JPS6056985A - Coumarin derivative - Google Patents

Abstract

NEW MATERIAL:A compound shown by the formula (X is methyl, formyl, p- toluenesulfonylhydrazinomethyl, diazomethyl, hydroxymethyl, etc.; Y is H, nitro, amino, 3-carboxyacryloylamino, etc.; with the proviso that the case where X is methyl and Y is H are omitted). EXAMPLE:6,7-Methylenedioxy-4-methyl-3-nitrocoumain. USE:A fluorescent labelled reagent. Having high relative fluorescent intensity even in an aqueous solution or a water-containing organic solvent in a wide pH range (1.5-10pH), slightly being influenced by pH. Having low sensitivity to light, requiring no light shielding. PREPARATION:A compound shown by the formula is obtainable by applying Pechmann reaction. For example, 3,4-methylenedioxyphenol is condensed with citric anhydride in the presence of a dehydrating catalyst such as conc. sulfuric acid, etc., to give a compound where X is carboxymethyl in the formula.

Description

[Detailed description of the invention] The present invention relates to novel marine derivatives.

The novel marine derivatives of the present invention are useful as fluorescent labeling reagents.

Conventionally, as coumarin derivatives, compounds having an amine group, methylamine group, dimethylamino group, or hydroxyl group at the 1st position, and hydroxyl groups at the 6th and 7th positions are known (Goodwi
n et al., Arch, Biocbem, Biophys;
2T, p. 152, 1950). However, 1
Coumarins having an amine group, methylamine group, or dimethylamino group at the position have a high relative fluorescence intensity in an organic solvent such as methanol or ethanol, but a low relative fluorescence intensity in an aqueous solvent.

In addition, coumarins having a hydroxyl group at the 1st position and hydroxyl groups at the 6th and 7th positions have a large relative fluorescence intensity in an alkaline aqueous solution, but the relative fluorescence intensity in a neutral or weak acid aqueous solution is lower than that in an alkaline aqueous solution. Compared to that, it has the disadvantage of being less than %aa. In addition, an amino group, a monoalkylamino group, at the 7th position,
Dialkylamino St-containing coumarins are highly sensitive to light and therefore have the disadvantage of requiring light shielding.

Furthermore, JC7-medoxy-4-bromomethylcoumarin is known (Dunges, Anal, Ohem;
49, pp. 442-445, 1977). However, this compound has the drawback of low relative fluorescence intensity.

Furthermore, N-[: S-(7'-azidocturin-4'
-carbonamide)-pentyloxycarbonylmethyl]
-Maleimide etc. are known (Japanese Patent Application Laid-Open No. 57-1467
No. 71). However, these compounds have the disadvantage that they have a high sensitivity to light and are unstable.

Furthermore, 6,7-methylenedioxycoumarin is known (Spath et al., Ber; vol. 10, p. 102,
(1937). However, there is no description of the use of this compound as a fluorescent labeling reagent.

Furthermore, 6,7-methylenedioxy-4-methylcoumarin is known (K-Fukui et al., J, sci, Hl
ro-shima Univ, ; A-M, 26
Vol. 131, 1963).

However, there is no description of the use of this compound as a fluorescent labeling reagent.

As a result of intensive research on coumarin derivatives, the present inventors discovered that the coumarin derivatives shown below are useful as fluorescent labeling reagents, and completed the present invention.

The present invention relates to a compound of the formula [wherein, Formylmethyl group, alkoxycarbonylmethyl group, hydrazinocarbonylmethyl group, cyanocarbonylmethyl group, diazoacetonyl group, haloacetonyl group, halomethyl group, phthalimidooxymethyl group, aminooxymethyl group, phthalimidomethyl group, aminomethyl group or incyanato A methyl group, Y represents a hydrogen atom, a nitro group, an amino group, a 3-carboxyacryloylamino group, a maleimide group, an acetylamino group, a haloacetylamino group, or a mineral isocyanate group. However, this excludes cases where X is a methyl group and Y is a hydrogen atom. ] Regarding a coumarin derivative having the following.

Generally, as a method for analyzing and quantifying compounds, spectroscopic methods that utilize visible or ultraviolet absorption spectra of compounds are widely used. When the absorbance coefficient of a compound is small, highly sensitive quantification becomes possible by converting the compound to a derivative with a large absorbance coefficient or a derivative having fluorescence. The compound having formula (1) t- has a molecular extinction coefficient of 320 to 370 am (sb at 10' L/mote cm, relative fluorescence intensity 1j).

It is large and has similar strength in aqueous solutions over a wide pH range (pHt, s to IQ). In addition, the above formula (1)
f. The compound line exhibits high relative fluorescence intensity even in aqueous organic solvents without being affected by pH. Furthermore,
A compound having the above formula (I)? ! I has low sensitivity to light and does not require light shielding. Furthermore, the above formula (
1) A converted compound derived from a compound having the following can be separated and quantified with high sensitivity by high performance liquid chromatography.

For example, by attaching a fluorescence detector to a high-performance liquid chromatography device, it is possible to detect compounds with carboxy groups, mercapto groups, carbonyl groups, alcohol u hydroxyl groups, hydrazino groups, amino groups, etc. with high sensitivity at the level of 1 loO femtomole. Can be separated and quantified. Therefore, the compounds having the above formula (I) are useful as fluorescent labeling reagents.

The compound of the present invention having the above formula CI) is Bechman (Pθ
chmann) reaction.

For example, it can be obtained by oxidizing a methyl group in a compound where X is a formyl group. A compound in which X is a p-toluenesulfonylhydrazinomethyl group can be obtained by reacting a formyl group with tosylhydrazine. A compound in which X is a diazomethyl group can be obtained by decomposing a p-toluenesulfonylhydrazinomethyl group with an alkali. A compound in which X is a hydroxymethyl group can be obtained by reducing a formyl group. Compound L in which X is a haloformyloxymethyl group is obtained by reacting a hydroxymethyl group with a trihalomethyl haloformate.

