3.14 (s, 3H), 3.80 (bro
ad s, 3H), 6.42 (t, J = 2.4Hz, 1H), 6.75 ~ 6.95 (m, 2H) ppm IR (liquid film): 2960, 1595, 1340, 1160cm ^-1

Reference Example 4

Embedded image

Titanium trichloride 4.5 at 0 ° C. under nitrogen atmosphere
g (29 mmol) and 0.57 g (15 mmol) of lithium aluminum hydride were sequentially added to 75 ml of anhydrous THF and stirred. 2.1m of triethylamine in this solution
1 (15 mmol) was added at room temperature, followed by heating under reflux for 15 minutes. A solution of t-butyl 3,5-dimethoxybenzoate (compound [6]) 1.43 g (6.01 mmol) and diisopropyl ketone 1.8 ml (12.7 mmol) dissolved in anhydrous THF 25 ml was added to this solution over 20 minutes. The mixture was added dropwise and heated under reflux for 2 hours.
After adding 100 ml of water to the reaction mixture at 0 ° C., the mixture was extracted with ethyl acetate. The extract layer was washed with water, dried over magnesium sulfate, and concentrated. The concentrate was applied to a silica gel column and poured out with a mixed solvent of hexane and dichloromethane at a ratio of 3: 1. 1-t-butoxy-2,2-diisopropyl-1-
(3,5-Dimethoxyphenyl) ethene (Compound [7]) was obtained as a colorless oily substance at a yield of 23.4% (450 mg).

¹ H NMR (400 MHz, CDCl ₃ ): δ0.89 (d, J = 6.8 Hz, 6
H), 1.11 (s, 9H), 1.24 (d, J = 6.8Hz, 6H), 2.44 (sept, J = 6.8
Hz, 1H), 2.64 (sept, J = 6.8Hz, 1H), 3.79 (s, 6H), 6.38 (t, J =
2.0Hz, 1H), 6.46 (d, J = 2.0Hz, 2H) ppm IR (liquid film): 2960, 1590cm ^-1 Mass (m / z,%): 320 (M ⁺ , 4), 264 (100) , 249 (36), 221 (5
7), 165 (48), 137 (19), 122 (20)

Reference Example 5

[Chemical 9]

Under a nitrogen atmosphere, at 0 ° C. sodium hydride (6
0%), 80 mg (2.0 mmol) of anhydrous DMF2m
0.15 ml of ethanethiol in the solution which was suspended in 1 and stirred
(2.0 mmol) was added dropwise. Anhydrous DMF in this solution
Compound [7] 30 synthesized in Reference Example 4 dissolved in 2 ml
0 mg (0.938 mmol) solution was added, followed by 3
Heated to reflux for hours. The reaction mixture was poured into saturated saline and extracted with ethyl acetate. The extract layer was washed with saturated brine, dried over magnesium sulfate, and concentrated. The concentrate was applied to a silica gel column and was poured out with a mixed solvent of hexane and ethyl acetate at a ratio of 5: 1 to give 1-t-butoxy-2,2-diisopropyl-1- (3-hydroxy-5-methoxyphenyl) ethene (compound [8]) was 244 mg, and the yield was 85.
Obtained as a pale yellow oil at 1%.

¹ H NMR (400 MHz, CDCl ₃ ): δ0.89 (d, J = 6.8 Hz, 6
H), 1.11 (s, 9H), 1.22 (d, J = 6.8Hz, 6H), 2.44 (sept, J = 6.8
Hz, 1H), 2.64 (sept, J = 6.8Hz, 1H), 3.77 (s, 3H), 4.86 (broa
ds, 1H), 6.33 (t, J = 2.0Hz, 1H), 6.39 (d, J = 2.0Hz, 1H), 6.4
5 (dd, J = 2.0 and 1.0Hz, 1H) ppm IR (liquid film): 3300, 2950, 1590cm ^-1

Reference Example 6

[Chemical 10]

230 mg (0.75 mmol) of the compound [8] synthesized in Reference Example 5 at 0 ° C. under a nitrogen atmosphere was added to anhydrous D
It was dissolved in 2 ml of MF and stirred. To this solution were sequentially added 2.0 ml (14 mmol) of triethylamine and 0.20 g (1.3 mmol) of t-butyldimethylchlorosilane, and subsequently stirred at room temperature overnight. The reaction mixture was poured into water and extracted with ethyl acetate. The extract layer was washed with saturated brine, dried over magnesium sulfate, and concentrated. The concentrate was applied to a silica gel column and poured out with a mixed solvent of hexane and ethyl acetate at a ratio of 5: 1 to give 1-t-butoxy-1-.
[3- (t-Butyldimethylsiloxy) -5-methoxyphenyl] -2,2-diisopropylethene (Compound [9]) was obtained as a colorless oil in 250 mg, yield 79.2%.

¹ H NMR (400 MHz, CDCl ₃ ): δ0.18 (s, 6H), 0.88
(d, J = 6.8Hz, 6H), 0.98 (s, 9H), 1.11 (s, 9H), 1.23 (d, J = 6.
8Hz, 6H), 2.42 (sept, J = 6.8Hz, 1H), 2.62 (sept, J = 6.8Hz, 1
H), 3.77 (s, 3H), 6.32 (t, J = 2.4Hz, 1H), 6.39 (dd, J = 2.4 an
d 1.5Hz, 1H), 6.48 (dd, J = 2.4 and 1.5Hz, 1H) ppm IR (liquid film): 2955, 2860, 1590, 1365, 1155cm ^-1 Mass (m / z,%): 420 (M ⁺ , 4), 364 (100), 349 (16), 321 (1
9), 265 (19)

Example 2

[Chemical 11]

Compound [9] 100 m synthesized in Reference Example 6
g (0.238 mmol) and 5 mg of TPP were dissolved in 10 ml of dichloromethane, and the mixture was stirred at -78 ° C under an oxygen atmosphere. This solution was irradiated with a Na lamp (940W) for 2 hours. The reaction mixture was concentrated, and the concentrate was applied to a silica gel column and poured out with a 10: 1 mixed solvent of hexane and ethyl acetate to give 3-t-butoxy-3- [3-
(T-Butyldimethylsiloxy) -5-methoxyphenyl] -4,4-diisopropyl-1,2-dioxetane (Compound [10]) was obtained as a pale yellow oily substance at 97 mg in a yield of 90.1%. .

