JP3027660B2 - Fluorescent compound - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluorescent compound, and more particularly to a molecule having a ureide bond, a molecule having a carboxyl group, and a molecule thereof, which are important in the fields of medicine, biochemistry, food, and the chemical industry. The present invention relates to a novel fluorescent compound suitable for use in qualitative and quantitative analysis of molecular salts.

[0002]

2. Description of the Related Art Urea bond (-NHCONH)
Molecules having-), molecules having a carboxyl group (-COOH), and salts of these molecules are important substances in fields such as medicine, biochemistry, food, and the chemical industry. Among them, barbital-related compounds and hydantoin-related compounds, and molecules having ureido bonds represented by their sodium salts and their salts have strong biological activities, and are especially Widely used in each country. In Japan,
Phenobarbital and sodium phenobarbital (as hypnotic, sedative, antiepileptic), barbital (sleep, sedative), amobarbital (anticonvulsant, sedative, hypnotic), phenytoin and sodium phenytoin (antiepileptic), and thiopental sodium and Thiamylal sodium (anesthetic) is listed in the Japanese Pharmacopoeia.

[0003] While these drugs have very remarkable effects, they also have strong side effects. Therefore, the analysis is extremely important for monitoring the concentration of these drugs in blood and urine and for monitoring the content of these drugs in pharmaceutical tablets and injections.

In addition to these drugs, chemicals having monoureido and diureide bonds are widely used in living organisms,
Present in the natural and chemical industries, its analysis is very important due to the need to monitor ecosystems and the environment and control synthetic processes and wastewater management in the chemical industry.

[0005] Taking barbital and hydantoin-related drugs as examples, these drugs are quantified in the Japanese Pharmacopoeia by neutralization titration without using a special analytical instrument. However, this method has a low sensitivity and is strongly influenced by coexisting substances. Therefore, in a special case where the concentration of the drug in the analysis sample is high and there is only a very limited coexisting substance, It is limited to its own purity test, measurement of contents in tablets and injections, and cannot be applied to measurement of blood and urine concentrations.

[0006] In order to enable the analysis of a trace amount, a high-performance liquid chromatograph method using an ultraviolet / visible spectrophotometer as a detector, a gas chromatograph method using a flame ionization detector or a mass spectrometer, etc. are being studied. . These methods are relatively sensitive and are not susceptible to coexisting substances,
All of them require a relatively large stationary type device, consume a large amount of solvent and carrier gas as a mobile phase, and take a long analysis time.
Need a minute. Therefore, practically, a simple and quick analysis method using a light or small device is desired.

[0007] Such a problem relating to analysis is not limited to the above-mentioned barbital and hydantoin drugs, but also applies to other molecules and salts generally having a ureide bond which are important in the fields of medicine, biology and chemical industry. .

[0008] Molecules having a carboxyl group and their salts (eg, acetic acid, oxalic acid, citric acid and their sodium salts) are important molecules particularly in the fields of living organisms, foods, chemical industries and the like. The importance of quality control and control of the manufacturing process, and in particular in recent years the monitoring of ecosystems and the environment, has increased the need for their analysis. Problems similar to those described above also exist in the method for analyzing these molecules having a carboxyl group and salts thereof, and a simple and quick analysis method using a lightweight and small-sized apparatus is desired.

[0009]

SUMMARY OF THE INVENTION An object of the present invention is to eliminate the above-mentioned disadvantages of the prior art,
It is an object of the present invention to establish a simple and quick means for analyzing a molecule having a ureide bond, a molecule having a carboxyl group, and salts thereof with high sensitivity.

As a result of repeated studies, the present inventors have found a novel compound whose fluorescence properties can be changed with high sensitivity when a molecule having a ureide bond, a molecule having a carboxyl group, or a salt thereof is captured. The above-mentioned object was achieved by utilizing.

[0011] That is, the present invention relates to a pair of (pyrido-2)
-Yl) aminocarbonyl group [(pyrid-2-yl) aminocar
bonyl] is attached to the molecular skeleton bonded at an appropriate distance to provide a fluorescent compound having a structure in which a fluorescent atomic group is present. For ease of understanding, the "pair of (pyrido-
A molecular skeleton in which a 2-yl) aminocarbonyl group is bonded at an appropriate distance is shown as follows.