In addition, compounds in which X is a carboxymethyl group include 3,4
-methylenedioxyphenol t- As a raw material compound,
It is obtained by condensation with anhydrous citric acid in the presence of a dehydration catalyst such as concentrated sulfuric acid. Compounds in which X is a haloformylmethyl group can be obtained by reacting a carboxymethyl group with an organic acid halide. Compounds in which X is an alkoxycarbonylmethyl group can be obtained by reacting a haloformyl group with an alcohol. A compound in which X is a hydrazinocarbonylmethyl group can be obtained by reacting an alkoxycarbonylmethyl group with hydrazine hydrate. A compound in which X is a cyanocarbonylmethyl group can be obtained by reaction with a haloformylmethyl-based trialkylsilyl cyanide. Compounds in which X is a diazoace)nyl group can be obtained by reacting a haloformyl group with diazomethane. Compounds in which X is a haloacetonyl group can be obtained by reacting a diazoacetonyl group with hydrohalic acid.

A compound in which x is a halomethyl group has OX obtained by reacting a carboxymethyl group with a halogen.
It is obtained by reacting the compound halimethyl group, which is a thalimidooxymethyl group, with N-hydroxyphthalimide.

A compound in which X is an aminooxymethyl group can be obtained by hydrolyzing a phthalimidoxymethyl group with an acid. Compounds in which X is a 7thalimidomethyl group are obtained by reacting a halomethyl group with an alkali metal salt of phthalimide. Compounds where X is an aminomethyl group can be obtained by hydrolyzing a phthalimidomethyl group with an acid. Compounds where X is an isocyanatomethyl group can be obtained by reacting an aminomethyl group with a trino-lomethyl haloformate. can get.

Next, a compound in which Y is a nitro group can be obtained by reacting a compound in which Y is a hydrogen atom with t-concentrated nitric acid. Y
Compounds in which is an amino group can be obtained by reducing the nitro group. A compound in which Y is a 3-carboxyacryloylamino group can be obtained by reacting an amino group with maleic anhydride. A compound in which Y is a maleimide group can be obtained by ring-closing a 3-carboxyacryloylamino group. A compound in which Y is an acetylamino group can be obtained by reaction with amino-based acetic anhydride.

A compound in which Y is a haloacetylamino group can be obtained by reacting an amine group with a haloacetyl halide. Compounds in which Y is an isocyanate group are prepared by reacting the amino group with trihalomethyl haloformate.

Next, the present invention will be explained in more detail with reference to examples.
The present invention is not limited to these examples.

Example 1.6.1-methylenedioxy-4-methyl-3
-Nitrocoumarin 6.1-methylenedioxy-4-methylcoumarin 6.1
After dissolving 2Ii in 400% acetic anhydride, the temperature was maintained at 5°C or lower. Concentrated salt f11 in this! (d = 1.42)
A mixture of 3.6- and acetic anhydride 50- was slowly added under stirring. After the addition was completed, the mixture was left to cool for about 2 hours. When the crystals Ii were separated and washed with water-cooled ether, crude crystals 6.8
g was obtained. When the obtained crude crystals were recrystallized using acetonitrile, the title compound 6.0.9 having a melting point of 234 to 235°C was obtained as a yellow cylindrical product.

Elemental analysis value: Total as a, 1u, o6n, 1L 1 荏 a, 53.02;) I, 2
．． 83; IJ, 5.62 Actual value 0, 53.3
0; H, 2,63; N, 5.311 Example 2
．． 6.7-Methylenedioxy-4-methyl-3-aminocoumarin 5 g of 6,7-methylenedioxy-4-methyl-3-nitrocoumarin obtained in Example 1 was dissolved in 1.6 t of tetrahydrofuran. After that, 10% palladium-carbon 4.
0.9' was added, and hydrogen gas was bubbled through the mixture for about 2 hours while stirring at room temperature. After the catalytic reduction reaction, insoluble matter was filtered off from the reaction mixture, and the resulting filtrate was filtered under reduced pressure to yield 3.3 g of crude crystals.
was gotten. When the obtained crude crystals were recrystallized using dichloromethane, the title compound 3.0.9 having a melting point of 251-252°C was obtained as white needle-like crystals.

Element minute value Calculated value as C11JO414 0, 60.27; H, 4, 14; N
, 6.39 Actual value c, 60.41; H, 3
, 96;
310 μm of maleic anhydride was added to 50 (1 g) of methyl-3-aminocoumarin and heated in a boiling water bath for 10 minutes.

Next, 1,0-dimethylformamide was added and heated for 20 minutes. After the reaction is complete, add saturated carbonic acid to the reaction mixture)
After adding an aqueous solution of IJum and washing with dichloromethane, 2N hydrochloric acid was added to make it acidic. Collect the white precipitate that formed.

When recrystallized using dichloromethane, the melting point is 180-18.
The title compound 382■ having a temperature of 4° C. was obtained as white button-shaped crystals.

Calculated value c as elemental analysis value 0+sHt+07N,
56.78; H, a, so; N, 4.4
2 Actual measurement values a, se, ay;, H, 349; N
, 4.11 Example 4, 6.1-methylenedioxy-
4-Methyl-3-maleimidocoumarin To 6,7-methylenedioxy-4-methyl-3-(3-carboxyacryloylamino)coumarin 31T119 obtained in Example 3, 200 my of sodium acetate, boric acid TO*9 and Acetic anhydride 2- was added and heated at 90°C for 10 minutes. After the reaction was completed, the reaction mixture was poured into 50ml of ice-cold water, and the resulting yellow brown precipitate was collected and dried. After dissolving the obtained crude crystals in acetonitrile 4g, activated carbon was added and heated to decolorize, then allowed to cool and the precipitated crystals were collected to obtain the title compound IB0.2 having a melting point of 256-25T'C.
M9 was obtained as light yellow columnar crystals.