1: 1 rotamer mixture ¹ H NMR (400 MHz, CDCl ₃ ): 0.14 to 0.23 (m, 6H), 0.43 (d, J = 7.3)
Hz, 1.5H), 0.44 (d, J = 7.3Hz, 1.5H), 0.89 (d, J = 6.8Hz, 3H),
0.98 (S, 4.5H), 0.99 (S, 4.5H), 1.17 (d, J = 6.8Hz, 3H), 1.2
2 (S, 4.5H), 1.22 (S, 4.5H), 1.31 (d, J = 6.8Hz, 3H), 2.34〜
2.45 (m, 1H), 2.55 to 2.68 (m, 1H), 3.76 (S, 1.5H), 3.81
(S, 1.5H), 6.37 (S with fine coupling, 0.5H), 6.39 (S
with fine coupling, 0.5H), 6.54 (S with fine couplin
g, 0.5H), 6.63 (S with fine coupling, 0.5H), 6.87 (S w
ith fine coupling, 0.5H), 6.94 (S with fine couplin
g, 0.5H) ppm IR (liquid film): 2960, 1595, 1455, 1335, 1255, 116
0cm ^-1 Mass (m / z,%): 420 (M ⁺ -32,2), 364 (30), 338 (30), 265 (1
4), 225 (100)

Reference Example 7

[Chemical 12]

Titanium trichloride 4.5 at 0 ° C. under nitrogen atmosphere
g (29 mmol) and 0.57 g (15 mmol) of lithium aluminum hydride were sequentially added to 70 ml of anhydrous THF and stirred. 2.1m of triethylamine in this solution
1 (15 mmol) was added and the mixture was heated to reflux. 1.47 g of methyl 5-methoxy-3-biphenylcarboxylate (compound [11]) dissolved in 25 ml of anhydrous THF was added to this solution.
(6.07 mmol) and diisopropyl ketone 2.
A 6 ml (18.4 mmol) solution was added dropwise over 20 minutes, and the mixture was further heated under reflux for 4 hours and 30 minutes. The reaction mixture was poured into water and extracted with ethyl acetate. Wash the extract layer with water,
The extract was dried over magnesium sulfate and concentrated. The concentrate was applied to a silica gel column and poured out with a mixed solvent of hexane and dichloromethane of 20: 1. 1,1-diisopropyl-
0.87 g of 2-methoxy-2- (5-methoxybiphenyl-3-yl) ethene (compound [12]), yield 4
Obtained as a colorless oil at 4.2%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.96 (d, J = 6.8 Hz, 6
H), 1.26 (d, J = 6.8Hz, 6H), 2.35 (sept, J = 6.8Hz, 1H), 2.55
(sept, J = 6.8Hz, 1H), 3.24 (s, 3H), 3.87 (s, 3H), 6.79 (dd, J
= 2.4 and 1.5Hz, 1H), 7.08 (dd, J = 2.4 and 1.5Hz, 1H),
7.10 (t, J = 1.5Hz, 1H), 7.35 (t with fine coupling, J
= 7.3Hz, 1H), 7.44 (t with fine coupling, J = 7.3Hz, 2H),
7.60 (d with fine coupling, J = 7.3Hz, 2H) ppm IR (liquid film): 2955, 2870, 2830, 1590cm ^-1

Reference Example 8

[Chemical 13]

80 mg (20 mmol) of sodium hydride (60%) was added to anhydrous DMF3 at 0 ° C. under a nitrogen atmosphere.
0.15 of ethanethiol was added to the solution suspended in ml and stirred.
ml (2.0 mmol) was added dropwise. Anhydrous D in this solution
Compound synthesized in Reference Example 7 dissolved in 3 ml of MF [1
2] A solution of 260 mg (0.80 mmol) was added, followed by heating under reflux for 8 hours. The reaction mixture was poured into saturated aqueous ammonium chloride solution and extracted with ethyl acetate. The extract layer was washed with saturated brine, dried over magnesium sulfate, and concentrated. The concentrate was subjected to preparative TLC to give 1 of hexane and ethyl acetate.
When developed with a mixed solvent of 0: 1, 190 m of 1- (5-hydroxybiphenyl-3-yl) -2,2-diisopropyl-1-methoxyethene (compound [13]) was obtained.
g as a yellow oil in a yield of 76.4%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.95 (d, J = 6.8 Hz, 6
H), 1.26 (d, J = 6.8Hz, 6H), 2.34 (sept, J = 6.8Hz, 1H), 2.55
(sept, J = 6.8Hz, 1H), 3.23 (s, 3H), 4.85 (s, 1H), 6.72 (dd, J
= 2.4 and 1.5Hz, 1H), 7.02 (dd, J = 2.4 and 1.5Hz, 1H),
7.09 (t, J = 1.5Hz, 1H), 7.35 (tt, J = 7.3 and 1.5Hz, 1H),
7.43 (t with fine coupling, J = 7.3Hz, 2H), 7.58 (dd wit
h fine coupling, J = 7.3 and 1.5Hz, 2H) p pmIR (liquid film): 3355, 2960, 2870cm ^-1