[0012]

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In the formula [Chemical formula 9], M represents a distance between a pair of a (pyrid-2-yl) aminocarbonyl group represented by the following [Chemical formula 10], preferably equivalent to 2 to 5 carbon atoms. Groups or functional groups separated by M may be an aromatic group including a benzene ring, a heterocyclic group including a pyridine ring, or an atom group or a functional group derived from an alicyclic or chain compound. (Note that, in the chemical structural formulas shown in this specification, carbon atoms and hydrogen atoms may be omitted according to a custom.)

[0014]

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As described above, the fluorescent compound of the present invention comprises:
It provides a suitable space for trapping molecules having a ureido bond, molecules having a carboxyl group, and salts thereof between (pyrid-2-yl) aminocarbonyl groups, and has two NH groups and two pyridine nitrogens. The atom acts as a trapping site for a molecule having a ureido bond through a hydrogen bond, and this trapping has a structure suitable for greatly changing the fluorescence characteristics of the fluorescent group disposed in the compound. I have. Thus, by using the fluorescent compound of the present invention, a molecule having a ureido bond, a molecule having a carboxyl group, or a salt thereof can be analyzed simply, quickly and with high sensitivity.

According to a particularly preferred embodiment of the present invention, the fluorescent compound of the present invention is represented by the following general formula (1).

[0017]

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In the formula, at least one of X ₁ , X _{2 and} X ₃ is an atomic group to which a fluorescent molecule is bonded. Preferred examples of the atomic group to which the fluorescent molecule is bonded include the following [Chemical formula 2], [Chemical formula 3], [Chemical formula 4], [Chemical formula 5], [Chemical formula 6], [Chemical formula 7] or [Chemical formula 8] Is mentioned.

[0019]

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[0020]

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[0021]

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[0022]

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[0023]

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[0024]

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[0025]

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Among these fluorescent molecules, pyrene as shown in [Chemical Formula 2] and [Chemical Formula 3], or anthracene as shown in [Chemical Formula 4] or [Chemical Formula 5] is preferred in terms of sensitivity and selectivity. Particularly preferred. That is, pyrene and anthracene have light absorption and fluorescence spectra in a relatively long wavelength region, are less susceptible to other light-absorbing or fluorescent impurities mixed in the sample, and select the target molecule or salt. Analysis is possible. In addition, pyrene and anthracene emit very strong fluorescence, so that highly sensitive analysis is possible by using a fluorescent group containing them.

If at least one of X ₁ , X ₂ or X ₃ contains a fluorescent molecule, it functions as a fluorescent substance, but X ₁ , X ₂ or X ₃ It is preferred that at least two of them contain the same or different fluorescent molecules. The reason is that firstly, by containing a plurality of fluorescent molecules, the fluorescence emission ability is increased, so that a more sensitive analysis can be performed. Furthermore, by arranging a plurality of appropriate fluorescent molecules, not only the fluorescence intensity when reacting with the target molecule to be measured, but also a fluorescence spectrum having a characteristic shape reflecting the structure of each molecule is provided. This is because qualitative analysis can be performed simultaneously with quantitative analysis using this. For example, according to a preferred embodiment of the present invention, two pyrene molecules are used as a fluorescent molecular group, and the ratio of the fluorescence from the monomer of the pyrene molecule to the excimer fluorescence from the aggregated molecule captures the guest molecule (the molecule to be measured). ) Using a system that changes depending on the shape of
Can perform qualitative analysis.

Y in the above formulas [Chemical Formula 2] to [Chemical Formula 8] is an atomic group or a functional group connecting a (pyrid-2-yl) aminocarbonyl group and a fluorescent molecule. It is selected from any atomic group or functional group that does not impair the function of the fluorescent molecule. From the viewpoint of chemical stability, Y is preferably a chain hydrocarbon group (particularly an alkyl group) which may contain an ether group, an ester group, a carbonyl group, or an amide group. If Y has an excessively long chain, the change in fluorescence characteristics upon reaction with the target molecule is small. Therefore, it is preferable that Y has a length corresponding to 1 to 8 carbon atoms. In particular, an atomic group containing an amide group is preferably used because it has an effect of promoting the ability to capture a target molecule. Thus, examples of particularly preferred _{Y, -NH-CO-CH 2} -CH 2 -CH
_And atomic groups such as _2- .