Assuming elemental analysis value 0+5HpO6N, combination value a, 60.2Q; H, 3,03; x
, 4.68 Actual value a, so, oa; H,
2,99; N, 5.08 Example 5, 6, T-methylenedioxy-4-methyl-3-acetylaminocoumarin 6,1-methylenedioxy-4-methyl-3 obtained in Example 2 - Aminocoumarin 500 μl was suspended in 500 μg of acetic anhydride, and then heated in an oil bath at 120° C. for 10 minutes. After the reaction was completed, the reaction mixture was allowed to cool, and tabular white crystals were precipitated. The precipitated crystals were collected, washed with water-cooled ether, and dried.

When the obtained crystals were recrystallized using acetonitrile, 460 mg of the title compound having a melting point of 151-152°C was obtained as white needle-like crystals.

Elemental analysis value Calculated value a as 0+5111506N
, sa, rr; H, 4,24; N, s, a
a Actual value 0, 59.68; H, 4,21; N
, 5.25 Example 6.6. T-methylenedioxy-
4-Methyl-3-bromoacetylaminocoumarin 6, T-methylenedioxy-4-methyl-3-aminocoumarin 438 obtained in Example 2 was converted into dry tetrahydro 7rane 120-, then bromoacetylbromide 404Q
and triethylamine 20389 at room temperature.
The mixture was heated and separated for 1 minute and then for 1 hour. After the reaction was completed, the solvent was distilled off from the reaction mixture under reduced pressure. The obtained residue was dissolved in a small amount of dichloromethane, and then subjected to column chromatography using silica gel, and purified by elution with dichloromethane. The obtained Ic crude crystals were recrystallized using acetonitrile to obtain the title compound 238, having a melting point of 212-214C, as pale yellow plate crystals.

Elemental analysis value Calculated value as 0+5H160sNBr I
J, 45.89; H, 2,96; N, 4
．． 12 Actual value 0, 45.20; H, 2J8;
N, 4.09 Example 76. 1.1 g of 1-methylenedioxy-4-methyl-3-incyanatocoumarin trichloromethylchloroforme was added to 10-ethyl acetate, cooled on ice, and 1 g of activated carbon was added. ,
The temperature was then raised to 25-30° C. for about 10 minutes while stirring. This solution was then cooled on ice again.

Then, to this solution, 1. s, r-methylenedioxy-4-methyl-3-aminocoumarin obtained in Example 2 was added.
About 30% of a solution of 03.9 dissolved in ethyl acetate 150%
It was added dropwise over a period of minutes. After the dropwise addition was completed, the mixture was gradually heated while stirring on an oil bath, and then heated under reflux for about 1 hour. After the reaction was completed, the reaction mixture was filtered to remove activated carbon. The obtained filtrate was concentrated under reduced pressure and allowed to cool, yielding pale yellow needle-shaped crude crystals. When the obtained crude crystals are recrystallized using ethyl acetate, the title compound having a melting point of 210 to 271° C.0.95
II was obtained as pale yellow needles.

Elemental analysis value Calculated value as Cl2H705N 0,5878; H, 2,88; N,
5.71 Actual value c, 5B, 20; H, 2,
79; N, 5.56 Actual tjffi Example 8. G, 7
-methylenedioxy-4-formylcoumarin 6,1-methylenedioxy-4-methylcoumarin 6.0
1 f: After dissolving in p-xylene 250-, well-pulverized gelene dioxide 6oI was added and heated under reflux on an oil bath for about 8 hours while stirring vigorously.

After the oxidation reaction was completed, insoluble matter was immediately filtered off from the reaction mixture. The insoluble matter was washed with heated p-xylene, and this washing liquid was combined with the filtrate.

The filtrate was concentrated under reduced pressure, and the residue was then diluted with 80% methanol.
0- was added, then activated carbon was added to decolorize the mixture, and then the mixture was allowed to cool and the precipitated crystals were collected to obtain 5.4 g of the title compound as pale yellow needle crystals having a melting point of 234-235°C.

Elemental analysis value Calculated value as CllH601i 0, 60.56; H, 2,77 actual value
a, 60.39; H, 2,64 Example 96.1
-Methylenedioxy-4-(p-toluenesulfonyl)hydrazinomethylcoumarin 6,1-methylenedioxy-4-formylcoumarin obtained in Example 8 Dissolved in 1.09.9 methanol and 400 g After that, tosylhydrazine 2.0.9 under stirring
i Added little by little. The reaction mixture was heated with t-eo'c for 30 minutes and then left overnight in water cooling.

The precipitated bright yellow needle-like crystals were collected to obtain crude crystals of 1.34 II.
was gotten. The obtained crude crystals were recrystallized using dichloromethane to obtain the title compound 1.20 If having a melting point of 188-190°C as yellow flocculent crystals.

Elemental analysis value Calculated value based on Cl8H1406N 2 days e
, 55.96; H, 3,62; N, 7.
25;S, 8.29 Actual value a, 55.70;)l, 3.45
; N, 6.82; S, 7.71 Example 10.6.7-Methylenedioxy-4-diazomethylcoumarin 6,T-methylenedioxy-4-(obtained in Example 9)
After dissolving 500 μl of p-)luenesulfonyl)hydrazinomethylcoumarin in 200 dlcl of dichloromethane,
．． IN aqueous sodium hydroxide solution 12,5- was added and stirred at room temperature for 2 hours. After the reaction was completed, the reaction mixture was separated into two layers, the aqueous layer being almost colorless and the organic solvent layer being yellow. The aqueous layer was removed, and the organic solvent layer was washed three times with 20 ml of water, and then dehydrated by adding sodium sulfate. When the organic solvent layer is concentrated under reduced pressure and allowed to cool, the precipitated crystals are collected, and the decomposition point is 282-28.
The title compound 245Q with a temperature of 4° C. was obtained as yellow-orange columnar crystals.