Reference Example 9

Embedded image

In a nitrogen atmosphere, 175 mg (0.56 mmol) of the compound [13] synthesized in Reference Example 8 was dissolved in 4 ml of anhydrous DMF and stirred at 0 ° C. 3.0 ml (21.5 mmol) of triethylamine and 430 mg (2.85 mmo of t-butyldimethylchlorosilane) were added to this solution.
1) were sequentially added, followed by stirring at room temperature for 24 hours. The reaction mixture was poured into saturated saline and extracted with ethyl acetate. The extract layer was washed with saturated brine, dried over magnesium sulfate, and concentrated. The concentrate was subjected to preparative TLC and developed with a mixed solvent of hexane and ethyl acetate at a ratio of 20: 1.
-Butyldimethylsiloxy) biphenyl-3-yl]-
2,2-Diisopropyl-1-methoxyethene (Compound [14]) was obtained as a colorless amorphous solid in a yield of 209 mg and a yield of 87.3%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.23 (s, 6H), 0.94
(d, J = 6.8Hz, 6H), 1.01 (s, 9H), 1.26 (d, J = 6.8Hz, 6H), 2.3
4 (sept, J = 6.8Hz, 1H), 2.54 (sept, J = 6.8Hz, 1H), 3.23 (S, 3
H), 6.71 (broad s, 1H), 7.02 (broad s, 1H), 7.11 (braod
s, 1H), 7.34 (t, J = 7.3Hz, 1H), 7.43 (t, J = 7.3Hz, 2H), 7.58
(d, J = 7.3Hz, 2H) ppm IR (KBr): 2955, 1585cm ^-1

Example 3

[Chemical 15]

Compound [14] 103 synthesized in Reference Example 9
mg (0.243 mmol) and 5 mg of TPP were dissolved in 10 ml of dichloromethane, and under an oxygen atmosphere, -78 ° C.
It was stirred at. This solution was irradiated with a Na lamp (940 W) for 2 hours. The reaction mixture was concentrated, and the concentrate was subjected to preparative TLC and developed with a mixed solvent of hexane and ethyl acetate of 20: 1. 3- [5- (t-butyldimethylsiloxy) biphenyl-3-yl)]- 4,4-Diisopropyl-3-methoxy-1,2-dioxetane (Compound [15]) was obtained as a pale yellow oily substance at a yield of 98 mg in a yield of 88.5%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.23 (s, 6H), 0.51
(d, J = 7.3Hz, 3H), 0.94 (d, J = 7.3Hz, 3H), 1.01 (s, 9H), 1.20
(d, J = 7.3Hz, 3H), 1.32 (d, J = 7.3Hz, 3H), 2.47 (sept, J = 7.3
Hz, 1H), 2.66 (sept, J = 7.3Hz, 1H), 3.18 (s, 3H), 6.81 ~ 7.
60 (m, 8H) ppm IR (liquid film): 3055, 3020, 2960, 2860, 1595cm ^-1 Mass (m / z,%): 424 (M ⁺ , 8), 342 (38), 286 (32) , 285 (10
0), 253 (12), 225 (12), 114 (13)

Reference Example 10

Embedded image

Titanium trichloride 4.5 at 0 ° C. under nitrogen atmosphere
g (29 mmol) and 0.57 g (15 mmol) of lithium aluminum hydride were sequentially added to 75 ml of anhydrous THF and stirred. 2.1m of triethylamine in this solution
1 (15 mmol) was added at room temperature, followed by heating under reflux for 15 minutes. 5 dissolved in 25 ml of anhydrous THF in this solution
A solution of 0.85 g (3.0 mmol) of t-butyl methoxy-3-biphenylcarboxylate (compound [16]) and 0.9 ml (6.4 mmol) of diisopropyl ketone was added dropwise over 20 minutes, and heating was continued for another 30 minutes. Refluxed. After adding 100 ml of water to the reaction mixture at 0 ° C., the mixture was extracted with ethyl acetate. The extract layer was washed with water, dried over magnesium sulfate, and concentrated. The concentrate was applied to a silica gel column and poured out with a mixed solvent of hexane and dichloromethane at a ratio of 3: 1. 1-t-butoxy-2,2-diisopropyl-
1- (5-Methoxybiphenyl-3-yl) ethene (Compound [17]) was obtained as a colorless oily substance in 400 mg in a yield of 36.5%.

¹ H NMR (400 MHz, CDCl ₃ ): δ0.92 (d, J = 6.8 Hz, 6
H), 1.13 (s, 9H), 1.26 (d, J = 6.8Hz, 6H), 2.49 (sept, J = 6.8
Hz, 1H), 2.67 (sept, J = 6.8Hz, 1H), 3.86 (s, 3H), 6.83 (dd, J
= 24 and 1.5Hz, 1H), 7.04 (t, J = 2.0Hz, 1H), 7.15 (t, J = 1.5
Hz, 1H), 7.34 (tt, J = 5.4 and 2.0Hz, 1H), 7.43 (td, J = 7.3 a
nd 1.5Hz, 2H), 7.59 (dd, J = 7.3Hz and 1.5Hz, 2H) ppm IR (liquid film): 2960, 2870, 1590cm ^-1

Reference Example 11

[Chemical 17]