As described above, X ₁ , X ₂ or X ₃
Among them, those that do not bind a fluorescent molecule are selected from any atoms or atomic groups that do not impair the function of the fluorescent compound. Such atoms or atomic groups include, for example,
Alkyl, alkyl ether, alkyl ester, alkyl amide, hydrogen atom and the like can be used. In addition, nitrobenzene, cyanobenzene, pyridine,
An alkylamine or the like may be bonded. Since the fluorescence quenching ability of these quenchers changes depending on the capture of the target molecule, a large change in fluorescence intensity can be obtained before and after the measurement of the target molecule. Further, instead of the quencher described above, it is also possible to bind to molecules such as indoles and N, N-dialkylaniline, which interact with fluorescent molecules and exhibit exciplex emission. It is. When these molecules are introduced, the compound emits both the intrinsic fluorescence of the fluorescent molecule and the exciplex fluorescence, and the intensity of both emission changes before and after the measurement of the target molecule. Analysis of the target molecule can be performed.

The molecule having a ureide bond suitable for measurement by utilizing the fluorescent compound of the present invention is, in particular, a cyclic diacyl urea represented by the following formula [Chemical Formula 11]. Among them, cyclic ureides derived from monocarboxylic acids represented by the following formula [Formula 12] or cyclic ureides derived from dicarboxylic acids represented by the following formula [Formula 13] are targeted.

[0031]

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[0032]

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[0033]

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A typical example of [Chemical formula 12] is a hydantoin-related compound (for example, phenytoin) used as a drug as described above, and a typical example of [Chemical formula 13] is a barbital-related compound. According to the present invention, a fluorescent compound suitable for analyzing such a molecule having a ureido bond or a salt thereof particularly includes a compound represented by the following general formula:
4].

[0035]

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The molecule having a carboxyl group suitable for measurement using the fluorescent compound of the present invention is, in particular, HOOC (CH ₂ ) _n COOH [n is an integer of 1 to 6]. An aliphatic dicarboxylic acid as represented. According to the present invention, a fluorescent compound that is particularly suitable for analyzing a molecule having a carboxyl group or a salt thereof includes a compound represented by the following general formula [Formula 15].

[0037]

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According to the method established by the present inventors, the fluorescent compound of the present invention can be synthesized by the following relatively simple method.

That is, phthalic acid or substituted phthalic acid is reacted with 2-aminopyridine or substituted 2-pyridine in an appropriate solvent, and the two are linked by an amide bond.
Subsequently, a molecule containing an appropriate fluorophore is reacted in a solvent to bind to a pyridine ring or a phthalic acid residue. Alternatively, 2-aminopyridine or substituted 2-aminopyridine is first coupled to a molecule containing a suitable fluorophore, followed by:
It can also be synthesized by linking this to phthalic acid or substituted phthalic acid via an amide bond.

Here, as a method for linking phthalic acids and 2-aminopyridines, various commonly known organic synthesis methods for linking a carboxylic acid and an amine via an amide bond can be applied. A method is preferred in which phthalic acid is converted to an acid chloride and reacted with 2-aminopyridines in an organic solvent in the presence of a suitable base. According to this method, the reaction proceeds promptly and quantitatively, and the target product can be obtained in good yield while minimizing generation of by-products. In this case, as the base, a tertiary amine such as triethylamine or pyridine is suitable. Any solvent can be used as long as it does not inhibit the desired reaction, but a dry solvent from which water is removed as much as possible is desirable, and a low-boiling solvent such as anhydrous tetrahydrofuran or anhydrous dichloromethane is particularly preferable. It is suitably used for the purpose of simplifying the post-processing operation.

Pyrene as a fluorescent molecule of X ₁ and X ₂ ;
Further, —CH ₂ CH ₂ CH ₂ CON as an atomic group Y connecting pyrene and a (pyrid-2-yl) aminocarbonyl group is used.
FIG. 1 illustrates the synthesis method further in accordance with a preferred example of the fluorescent compound of the present invention using a H-group and a hydrogen atom as X ₃ .

That is, in the presence of about 2 molar equivalents of triethylamine in dry tetrahydrofuran solvent, 2 molar equivalents or more of 2,6-
Compound 3 is obtained by reacting with diaminopyridine. on the other hand,
Compound 5 is prepared by reacting pyrene butyric acid with excess oxalyl chloride in dry dichloromethane. Compound 3
The desired compound 6 can be obtained by reacting 2 mole equivalents or more of compound 5 in the presence of about 2 mole equivalents of triethylamine in dry tetrahydrofuran with respect to 1 mole of the above.