Calculated value as elemental analysis value 0.1H604N c, 57.40; H, 2,63; N
, 12.17 Actual value a , 57.29 ;・H
, 2,45; N, 11.50 Example 11.6.7
-methylenedioxy-4-hydroxymethylcoumarin 6,7-methylenedioxy-4- obtained in Example 8
Suspend 1.5 g of formalic marine in 80% of fc methanol and add 5% of sodium borohydroxide while stirring at room temperature.
A solution prepared by dissolving 60 ml of methanol in 10 mg of methanol was added dropwise little by little. As the reaction progressed, the reaction solution became colorless and transparent, and then pale yellow crystals were precipitated. When all the precipitated crystals were collected and the obtained crude crystals were recrystallized using acetonitrile, the title compound 1.21 having a melting point of 011-119°C was obtained.
g was obtained as pale yellow needles.

Elemental analysis value Calculated value as UuHsOs a, eo, oo; H, 3,67 Actual value a, ss, as; H, 3,46 Example 12.
6.7-methylenedioxy-4-chloroformyloxymethyl coumarin (trichloromethyl chloroforme) 1.119 was added to ethyl acetate tag, cooled with water, and 10 m/9 ft of activated carbon was added.
Then, the temperature was raised to 25-30° C. for about 10 minutes while stirring. This solution was then cooled on ice again.

Next, 6,1-methylenedioxy-4-hydroxymethylcoumarin o, sag obtained in Example 11 was added to this solution.
and 200- toluene solution containing quinoline 383Q'
was added dropwise. After completion of the dropwise addition, the mixture was left under stirring overnight. After the reaction was completed, the precipitate was removed and concentrated under reduced pressure. When the resulting residue is washed with n-hexane, the melting point is 1.
The title compound o, yy y having 44-148 C was obtained as pale yellow crystals.

Calculated value a as elemental analysis value 012H7060z,
50.96; H, 2,50 actual measurement c, so
, so; Warmed to ~10°C for 30 minutes.The resulting pale yellow syrupy solution was cooled on ice and heated to 5°C.
34.5 F of pulverized 3,4-methylene dioxyphenol was gradually added under stirring.

After the addition was completed, the mixture was further stirred at 5° C. or lower overnight. After the reaction was completed, the reaction mixture was dispersed in 100 g of water. The yellowish brown precipitate produced was separated, washed with ice-cold water, and then dissolved in a 2.0 N hydroxide aqueous solution. Then 2. After adjusting the pH to 2.5 by adding ON sulfuric acid, the mixture was allowed to cool overnight. The generated precipitate was separated, washed with water, dried, then dissolved again in acetonitrile, decolorized with activated carbon, and cooled on ice. When the precipitated crystals were separated, the title compound IT, 0 having a melting point of 177-119°C was obtained.
11 was obtained as pale yellow columnar crystals.

Elemental analysis value Calculated value as C12H606 0, 58.07; H, 3,25 actual value
B, 57.98; H, 3,08 Example 14. ．． 6
．． 7-methylenedioxy-4-chloroformylmethylcoumarin 6 obtained in Example 13, T-methylenedioxy-4
-Carboxymethylcoumarin Loy? After dissolving in 200% of dry dimethoxyethane, 2.09% of oxalic acid chloride
- was added, and then nitrogen gas was blown in. After the reaction is complete,
The solvent was distilled off from the reaction mixture under reduced pressure to obtain the title compound as a pale yellow amorphous substance. This compound was confirmed by converting it into a methoxycarbonylmethyl compound (Example 15 below).

Example 15.6.7-Methylenedioxy-4-methoxycarbonylmethylcoumarin 6,7-methylenedioxy P4- obtained in Example 14
An excess amount of methanol was added to chloroformylmethylcoumarin 50011!9, and the mixture was left at room temperature for about 30 minutes. After the reaction was completed, the solvent was distilled off from the reaction mixture. When the obtained residue is recrystallized using acetonitrile, the melting point is 206~
The title compound 472Q with 207C was obtained.

Elemental analysis value a15"food 13 ([0, 59, 54: H, 3, 84 actual value 0
, 59.29; H, 3JO Example 16.6.7-
Methylenedioxy-4-hydrazinocarbonylmethylcoumarin'6.1-Methylenedioxy-4 obtained in Example 15
-Methoxycarbonylmethylcoumarin L 5.9 ft
After dissolving in methanol 25-, hydrazine hydrate 0.6
- was added and stirred at room temperature for 30 minutes.

The reaction mixture was allowed to cool overnight, and the precipitated crystals were collected. When the obtained crystals are recrystallized using acetonitrile, the melting point is 2.
The title compound θ, 9I with a temperature of 04-206°C! was obtained as white needle-like crystals.

Elemental analysis value Calculated value a as 0+2H1oOsN2
, 54.96; H, 3,84; N, 10.
68 Actual value a, 54.61; H, 3,78;
N', to, aa Example 1 γ, 6, 1-methylenedioxy-4-cyanocarbonylmethylcoumarin 6, T-methylenedioxy-4 obtained in Example 14
-10- of a dimethoxyethane solution containing 4q of zinc iodide and 400119 of trimethylsilyl cyanide was added to 1.07 g of chloroformylmethylcoumarin, and the mixture was allowed to stand overnight at room temperature. After the reaction was completed, the solvent from the reaction mixture was distilled off under reduced pressure. The obtained residue was subjected to column chromatography using silica gel and purified by elution with dichloromethane to obtain the title compound having a decomposition point of 184-186°C.
37yb: Obtained as pale yellow plate crystals.