Under a nitrogen atmosphere, at 0 ° C. sodium hydride (6
0%), 80 mg (2.0 mmol) of anhydrous DMF2m
0.15 ml of ethanethiol in the solution which was suspended in 1 and stirred
(2.0 mmol) was added dropwise. Anhydrous DMF in this solution
Compound [17] synthesized in Reference Example 10 dissolved in 2 ml
A solution of 100 mg (0.273 mmol) was added, followed by heating under reflux for 3 hours. The reaction mixture was poured into saturated saline and extracted with ethyl acetate. The extract layer was washed with saturated saline,
The extract was dried over magnesium sulfate and concentrated. The concentrate was applied to a silica gel column and poured out with a mixed solvent of hexane and ethyl acetate at a ratio of 5: 1, and 1-t-butoxy-1- (5-
85 mg of hydroxybiphenyl-3-yl) -2,2-diisopropyl-ethene (compound [18]), yield 8
Obtained as a colorless amorphous solid at 8.4%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.91 (d, J = 6.8 Hz, 6
H), 1.13 (s, 9H), 1.25 (d, J = 6.8Hz, 6H), 2.49 (sept, J = 6.8
Hz, 1H), 2.66 (sept, J = 6.8Hz, 1H), 4.91 (broad s, 1H), 6.7
6 (dd, J = 2.4 and 1.5Hz, 1H), 6.99 (t, J = 2.0Hz, 1H), 7.14
(d, J = 1.5Hz, 1H), 7.35 (dt, J = 7.3 and 2.4Hz, 1H), 7.43 (t
d, J = 7.3 and 1.5Hz, 2H), 7.58 (dd, J = 7.3 and 1.5Hz, 2H) p
pm IR (liquid film): 3350, 2960, 1590cm ^-1 Mass (m / z,%): 352 (M ⁺ , 3), 296 (60), 281 (33), 253 (10
0), 197 (70), 169 (32), 141 (31), 115 (28)

Reference Example 12

Embedded image

In a nitrogen atmosphere, at 0 ° C., 55 mg (0.156 mmol) of the compound [18] synthesized in Reference Example 11 was dissolved in 2 ml of anhydrous DMF and stirred. 1.0 ml (7.2 mmol) of triethylamine and 0.10 g (0.66 mmo) of t-butyldimethylchlorosilane were added to this solution.
1) were added sequentially, followed by stirring overnight at room temperature. The reaction mixture was poured into water and extracted with ethyl acetate. The extract layer was washed with saturated brine, dried over magnesium sulfate, and concentrated. The concentrate was applied to a silica gel column and poured out with a mixed solvent of hexane and ethyl acetate at a ratio of 5: 1. 1-t-butoxy-1- [5- (t-butyldimethylsiloxy) biphenyl-3-yl] -2, 2-Diisopropylethene (Compound [19]) was obtained as a colorless oily matter in 56 mg, yield 76.9%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.21 (s, 6H), 0.97
(d, J = 6.8Hz, 6H), 1.00 (s, 9H), 1.13 (s, 9H), 1.25 (d, J = 6.
8Hz, 6H), 2.47 (sept, J = 6.8Hz, 1H), 2.65 (sept, J = 6.8Hz, 1
H), 6.75 (dd, J = 2.0 and 1.5Hz, 1H), 6.99 (t, J = 2.0Hz, 1
H), 7.17 (t, J = 1.5Hz, 1H), 7.34 (t, J = 7.8Hz, 1H), 7.43 (t, J
= 7.8Hz, 2H), 7.58 (dd, J = 7.8 and 1.5Hz, 2H) ppm IR (liquid film): 2950, 1590, 1360, 1175cm ^-1 Mass (m / z,%): 466 (M ⁺ , 3), 410 (93), 395 (23), 365 (34),
311 (30), 149 (11), 111 (16), 73 (100)

Example 4

[Chemical 19]

Compound [19] 56 synthesized in Reference Example 12
mg (0.120 mmol) and 5 mg of TPP were dissolved in 10 ml of dichloromethane, and under oxygen atmosphere, at -78 ° C.
It was stirred at. This solution was irradiated with a Na lamp (940W) for 2 hours. The reaction mixture was concentrated, and the concentrate was applied to a silica gel column and poured out with a 10: 1 mixed solvent of hexane and ethyl acetate to give 3-t-butoxy-3- [5-.
(T-Butyldimethylsiloxy) -5-biphenyl-3
-Yl] -4,4-diisopropyl-1,2-dioxetane (compound [20]) was 42 mg, yield 70.2%.
To give a pale yellow oil.

1: 1 rotamer mixture ¹ H NMR (400 MHz, CDCl ₃ ): δ 0.17 to 0.27 (m, 6H), 0.43 (d, J =
6.8Hz, 1.5H), 0.43 (d, J = 6.8Hz, 1.5H), 0.90 (d, J = 6.3Hz, 3
H), 1.01 (s, 4.5H), 1.01 (s, 4.5H), 1.20 (d, J = 6.8Hz, 1.5
H), 1.21 (d, J = 7.3Hz, 1.5H), 1.23 (s, 4.5H), 1.25 (s,
4.5H), 1.34 (d, J = 7.3Hz, 1.5H), 1.34 (d, J = 7.3Hz, 1.5H),
2.35 ~ 2.70 (m, 2H), 6.91 (s with fine coupling, 0.5H),
7.07 (d with fine coupling, J = 7.8Hz, 1H), 7.22 ~ 7.66
(m, 6.5H) ppm IR (liquid film): 2965, 1595, 1335, 1260, 1000cm ^-1