FIG. 6 shows a reaction scheme for synthesizing a fluorescent compound (compound 9) having only one fluorescent atomic group in a molecule as another preferred example according to the present invention. FIG. 7 shows, as still another example of the compound of the present invention, a fluorescent compound (Compound 1) suitable particularly for analyzing a molecule having a carboxyl group or a salt thereof.
1 shows a reaction scheme for synthesizing 1).

The fluorescent compound of the present invention obtained as described above can be prepared by reacting with a molecule to be measured (analyte molecule), ie, a molecule having a ureide bond, a molecule having a carboxyl group, or a salt thereof. It is a compound whose molecule is precisely designed so that its fluorescence characteristics are greatly changed. The mechanism of action is thought to differ depending on the type and combination of the fluorescent groups used and the form of use. For example, a compound using a pyrene group as the fluorescent group (compound 6 in FIG. 1) is exemplified. Is assumed to be as shown in FIG.

That is, first, when Compound 6 is used by dissolving it in a highly soluble solvent such as chloroform or a mixed solvent of chloroform and hexane, Compound 6 itself emits fluorescence (about 380 nm) from the pyrene monomer and the pyrene excimer. Emits two types of fluorescence (about 480 nm), some of which are partially quenched by the pyridine ring. Molecules with ureido bonds are added,
When this is captured by the compound, a change occurs in the fluorescence and quenching processes, and the fluorescence spectrum changes.

For example, when barbital is added, excimer fluorescence decreases while monomer fluorescence increases remarkably. This is presumably because barbital is trapped by the receptor by forming six-point hydrogen bonds with the fluorescent compound, so that the quenching process is first suppressed and the excimer fluorescence emission process is also suppressed. That is, it is considered that the quenching process is suppressed by inactivating the lone electron pair on the nitrogen atom of the pyridine ring by the hydrogen bond, while the excimer fluorescence process is suppressed by a steric factor due to the barbital bond.

When ethylene urea is added,
Excimer fluorescence is greatly increased. This is because the quenching process is suppressed by the same effect as in the case of barbital,
This is probably because there is no steric effect that inhibits the association of two pyrene molecules in the molecule.

Further, the fluorescent compound of the present invention causes self-association when added to a nonpolar solvent such as hexane,
Its fluorescence is significantly reduced. At this time, when a guest molecule such as barbital is added, the fluorescent compound of the present invention dissociates by forming a complex with the guest molecule and is converted to a strong fluorescent property. Using such an action mechanism, compared to the case where the above-described fluorescent compound is completely dissolved in a highly soluble solvent and used,
Higher analytical sensitivity may be obtained.

When only one fluorescent atom group is present in the molecule as in the case of compound 9 shown in FIG. 6, the intramolecular quenching process is reduced as the molecule to be measured is captured.
The target molecule can be analyzed by utilizing the increase in the fluorescence from the atomic group.

Further, in the compound of the present invention having a structure in which a fluorescent atomic group and its quenching atomic group are combined, the change in the fluorescence intensity is accompanied by the capturing of the molecule to be measured, and the configuration of the fluorescent atomic group and the quenching atomic group is changed. Is considered to be caused by the change of

As will be understood from the above description, the compound of the present invention has a property of causing a sharp change in fluorescence characteristics when reacted with a molecule having a ureide bond, a molecule having a carboxyl group, or a salt thereof. Thus, as another aspect, the present invention provides a method for analyzing a molecule having a ureido bond, a molecule having a carboxyl group, or salts of those molecules, which comprises a fluorescent compound represented by the above formula [Formula 1]. A fluorescent probe is provided. There are various embodiments for use as a fluorescent probe, and there is no particular limitation. Generally, the fluorescent compound of the present invention is dissolved in a solution containing the molecule to be measured, and the fluorescence intensity is measured. Alternatively, the fluorescent compound of the present invention can be dissolved in an organic solvent insoluble in water, and the molecule to be measured can be extracted from the sample water to measure the fluorescence intensity of the organic layer. In still another embodiment, the fluorescent compound of the present invention is mixed with other film components to form a suitable film, for example,
It can also be used for optrodes. Further, the fluorescent compound of the present invention can be used not only as a single compound as it is, but also as a component of another compound, for example, a composite compound chemically bonded to an appropriate polymer chain.