Elemental analysis value Calculated value as Uj5H705N 0, 60.69; H, 2,75; N
, 5.45 Actual value 0, 80.32; H, 2,
87; N, 5.52 Example IB, 6,1-methylenedioxy-4-diazoacetonylcoumarin 6,1-methylenedioxy-4-obtained in Example 14
An excess amount of an ethereal solution of diazomethane was added to chloroformylmethylcoumarin 40019, and the method of Visagr et al. (Biochemistry, Vol. 10, p. 135) was applied.

(1911). It reacted violently with the addition of diazomethane. After the reaction was completed, the solvent was distilled off from the reaction mixture under a stream of chlorine to obtain a pale yellow amorphous substance. The obtained pale yellow amorphous substance was subjected to column chromatography using silica gel (solvent dichloromethane).
The title compound 240119 was obtained upon purification.

Calculated value a as elemental analysis value 01aH805N2,
sr, ss; ((, 2,94; N, 10.
29 actual measurement value C, 57, 12; 1 (, 3,00; P
I, 9.98IRxPect/'V Nu"'l
ff-' :ax 210G (0=N=N) Example 19.6.7-Methylenedioxy-4-bromoacetonylcoumarin 6,1-methylenedioxy-4- obtained in Example IB
Diazoacetonylcoumarin 136ml t-After dissolving in 2 parts of acetonitrile, 48% hydrobromic acid 84.4μt
was injected gently. Nitrogen gas was generated violently. After the reaction was completed, the reaction mixture was allowed to stand under water cooling to precipitate crystals. The precipitated crystals are separated, washed with water-cooled ether from the evening scene, and dried to yield the title compound 8 having a melting point of 212-214°C.
2.4■ was obtained as white flocculent crystals.

Elemental analysis value Calculated value as 0+5HpOsBr 0,
48.00; H, 2JT; Br, 24.5
8 actual measurements 0, 4B, 46; H, 2,70; B
r, 24.75 Example 20.6.7-Methylenedioxy-4-bromomethylcoumarin 6,7-methylenedioxy-4-obtained in Example 13
Carboxymethylcoumarin 4. ! J6 JiJ was suspended in 15 dK of glacial acetic acid and then heated to 120° C. in an oil bath. A solution of 3.2 y of bromine dissolved in 915 ml of rice vinegar was gradually added dropwise to this. After the completion of the dropwise addition, the reaction mixture was heated under reflux for about 2 hours, then allowed to cool, and the yellow-green crystals produced were separated. The obtained crystals were washed with ether and dried, then dissolved again in 400°C of acetonitrile under heating, decolorized with activated carbon, and cooled on ice.

The precipitated pale yellow needle crystals were collected and recrystallized using acetonitrile to obtain the title compound 3.911 having a decomposition point of 235-238°C.

Elemental analysis value Calculated value as 011H704Br 0,
46.64; H, 2,4T; Br, 28.
23 actual measurement value 0, 4B, 93; H, 2,44;
Br, 28.03 Example 21.6.7-methylenedioxy-4-7talimidoxymethylkubrin 6, T-methylenedioxy-4 obtained in Example 20
- 2.59 ml of bromomethylcoumarin was dissolved in dimethylformamide Sod. σ) The solution was preliminarily mixed with 12.2 td of triethylamine and 2.2 td of N-hydroxyphthalimide.
The mixture was added to a dimethylformamide solution in which 4111 had been dissolved while stirring under water cooling.

The reaction mixture was stirred for 30 minutes, the precipitated crystals were collected,
After washing with ice-cold ether and drying, crude crystals 2. T
9 was obtained. When the obtained crude crystals are crystallized using a dimethylformamide-acetonitrile mixed solvent, the melting point is 2.
The title compound 2.059 was obtained as white flocs with a temperature of 35-238°C.

Elemental analysis value Calculated value as 0.5H906N a, 6G, 20; H, 3,03; N
, 4.68 Actual value c,, so, oa; H
, 2,99; N, 5.08 Example 22.6.7-
Methylenedioxy-4-aminooxymethylcoumarin 6,1-methylenedioxy-4- obtained in Example 21
6N hydrochloric acid (20) and glacial acetic acid (20) were added to 1.341 of phthalimidoxymethyl coumarin, and the mixture was heated at 95 to 100°C for 1 hour. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and then neutralized.

The resulting residue was extracted using ethyl acetate.

When the bath medium was distilled off from the extract, 430 m9 of the title compound having a melting point of 170-112°C was obtained as white needle-like crystals.

Elemental analysis value 0. , HpO,N as calculated value 0, 56.17; H, 3,86; N
, 5.96 Actual value a, 56.33; H, 3
, 82; N, 5.91 example 23. 6. r-
Methylenedioxy-4-7thalimidomethylcoumarin fc obtained in Example 20 6. After dissolving 2.0.9' of T-methylenedioxy-4-bromomethylcoumarin in dimethylformamide rafnt, 1.41 g of potassium 7-talimide was added and stirred overnight at room temperature. After the reaction was completed, the reaction mixture was poured into 400 ml of water, and all the insoluble matter produced was collected. The aliquot was purified with acetonitrile 20 [1td.
The mixture was dissolved by heating and allowed to cool. When all the precipitated crystals are collected and then recrystallized using acetonitrile, the melting point is 248-2.
2.1 g of the title compound with a temperature of 49° C. were obtained as white crystals.