Reference Example 13

Embedded image

Titanium trichloride 4.5 at 0 ° C. under nitrogen atmosphere
g (29 mmol) and 0.57 g (15 mmol) of lithium aluminum hydride were sequentially added to 75 ml of anhydrous THF and stirred. 2.1m of triethylamine in this solution
1 (15 mmol) was added and the mixture was heated under reflux for 15 minutes. 1-Methoxy-3 dissolved in 25 ml of anhydrous THF was added to this solution.
-Fluorene-methyl carboxylate (compound [21]) 5
A solution of 98 mg (2.35 mmol) and 0.7 ml (4.94 mmol) of diisopropyl ketone was slowly added dropwise, and the mixture was heated under reflux for 3 hours. 100 ml of water was added dropwise to this solution at 0 ° C., and the mixture was extracted with ethyl acetate. The extract layer was washed with saturated brine, dried over magnesium sulfate, and concentrated. The concentrate was applied to a silica gel column and was poured out with a 3: 1 mixed solvent of hexane and dichloromethane.
1,1-diisopropyl-2-methoxy-2- (1-methoxyfluoren-3-yl) ethene (compound [2
2]) was obtained as a pale yellow oily substance in a yield of 36.9% (292 mg).

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.96 (d, J = 6.8 Hz, 6
H), 1.30 (d, J = 6.8Hz, 6H), 2.37 (sept, J = 6.8Hz, 1H), 2.55
(sept, J = 6.8Hz, 1H), 3.25 (s, 3H), 3.84 (s, 2H), 3.94 (s, 3
H), 6.73 (s with fine coupling, 1H), 7.31 (s, 1H), 7.31
(t with fine coupling, J = 7.3Hz, 1H), 7.37 (t, J = 7.3Hz,
1H), 7.56 (d, J = 7.3Hz, 1H), 7.76 (d, J = 7.3Hz, 1H) ppm

Reference Example 14

[Chemical 21]

Sodium hydride (60%), 0.26 g (6.5 mmol) was added to anhydrous DM at 0 ° C. under a nitrogen atmosphere.
0.5 ml (6.8 mmol) of ethanethiol was added dropwise to a solution suspended in 10 ml of F and stirred. To this solution, 282 mg (0.84 mmol) of the compound [22] synthesized in Reference Example 13 dissolved in 10 ml of anhydrous DMF was added at room temperature, followed by heating under reflux for 4 hours. The reaction mixture was poured into saturated aqueous ammonium chloride solution and extracted with ethyl acetate.
The extract layer was washed with saturated brine, dried over magnesium sulfate, and concentrated. The concentrate was applied to a silica gel column and flushed out with dichloromethane. As a result, 1- (1-hydroxyfluoren-3-yl) -2,2-diisopropyl-1-methoxyethene (Compound [23]) was obtained in a yield of 48.3 mg. 1
Obtained as a pale yellow amorphous solid at 7.9%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.95 (d, J = 6.8 Hz, 6
H), 1.28 (d, J = 6.8Hz, 6H), 2.35 (sept, J = 6.8Hz, 1H), 2.54
(sept, J = 6.8Hz, 1H), 3.24 (s, 3H), 3.86 (s, 2H), 6.68 (s wi
th fine coupling, 1H), 7.30 (s, 1H), 7.32 (t with fine
coupling, J = 7.3Hz, 1H), 7.38 (t, J = 7.3Hz, 1H), 7.57 (d,
J = 7.3Hz, 1H), 7.77 (d, J = 7.3Hz, 1H) ppm

Reference Example 15

[Chemical formula 22]

Under a nitrogen atmosphere, at 0 ° C., 46 mg (0.14 mmol) of the compound [23] synthesized in Reference Example 14 was dissolved in 1 ml of anhydrous DMF and stirred. To this solution were sequentially added 1 ml (7.2 mmol) of triethylamine and 120 mg (0.80 mmol) of t-butyl-dimethylchlorosilane, and subsequently stirred at room temperature for 1 hour. The reaction mixture was poured into saturated saline and extracted with ethyl acetate. The extract layer was washed with saturated brine, dried over magnesium sulfate, and concentrated. The concentrate was applied to a silica gel column and was poured out with a mixed solvent of hexane and ethyl acetate of 10: 1. 1- [1- (t
-Butyldimethylsiloxy) fluoren-3-yl]-
A crude product of 2,2-diisopropyl-1-methoxyethene (compound [24]) was obtained. Preparative TL
When subjected to C and developed with a mixed solvent of hexane and ethyl acetate of 10: 1, the compound [24] was 25 mg, and the yield was 4
Obtained as a pale yellow oil at 0.1%.

¹ H NMR (400 MHz, CDCl ₃ ): δ0.25 (s, 6H), 0.94
(d, J = 6.8Hz, 6H), 1.06 (s, 9H), 1.28 (d, J = 7.3Hz, 6H), 2.35
(sept, J = 6.8Hz, 1H), 2.55 (sept, J = 6.8Hz, 1H), 3.23 (s, 3
H), 3.83 (s, 2H), 6.65 (s with fine coupling, 1H), 7.27
~ 7.39 (m, 3H), 7.55 (d, J = 7.3Hz, 1H), 7.76 (d, J = 7.8Hz, 1
H) ppm

Example 5

[Chemical formula 23]