As will be apparent from the specific examples described below, the use of the fluorescent compound of the present invention not only allows the target molecule in the organic solvent sample but also the target molecule in the aqueous solution sample to be extremely simple, rapid and highly efficient. Can be analyzed for sensitivity. An organic solvent sample can be analyzed by simply mixing the fluorescent compound of the present invention in an appropriate organic solvent. In the case of aqueous samples, extraction and extraction are performed in one step using the solvent extraction technology.
Can analyze. The time required for these analysis operations is several minutes.

Hereinafter, the present invention will be described with reference to Examples to further clarify the features of the present invention.

【Example】

<< Preparation Example 1 >> According to the flowchart of FIG. 1, a fluorescent compound 6 (shown by the following formula [Formula 16]) suitable for analysis of a molecule having a ureido bond according to the present invention was prepared.

Preparation of Compound 3 : 13.1 g (120 mmol) of 2,6-diaminopyridine and 2.40 g (24 mmol) of triethylamine were dissolved in 400 ml of anhydrous tetrahydrofuran. To this solution, a solution of 2.44 g (12 mmol) of isophthaloyl dichloride in 100 ml of anhydrous tetrahydrofuran was added at room temperature under a nitrogen atmosphere.
The solution was added dropwise with stirring. After completion of the dropwise addition, stirring was further continued for 3 hours, and then the solvent was distilled off under reduced pressure. After the solid residue was sufficiently washed with water, the target product was separated and purified using silica gel column chromatography using a mixed solvent of chloroform and tetrahydrofuran as a developing agent. The product was further purified by a recrystallization method from a mixed solvent of tetrahydrofuran and hexane to obtain 3.59 g (yield 95%) of Compound 3: white crystals, mp 200-202 ° C; ¹ H NMR (THF-d ₈ ), δ = 5.06 (s, 4H),
6.20 (d, 2H, J = 8H Z), 738 (t, 2H, J = 8H Z), 7.57 (t, 1H, J = 8H Z),
7.63 (d, 2H, J = 8H Z), δ = 13 (dd, 2H, J = 8 and 2H _Z), 8.52 (t,
_{1H, J = 2H Z),} 9.41 (s, 2H), Calculated _{(C 18 H 16 N 6 O} 2): C, 62.06; H, 4.36; N, 24.12. Measured value: C, 6
2.10; H, 4.34; N, 24.04.

Preparation of Compound 5 : 1-pyrenebutyric acid 1.00
g (3.5 mmol) in 100 ml of anhydrous dichloromethane
Oxalyl chloride (1.0 ml) and N, N
-1 drop of dimethylformamide was added. The reaction mixture was stirred at room temperature for 4 hours. After the volatile components were distilled off by a rotary evaporator, the solid residue was dried in a high vacuum for 4 hours to obtain Compound 5.

Preparation of Compound 6 : 0.40 g of Compound 3
(1.1 mmol) and 0.35 g of triethylamine
(3.5 mmol) in 50 ml of anhydrous tetrahydrofuran
Was dissolved. To this solution was added dropwise a solution of compound 5 (3.5 mmol) in anhydrous tetrahydrofuran (50 ml) with stirring under a nitrogen atmosphere at room temperature. Add 3 more of this solution
After stirring for an hour, volatile components were distilled off using a rotary evaporator. The solid residue was dissolved in 30 ml of N, N-dimethylformamide, poured into 300 ml of a 0.05 M aqueous sodium hydroxide solution, and the resulting precipitate was collected by filtration. After washing the solid with water, the N, N-dimethylformamide 3
It was dissolved in 0 ml, poured into 300 ml of a mixed solvent of methanol and water (1: 1, v / v), and the resulting precipitate was collected by filtration and dried. The crude product was recrystallized from a mixed solvent of tetrahydrofuran and hexane to give pure compound 6 (formula [Chem. 16]
0.72 g (0.81 mmol, 71%): white powder, mp 265-267 ° C .; IR (KBr) v 3289,1685,167
^{4,1584cm -1; 1 HNMR (THF =} d 8) δ = 2.24 (m, 4H), 2.51 (t, 4H, J =
7H _Z ), 3.45 (t, 4H, J = 8H _Z ), 7.61 (t, 1H, J = 8H _Z ), 7.74 (t, 2H, J
= 8H _Z ), 7.89-8.17 (m, 22H), 8.43 (d, 2H, J = 8H _Z ), 8.45 (s, 1
. H), 9.16 (s, 2H), as 9.47 (s, 2H) calcd _{(C 58 H 44 N 6 O} 4): C, 78.36; H, 4.99; N, 9.45%. Actual value:
C, 78.29; H, 5.07; N, 9.47%.