Calculated value c as elemental analysis value 0,9H,,06N,
85.33; H, 318; N, 4.01 actual value a, 65.24; H, 2,98; N
, 3.96 Example 24.6.7-methylenedioxy-
4-Aminomethylcoumarin 6, T-methylenedioxy-4 obtained in Example 23
-A mixture of 125 g of 48% hydrobromic acid and 12 tRt of glacial acetic acid was added to 1.6 g of phthalimidomethylcoumarin, and the mixture was refluxed on an oil bath for about 3 hours. After the reaction was completed, the reaction mixture was poured into 100 ml of water.

The reaction mixture was washed with acetic acid ester under acidic conditions, made weakly alkaline by adding aqueous ammonia, and allowed to cool below 50°C to obtain the title compound 0.7219 having a melting point of 205 to 206 cvr as white flocculent crystals.

Elemental analysis value L! 1. Side calculation value c as H904N,
60.27; H, 4,14; N, 6.39
LOL Example 256. T-methylenedioxy-4-isocyanatomethylcoumarin trichloromethylchloroforme) 11F to ethyl acetate 1011LI! After cooling with water, 10~ of activated carbon was added, and then the temperature was raised to 25~30°C at about ta5) while stirring. This solution was then cooled on ice again. Next, 6,1-methylenedioxy-4 obtained in Example 24 was added to this solution.
-Aminomethylcoumarin1. of ethyl acetate 150m
/ was added dropwise over about 30 minutes. After the addition was completed, the reaction mixture was treated in the same manner as in Example T to obtain pale yellow needle-like crude crystals. When the resulting crude crystals were recrystallized using ethyl acetate, the title compound having a melting point of 210-212°C was obtained. T2f was obtained as pale yellow needles.

Elemental analysis value c12H705N Calculated value C, 5B
, 78; H, 2, 88; N, 5. r1 actual measurement value C, 5B,
42:H, 2, 92:N, 5.44 Next, test examples will be shown.

Test Example 1 Isolation of Captogril - Footprint 6. T-methylenedioxy-4-methyl-3-maleimidocoumarin (compound of Example 4) does not show much fluorescence itself, but has a mercapto group. It was found that a fluorescent reactant (excitation wavelength: 355 nm, fluorescence wavelength: 435 nm) was produced by reacting with a compound having the following. Therefore, which compound was used to separate and quantify captogril.

1) Sample preparation and analysis Captogril methanol solution (10μt/l117)
1, diluted with methanol to 100G, Son
200 μt of each of j250, 125, and 62.5 μf/m was placed in a test tube, and the solvent was distilled off under reduced pressure.

A test tube containing no captogril was prepared as a control.

Add ice-cold fresh plasma o and sm to each test tube and mix;
1111, 9.5* of ice-cooled 10 tidotrichloroacetic acid (containing 200 µ2/penalty of quenylmercabutane as an internal standard substance) was immediately added and mixed well.

After centrifugation (zsoorpm S 10 min) under water cooling,
Aliquot 50μt of it into another test tube, add 1ooμt1 of 0.5M phosphate buffer (pHr, a) and then 6.1-
50 μt of a 0.02% acetonitrile solution of methylenedioxy-4-methyl-3-maleimidocoumarin was added and stirred. After the reaction, a certain amount (10 μt) of the mixture was injected into a reversed phase column (Micro Bonder Quunk C48, manufactured by Nippon Waters Co., Ltd.) and acetonitrile-water-acetic acid (
37:82:1) was subjected to high performance liquid chromatography using a mobile phase.

The high-performance liquid chromatography equipment used was a model 6000A, 440-n9 manufactured by Nihon Tsutsu A-Tase Co., Ltd.
A Hitachi model 850-108 was used as a fluorescence detector.

An excitation wavelength of 355 nm and a fluorescence wavelength of 435 nm were used. The results are shown in Figure 1.

2) Results In Figure 1, arrow A indicates the injection time point of the class of sample indicated by each captogril concentration, arrow B indicates the chromatographic beak corresponding to the injected sample, and arrow C similarly indicates the time point of the sample derived from the corresponding internal standard. Chromatobeak is shown. The horizontal axis shows the elution time at each captogril concentration, and the vertical axis shows the fluorescence intensity.

As is clear from Figure 1, the concentration of captogril in plasma was
．． By using 1-methylenedioxy-4-methyl-3-maleimidocoumarin, at least 5 μt/
It is possible to reproducibly quantify concentration levels as low as ml without extraction operations.

3) Idea 6.1-methylenedioxy-4-methyl-3-maleimidocoumarin reacts with a compound having a mercapto group,
Generates a fluorescent reactant. Therefore, for example cysteine,
Glutathione, ergothioneine, coenzyme A, 6
- Useful for the separation and determination of mercabutoprine, penicillamine, 2-thiouracil, etc.

Test Example 2 Separation and quantification of organic acids using 6,7-methylenedioxy-4-diazomethylcoumarin 6.1-Methylenedioxy-4-diazomethylcoumarin (compound of Example 10) has 4j! It reacted with acid to produce a fluorescent ester.

That is, 8. After dissolving 0.1 mmol of T-methylenedioxy-4-diazomethylcoumarin in acetonitrile at 1°OmK, acetic acid such as acetic acid, propionic acid, n-butyric acid, etc.
．． 02 mmol was added and heated at 60° C. for 1 hour to obtain a fluorescent ester (excitation wavelength 355 nm, fluorescence wavelength 435 nm, in ethanol). This fluorescent ester was analyzed by thin layer chromatography using silica gel, and was analyzed using toluene-dioxane-acetic acid (70:25:5).
), it was separated and quantified as a new firefly C spot. Identification was performed by mass spectrometry.

Test Example 3 Classification and Quantification of Prostaglandins 6 Glostaglandins were separated and quantified using γ-methylenedioki7-4-diazomethylcoumarin.