Compound [24] 20 synthesized in Reference Example 15
mg (0.046 mmol) and 2 mg of TPP were dissolved in 6 ml of dichloromethane, and the mixture was stirred at -78 ° C under an oxygen atmosphere. This solution was irradiated with a Na lamp (940 W) for 2 hours. The reaction mixture is concentrated and the concentrate is collected T
When subjected to LC and developed with a mixed solvent of hexane and ethyl acetate of 20: 1, 3- [1- (t-butyldimethylsiloxy) fluoren-3-yl] -4,4-diisopropyl-3-methoxy-1, 2-Dioxetane (Compound [25]) was obtained as a pale yellow oil in an amount of 16 mg and a yield of 74.5%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.18 to 0.30 (m, 6
H), 0.49 (d, J = 7.3Hz, 3H), 0.94 (d, J = 6.8Hz, 3H), 1.06 (s,
9H), 1.22 (d, J = 6.8Hz, 3H), 1.34 (d, J = 7.3Hz, 3H), 2.40 ~
2.75 (m, 2H), 3.18 (s, 3H), 3.84 (broad s, 2H), 6.70 ~ 6.8
7 (m, 1H), 7.33 (td, J = 7.3 and 1.0Hz, 1H), 7.40 (t, J = 7.3H
z, 1H), 7.57 (d, J = 7.3Hz, 1H), 7.70 ~ 7.88 (m, 2H) ppm

Reference Example 16

[Chemical formula 24]

Titanium trichloride 4.5 at 0 ° C. under nitrogen atmosphere
g (29 mmol) and 0.57 g (15 mmol) of lithium aluminum hydride were sequentially added to 75 ml of anhydrous THF and stirred. 2.1m of triethylamine in this solution
1 (15 mmol) was added and the mixture was heated under reflux for 15 minutes. 684 mg (2.30 mmol) of methyl 1,9-dimethoxy-9-methyl-3-fluorenecarboxylate (compound [26]) and 2.0 ml (14.1 mmol) of diisopropyl ketone dissolved in 25 ml of anhydrous THF were added to this solution.
The solution of was slowly added dropwise, and the mixture was further heated under reflux for 4 hours and 30 minutes. 100 ml of water was added dropwise to this solution at 0 ° C., and the mixture was extracted with ethyl acetate. The extract layer was washed with saturated brine, dried over magnesium sulfate, and concentrated. The concentrate is applied to a silica gel column, hexane and dichloromethane 2: 1.
When it was run off with the mixed solvent of 1,1-diisopropyl-2-methoxy-2- (1,9-dimethoxy-9-methylfluoren-3-yl) ethene (compound [27])
Was obtained as a pale yellow oily matter in a yield of 53.4%.

¹ H NMR (400 MHz, CDCl ₃ ): δ0.98 (d, J = 6.8 Hz, 6
H), 1.29 (d, J = 6.8Hz, 6H), 1.82 (s, 3H), 2.37 (sept, J = 6.8
Hz, 1H), 2.57 (sept, J = 6.8Hz, 1H), 2.82 (s, 3H), 3.26 (s, 3
H), 3.93 (s, 3H), 6.72 (s, 1H), 7.16 (s, 1H), 7.29 ~ 7.40
(m, 2H), 7.45 to 7.50 (m, 1H), 7.57 to 7.63 (m, 1H) ppm

Reference Example 17

[Chemical 25]

80 mg (2.0 mmol) of sodium hydride (60%) was added to anhydrous DMF at 0 ° C. under a nitrogen atmosphere.
0.1 ml of ethanethiol was added to the solution which was suspended in 2 ml and stirred.
5 ml (2.0 mmol) was added dropwise. To this solution, 97 mg (0.26 mmol) of the compound [27] synthesized in Reference Example 16 dissolved in 2 ml of anhydrous DMF was added at room temperature,
Then, the mixture was heated under reflux for 3 hours. The reaction mixture was poured into saturated aqueous ammonium chloride solution and extracted with ethyl acetate. The extract layer was washed with saturated brine, dried over magnesium sulfate, and concentrated. When the concentrate was subjected to preparative TLC and developed with dichloromethane, 1- (1-hydroxy-9-methylfluorene-
3-yl) -2,2-diisopropyl-1-methoxyethene (compound [28]) was obtained as 21 mg in a yield of 24.5% as a colorless amorphous solid.

¹ H NMR (400 MHz, CDCl ₃ ): δ0.95 (d, J = 6.8 Hz, 6
H), 1.27 (d, J = 6.8Hz, 6H), 1.61 (d, J = 7.3Hz, 3H), 2.35 (se
pt, J = 6.8Hz, 1H), 2.56 (sept, J = 6.8Hz, 1H), 3.24 (s, 3H),
4.08 (q, J = 7.3Hz, 1H), 4.82 (s, 1H), 6.61 (d, J = 1.0Hz, 1H),
7.23 ~ 7.39 (m, 3H), 7.51 (dd, J = 6.8 and 1.5Hz, 1H), 7.72
(d with fine coupling, J = 6.8Hz, 1H) ppm

Reference Example 18

[Chemical formula 26]

34.9 mg (0.10 mmol) of the compound [28] synthesized in Reference Example 17 was dissolved in 1 ml of anhydrous DMF and stirred at room temperature under a nitrogen atmosphere. To this solution 0.1 ml (0.72 mmol) triethylamine and t-
Butyldimethylchlorosilane 40 mg (0.27 mmo
1) were sequentially added and stirred for 1 hour. The reaction mixture was poured into saturated saline and extracted with ethyl acetate. The extract layer was washed with saturated brine, dried over magnesium sulfate, and concentrated. The concentrate is applied to a silica gel column and hexane and ethyl acetate are mixed 1: 1.
When it was run out with the mixed solvent of 1- [1- (t-butyldimethylsiloxy) -9-methylfluoren-3-yl] -2,2-diisopropyl-1-methoxyethene (Compound [29]), 37 mg. It was obtained as a pale yellow oily substance in a yield of 79.2%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.23 (s, 3H), 0.30
(s, 3H), 0.93 (d, J = 6.8Hz, 3H), 0.96 (d, J = 7.4Hz, 3H), 1.0
6 (s, 9H), 1.28 (d, J = 7.4Hz, 3H), 1.28 (d, J = 6.8Hz, 3H), 1.5
8 (d, J = 7.0Hz, 3H), 2.34 (sept, J = 7.4Hz, 1H), 2.56 (sept,
J = 6.8Hz, 1H), 3.23 (s, 3H), 4.00 (q, J = 7.0Hz, 1H), 6.62
(d, J = 1.2Hz, 1H), 7.28 (d, J = 1.2Hz, 1H), 7.28 ~ 7.38 (m, 2
H), 7.49 (d with fine coupling, J = 6.8Hz, 1H), 7.71 (dw
ith fine coupling, J = 6.8Hz, 1H) ppm