1) Preparation and analysis of sample amount Prostaglandins (Prostaglandidi F, 6
10 acetonitrile solutions containing L Oμ2 of each of m '2t'''+ and 6-keto prostaglandin F,
Add 200 μt of 0.02% acetonitrile solution of 61T-methylenediochyc-4-diazomethylcoumarin to 0 μt, seal with a Teflon-coated cap, and incubate at 80°C for 3 hours.
Stirred for 0 minutes. 10 μt of the solution was injected into a reverse phase column (Micro Bondervac C1B, manufactured by Nippon Waters Co., Ltd.) and subjected to high performance liquid chromatography using acetonitrile-water-acetic acid (50:50:1) as a mobile phase. The same equipment as used in Test Example 1 was used. The results are shown in Figure 2.

2) Results In Figure 2, the arrows indicate the injection points of the sample, and the numbers 1,
The chromatographic peaks indicated by 2.3 and 4 are 1.6-kedoga staglandein'1d2, glostagrandein'2ct3, prostaglandin F, a4, and glostagrandein E, respectively.

shows. The horizontal axis shows elution time, and the vertical axis shows fluorescence intensity. As the elution time progressed, it was successfully separated and quantified.

Test Example 46. Separation and quantification of alcohols using 1-methylenedioxy-4-diazomethylcoumarin 6. T-methylenedioxy-4-diazomethylcoumarin reacts with alcohols to produce C-based ethers. did.

That is, 0.1 mmol of methanol, ethanol, globanol, n-butanol and 6. Added 0.5 mmol of T-methylenedioxy-4-diazomethylcoumarin, then added 20 wu3 of dichloromethane and 1 mg of 1.0M mixed dk of tetrafluoride porcelain to this solution, and stirred at room temperature for 1 hour. did. Next, this solution was subjected to thin layer chromatography using silica gel and developed with toluene-dioxane-acetic acid (rO: 25:5), and the Rf values were 0.72 for methanol, 0.76 for ethanol, and 0.76 for propanoyl, respectively. It was separated and quantified as a fluorescent substance of 0.82 and n-butanol 0.88. Identification was performed by mass spectrometry.

Test example 56. Relative fluorescence intensity of T-methylenedioxy-4-bromomethylcoumarin derivative About 1 micromole of acetic acid, e, r = 002 solution of methylenedioxy-4-bromomethylcoumarin (compound of Example 20) in acetonitrile 0 .2ru! and 1B-crown-6 o, rs
myGold-containing potassium carbonate saturated acetonitrile solution 01
sru& was added and stirred at 40°C for 30 minutes. then 1
The reaction was stopped by adding 9 G and 1 ru of vinegar. The relative fluorescence intensity of the obtained acetate ester is shown below. That is, when the fluorescence intensity of 1-methoxy-4-acetyloxymethylcoumarin in each solvent is taken as 1, the relative fluorescence intensity was as follows.

From this, 6. Compared to T-methoxy-4-bromomethylcoumarin, T-methylenedioxy-4-7'lomomethylcoumarin has a relative fluorescence intensity over a wide pH range (pH 1
, 5 to 10), and is also more than twice as large as the melting point of M solvents frequently used in high performance liquid chromatography, such as methanol and acetonitrile, or a mixture of these and water. RrJ- This shows that a #photosensitive ester with higher sensitivity can be obtained.

Test Example 6 Separation and determination of fatty acids 6 Using T-methylenedioxy-4-bromomethylcoumarin, fatty acids were separated and determined.

1) Preparation and analysis of samples Various fatty acids were prepared according to the method of Test Example 5. A constant meter of this solution was added to a reverse phase column (Micro Bonder Pack C18).
, Nippon Waters 1tl! ), and each fatty acid was subjected to high performance liquid chromatography using the following mobile phase. The same equipment as used in Experiment 1 was used. The results are shown in FIGS. 3 and 4.

2) Results Figure 3 #i shows the separation/isolation I pattern when using the following mobile phase. Arrows indicate sample injection time points.

Hinoki 1111 is U at each acetonitrile θ degree of mobile phase.
It shows the release time, and the vertical axis shows the strength of fireflies.

FIG. 4 shows the retention factor (k) for the column when acetonitrile-water-acetic acid was used as a reaction and the acetonitrile concentration was changed sequentially at an acetic acid concentration of 1.5%. The horizontal axis shows the acetonitrile concentration of the mobile phase, and the vertical axis shows the retention factor (ko).

The numbers 1 to 13 in FIGS. 3 and 4 indicate the numbers of the fatty acids mentioned above.

It can be seen from FIGS. 3 and 4 that both were well separated and determined.

It has also been shown that it is possible to separate and distribute carvone type 9 at a high sensitivity of about 100 femtomoles per injection needle.

Test Example 1 Separation and Quantification of Carbonyl Compound 6. T-methylenedioxy-4-aminooxymethylcoumarin (compound of Example 22) was dissolved in methanol, in the presence of an acid catalyst such as hydrochloric acid, acetic acid, or trichloroacetic acid. A fluorescent derivative (excitation wavelength 358 nm, fluorescence wavelength 430 nm) is produced by reacting with a carbonyl compound in a solvent such as ethanol or benzene.
)was gotten.

That is, for about 10 nanomoles of carbonyl compound, 6.
A 0.02% mixed solvent of T-methylenedioxy-4-aminooxymethylcoumarin (the mixed solvent was 1% glacial acetic acid-
Methanol solution) 0.2 md was added and heated at 50°C for 30 minutes to obtain a fluorescent derivative. The obtained derivative was injected into a reverse phase column (Micro Bonder Pack C18, Nippon Waters Co., Ltd.) and applied to a high-performance liquid chromatogram, 7-fee, using each mobile phase.The apparatus was the same as that used in Test Example 1. things were used.

Table 1 shows the results. The numbers indicate the retention factor (k >) for the column.