Example 6

[Chemical 27]

Compound [29] 19 synthesized in Reference Example 18
mg (0.042 mmol) and 2 mg of TPP were dissolved in 10 ml of dichloromethane, and under an oxygen atmosphere, -78 ° C.
It was stirred at. This solution was irradiated with a Na lamp (940 W) for 2 hours. The reaction mixture was concentrated, and the concentrate was subjected to preparative TLC and developed with a mixed solvent of hexane and ethyl acetate of 10: 1 to give 3- [1- (t-butyldimethylsiloxy) -9-methylfluoren-3-yl. ] -4,
4-Diisopropyl-3-methoxy-1,2-dioxetane (Compound [30]) was obtained as a pale yellow oily substance in a yield of 18 mg and a yield of 88.4%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.16 to 0.40 (m, 6
H), 0.49 (d, J = 6.8Hz, 3H), 0.94 (d, J = 6.8Hz, 3H), 1.06 (s,
9H), 1.21 (d, J = 7.4Hz, 3H), 1.33 (d, J = 7.4Hz, 3H), 1.57
(d, J = 7.2Hz, 3H), 2.40 ~ 2.56 (m, 1H), 2.60 ~ 2.77 (m, 1H),
3.19 (s, 3H), 4.01 (q, J = 7.2Hz, 1H), 6.69 ~ 6.86 (m, 1
H), 7.30 ~ 7.41 (m, 2H), 7.51 (d, J = 7.2Hz, 1H), 7.65 ~ 7.8
5 (m, 1H) ppm

Reference Example 19

[Chemical 28]

Titanium trichloride 4.5 at 0 ° C. under nitrogen atmosphere
g (29 mmol) and 0.57 g (15 mmol) of lithium aluminum hydride were sequentially added to 75 ml of anhydrous THF and stirred. 2.1m of triethylamine in this solution
1 (15 mmol) was added and the mixture was heated under reflux for 15 minutes. 1-Methoxy-dissolved in 25 ml of anhydrous THF
1.02 g (3.62 mmol) of methyl 9,9-dimethyl-3-fluorenecarboxylate (compound [31]) and 2.01 ml (14.2 mm) of diisopropyl ketone.
solution) was slowly added dropwise, and the mixture was heated under reflux for 3 hours. 100 ml of water was added dropwise to this solution at 0 ° C., and the mixture was extracted with ethyl acetate. The extract layer was washed with saturated brine, dried over magnesium sulfate, and concentrated. The concentrate is applied to a silica gel column, hexane and dichloromethane 3: 1.
When it was poured out with a mixed solvent of 1,1-diisopropyl-2-methoxy-2- (1-methoxy-9,9-dimethylfluoren-3-yl) ethene (compound [32])
Was obtained as a colorless amorphous solid in a yield of 23.2%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.96 (d, J = 6.8 Hz, 6
H), 1.29 (d, J = 6.8Hz, 6H), 1.58 (s, 6H), 2.36 (sept, J = 6.8
Hz, 1H), 2.57 (sept, J = 6.8Hz, 1H), 3.25 (s, 3H), 3.90 (s, 3
H), 6.69 (s, 1H), 7.22 (s, 1H), 7.28 ~ 7.35 (m, 2H), 7.38 ~
7.44 (m, 1H), 7.65 ~ 7.72 (m, 1H) ppm

Reference Example 20

[Chemical 29]

80 mg (2.0 mmol) of sodium hydride (60%) was added to anhydrous DMF at 0 ° C. under a nitrogen atmosphere.
0.1 ml of ethanethiol was added to the solution which was suspended in 2 ml and stirred.
5 ml (2.0 mmol) was added dropwise. 205 mg (0.56) of the compound [32] synthesized in Reference Example 19 was added to this solution.
mmol) at room temperature, followed by heating under reflux for 1 hour 30 minutes. The reaction mixture was poured into water and extracted with ethyl acetate.
The extract layer was washed with saturated brine, dried over magnesium sulfate, and concentrated. The concentrate was applied to a silica gel column and flushed with dichloromethane to give 1- (1-hydroxy-9,
9-Dimethylfluoren-3-yl) -2,2-diisopropyl-1-methoxyethene (compound [33]) was 7
Obtained as a colorless amorphous solid in 6 mg, yield 38.6%.

¹ H NMR (400 MHz, CDCl ₃ ): δ0.95 (d, J = 6.8 Hz, 6
H), 1.27 (d, J = 6.8Hz, 6H), 1.63 (s, 6H), 2.34 (sept, J = 6.8
Hz, 1H), 2.56 (sept, J = 6.8Hz, 1H), 3.23 (s, 3H), 4.70 (s, 1
H), 6.54 (d, J = 1.0Hz, 1H), 7.21 (d, J = 1.0Hz, 1H), 7.30 ~ 7.
36 (m, 2H), 7.40 ~ 7.45 (m, 1H), 7.65 ~ 7.71 (m, 1H) ppm

Reference Example 21

Embedded image

In a nitrogen atmosphere, 69.2 mg (0.20 mmol) of the compound [33] synthesized in Reference Example 20 was dissolved in 1 ml of anhydrous DMF and stirred at 0 ° C. To this solution 0.1 ml (0.72 mmol) triethylamine and t-
Butyldichlorosilane (50 mg, 0.33 mmol) was sequentially added, and the mixture was subsequently stirred at room temperature for 4 hours and 30 minutes. The reaction mixture was poured into saturated saline and extracted with ethyl acetate. The extract layer was washed with saturated brine, dried over magnesium sulfate, and concentrated. The concentrate was applied to a silica gel column and poured out with a mixed solvent of hexane and ethyl acetate at a ratio of 5: 1.
A crude product of [1- (t-butyldimethylsiloxy-9,9-dimethyl-fluoren-3-yl] -2,2-diisopropyl-1-methoxyethene (compound [34]) was obtained. The product was subjected to preparative TLC and developed with a mixed solvent of hexane and ethyl acetate at a ratio of 10: 1 to obtain 64.5 mg of compound [34] as a pale yellow oily substance in a yield of 70.3%.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.34 (s, 6H), 0.96
(d, J = 6.8Hz, 6H), 1.08 (s, 9H), 1.28 (d, J = 6.8Hz, 6H), 1.6
0 (s, 6H), 2.34 (sept, J = 6.8Hz, 1H), 2.59 (sept, J = 6.8Hz, 1
H), 3.23 (s, 3H), 6.62 (d, J = 1.0Hz, 1H), 7.23 (d, J = 1.0Hz, 1
H), 7.27 ~ 7.35 (m, 2H), 7.37 ~ 7.45 (m, 1H), 7.64 ~ 7.72
(m, 1H) ppm

Example 7

[Chemical 31]

Compound [34] 57 synthesized in Reference Example 21
mg (0.12 mmol) and 2 mg of TPP were dissolved in 10 ml of dichloromethane, and the mixture was stirred at -78 ° C under an oxygen atmosphere. This solution was irradiated with a Na lamp (940 W) for 2 hours. The reaction mixture is concentrated and the concentrate is collected T
When subjected to LC and developed with a mixed solvent of hexane and ethyl acetate of 20: 1, 3- [1- (t-butyldimethylsiloxy) -9,9-dimethylfluoren-3-yl] was obtained.
-50 mg of -4,4-diisopropyl-3-methoxy-1,2-dioxetane (compound [35]), yield 82.1.
% As a pale yellow oil.

¹ H NMR (400 MHz, CDCl ₃ ): δ 0.35 (s, 6H), 0.47
(d, J = 6.8Hz, 3H), 0.93 (d, J = 6.8Hz, 3H), 1.08 (s, 9H), 1.21
(d, J = 7.3Hz, 3H), 1.33 (d, J = 7.3Hz, 3H), 1.59 (s, 6H), 2.4
0 ~ 2.53 (m, 1H), 2.60 ~ 2.77 (m, 1H), 3.20 (s, 3H), 6.70 ~
6.86 (m, 1H), 7.30 ~ 7.45 (m, 3H), 7.64 ~ 7.80 (m, 2H) ppm

Test Example The compound [15] obtained in Example 3, the compound [25] obtained in Example 5, the compound [30] obtained in Example 6
And 1 ml of a 1.8 × 10 ⁻⁵ M DMSO solution of each of the compound [35] obtained in Example 7 and 1.0 × 10 ⁻³ M DM of tetrabutylammonium fluoride.
The solution was added to 2 ml of SO solution at 20 ° C., and the luminescence at that time was measured by a fluorescence analyzer. Table 1 shows the measured emission wavelength (λ _max ), emission lifetime (emission half-life), and emission quantum yield.

[0111]

[Table 1]

[0112]

The 1,2-dioxetane derivative of the present invention is characterized by excellent chemical stability and high luminescence sustainability. Therefore, there is no need to refrigerate when storing, and it is possible to save time and effort such as preparation and temperature control when starting light emission. Further, since the stable light emission continues after the light emission is started, the obtained data is stable and the reproducibility is high. Further, it is not necessary to use a highly sensitive measuring device when measuring the light emission amount, and the measurement can be performed using an inexpensive device.

Claims (5)

[Claims] 1. A 1,2-dioxetane derivative represented by the following general formula [I]: Embedded image (However, in the formula, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R
⁶ are each independently a hydrogen atom or an alkyl group (provided that R is
All of ^{1 to} R ⁶ may not be hydrogen atoms at the same time, and R ¹ and R ² and R ⁴ and R ⁵ may be integrated together to form a cyclic alkyl group), R ⁷ is an alkyl group, R ⁸ alkoxyl ^{group, -OSi (R 9 R 10 R} 11) ( wherein, R
⁹ , R ¹⁰ and R ¹¹ each independently represent an alkyl group) or a phosphate group, and X and X ′ are each independently substituted by a hydrogen atom, an alkoxyl group, a phenyl group (a halogen, an alkyl group or an alkoxyl group). ), A halogen or an alkyl group. (However, X and X'may not be hydrogen atoms at the same time, and X and X'may together form a ring.)) 2. The 1,2-dioxetane derivative according to claim 1, wherein X is an alkoxyl group and X ′ is a hydrogen atom. 3. The 1,2-dioxetane derivative according to claim 2, wherein X is a methoxy group. 4. The 1,2-dioxetane derivative according to claim 1, wherein X is a phenyl group and X ′ is a hydrogen atom. 5. The following formula [II] in which X and X ′ are integrated.
A 1,2-formation according to claim 1, which forms a ring as shown in
Dioxetane derivative. Embedded image (However, in the formula [II], R ¹⁴ and R ¹⁵ each independently represent hydrogen or a lower alkyl group.)