Table 1 High performance liquid chromatography of carbonyl compounds Carbonyl compounds Mobile phase 1IInI CU, CIO1,8 CH, COCH2C0OH2,5 Corti8one j, HydroCortisone 1,6Cortico
sterone 2.4 Mobile phase I Niacetonitrile-water-acetic acid (35:65:1.5) Mobile phase ■ Niacetonitrile-water-acetic acid (66:35:1.5) For mobile phase: Methanol-water-Vick B7 ( T8:
22: 0.1) Table 1 shows that aliphatic and aromatic aldehydes, ketones, keto acids, ketosteroids, etc. can be separated and quantified with a high sensitivity of 0.2 picomole. There is.

Test Example 8 Separation and determination of alcohols 116, Separation and determination of alcohols using T-methylenedioxy-4-methoxycarbonylmethylcoumarin (compound of Example 15)
I made a decision.

1) Preparation and Analysis of Samples 0.0 μV of dry dichloromethane containing 5 μV each of alcohols.
51 solution, 6. 05 ml of 002 tidichloromethane solution of γ-methylenedioxy-4-methoxycarbonylmethylcoumarin was added, and the mixture was stirred at room temperature for 5 minutes or more. That 10μ
t was injected into a reverse phase column (Micro Bonder Pack C2 manufactured by Nippon Waters Co., Ltd. 8), and acetonitrile-water-acetic acid (40: fJ
It was subjected to high performance liquid chromatography at a flow rate of 1.011 V/min using O71,5) as a mobile phase. The device is Test Example 1
The same one used in was used. The results are shown in Figure 5.

2) Results In Figure 5, the arrows indicate the injection time points of the sample, and the numbers 1-
The chromatographic peaks indicated by 8 are respectively 1, methanol 2, ethanol 3, n-gulo/knol 4. 1-pronotol 5, n-butanol 6.8-butanol 7, t-butanol 8, octatool. show.

The horizontal axis shows elution time (minutes), and the vertical axis shows fluorescence intensity.

As the elution time progressed, it was successfully separated and quantified.

Test Example 9 Separation and quantitative determination of alcohols 6, T-methylenedioxy-4-methyl-3-isocyanatocoumarin (compound of Example T) and 6. T-
Separation of alcohols using methylenedioxy-4-incyanatomethylcoumarin (compound of Example 25)
Quantification was performed.

1) Preparation and analysis of samples To toluene solutions o and sm containing 0.1 mmol each of alcohols, add 0.5- toluene solution containing 2 mmol of the listed compound, seal tightly, and heat at 80°C for 2 hours. It was warm. Then, a certain amount (2 .mu.t) was collected, subjected to thin layer chromatography using silica gel, and developed with toluene-dioxane-glacial acetic acid (70:25:5).

2) Results■6. In the case of T-methylenedioxy-4-methyl-3-isocyanatocoumarin Type of alcohol Rf value Methanol 0.44 Ethanol #0.52 n-Glonoinol 0.51 n-Butanol 0.81 Nclohexanol 0.62 ■6 ．． In the case of T-methylenedioxy-4-incyanatomethylcoumarin, type of alcohol: Rf value Methanol 0.51 Ethanol 0.80 n-Glopanol 0.63 n-Butanol O, SS Nclohexanol 0.64 Both have blue fluorescence It was isolated and identified as a substance. The analysis was carried out using a mass spectrometer manufactured by Iidaisha.

6.1-methylenedioxy-4-methyl-3-incyanatocoumarin and 6.7-methylenedioxy-4-
Incyanate methylcoumarins themselves had weak fluorescence, but the products produced by reaction with alcohols showed strong fluorescence.

[Brief explanation of drawings]

Figure 1 Halo, 7-methylenedioxy-4-methyl-3
- Shows the separation and constant of captogril using maleimide. The horizontal axis shows the elution time at each captogril concentration, and the vertical curve 1 shows the fluorescence intensity. Figure 2 shows 6. The separation and quantification of glostagrandeins using γ-methylenedioxy-4-diazomethylcoumarin is shown. The horizontal axis shows elution time, and the vertical axis shows fluorescence intensity. Figure 3 shows 6. Figure 4 shows the separation and determination of fatty acid 4 using T-methylene dioxy/-4-phyromomethylcoumarin. The horizontal axis shows the elution time at each acetonitrile concentration of the mobile phase, and the vertical axis shows the fluorescence intensity. Figure 4 shows 6. The separation and quantification of fatty acids using γ-methylenedioxy-4-bromomethylcoumarin is shown. The horizontal axis shows the acetonitrile concentration of the mobile phase, and the vertical axis shows the retention factor (k). Figure 5 H8. Separation of alcohols using 1-methylenedioxy-4-methoxycarbonylmethylcoumarin
Indicates quantification. The horizontal axis shows elution time, and the vertical axis shows fluorescence intensity. Patent applicant Sankyo Co., Ltd. Agent Patent attorney Shoji Kashiyama 200 100 50 Figure 1

Claims (1)

[Claims] A coumarin derivative having the general formula. However, in the formula, x is a methyl group, formyl group, p-)luenesulfonylhydrazinomethyl group, diazomethyl group, hydroxymethyl group, haloformyloxymethyl group, carboxymethyl group, haloformylmethyl group, alkoxycarbonylmethyl group , hydrazinocarbonylmethyl group, cyanocarbonylmethyl group, diazoacetonyl group, haloacetonyl group,
Halomethyl group, phthalimidooxymethyl group, aminooxymethyl group, phthalimidomethyl group, aminomethyl group or isocyanatomethyl group, Y is a hydrogen atom, nitro group, amino group, 3-carboxyacryloylamino group, maleimide group, acetylamino group, haloacetylamino group or isocyanate group. However, this excludes cases where X is a methyl group and Y is a hydrogen atom.