[0057]

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Preparation of comparative compound : For comparison, compound 7 represented by the following formula [Formula 17] was prepared. 0.29 g (0.83 mmol) of compound 3 (FIG. 1) and 0.62 g (6.1 mmol) of triethylamine were dissolved in 30 ml of anhydrous tetrahydrofuran. To this solution, a solution (30 ml) of 0.45 g (3.73 mmol) of 1-pentanoyl chloride in anhydrous tetrahydrofuran was added at room temperature under a nitrogen atmosphere, and stirring was continued for 4 hours. The volatile components were distilled off under reduced pressure, and the solid residue was treated with a 0.05 M aqueous sodium hydroxide solution,
Washed with 0.05M aqueous hydrochloric acid, followed by water. This solid was recrystallized from a mixed solvent of tetrahydrofuran and hexane to give 0.33 g (0.63 mmol) of pure compound 7.
1, 76%): white powder, mp 232-234 ° C .; IR (KBr)
_{^{ν3289,1671,1651,1588cm -1; 1 HNMR (CDCl 3}} ) δ = 0.96 (t, 6
H, J = 7H _Z ), 1.44 (m, 4H), 1.76 (m, 4H), 2.42 (t, 4H, J = 8H _Z ), 7.
67 (s, 1H), 7.67 (t, 1H, J = 8H Z), 7.79 (t, 2H, J = 8H Z), 8.00 (d,
2H, J = 8H _Z ), 8.06 (d, 2H, J = 8H _Z ), 8.12 (dd, 2H, J = 8 and 2
_{. H Z), 8.41 (s} , 2H), as 8.46 (s, 2H) calcd _{(C 28 H 32 N 6 O} 4): C, 65.10; H, 6.24; N, 16.27%. Actual value:
C, 64.95; H, 6.30; N, 16.56%.

[0059]

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<< Application Example 1 >> In a 5 ml volumetric flask, 1 ml of chloroform and about 3 ml of cyclohexane were added, and 10 μl of a 1 mM solution of compound 6 in tetrahydrofuran was added thereto.
Was injected and dissolved. To this solution, 10 μl of a tetrahydrofuran solution of a molecule having various ureido bonds or a mixed solution of tetrahydrofuran and methanol was injected, and the mixture was diluted to exactly 5 ml with cyclohexane. Intensity and intensity ratio (Im / Iex) of fluorescence (Im) from pyrene monomer and fluorescence (Iex) from excimer in these solutions
Was measured under the following conditions.

Conditions: Spectrofluorometer: Hitachi F-4500 type spectrofluorometer cell: 0.5 cm quartz cell made of quartz Excitation wavelength: 345 nm Fluorescence wavelength: Im: 378 nm , Iex: 480 nm Temperature... 25 ° C. The results are shown in Table 1. FIG. 3 shows the change in the fluorescence spectrum of Compound 6 when barbital and ethylene urea were added as molecules having a ureido bond.

As a comparative example, instead of compound 6,
Table 1 also shows the results of a test conducted using Compound 7 in the same manner. However, since Compound 7 does not emit fluorescence at the same wavelength as Compound 6, the excitation wavelength is 305 nm,
The fluorescence intensity (Ir) was measured at a fluorescence wavelength of 480 nm.

[0063]

[Table 1]

As shown in Table 1, compound 6 according to the present invention
Responds sharply to molecules with ureido bonds, greatly altering the intensity of pyrene monomer and excimer fluorescence,
It is clear that these molecules are excellent fluorescent probes. Further, Im / I is determined according to each analyte molecule.
Since the ex value is changed, qualitative analysis is also possible using this. In contrast, Comparative Compound 7 has little fluorescence and is not suitable for analyzing a molecule having a ureido bond.

<< Application Example 2 >> Following the same method as in Application Example 1, various concentrations of barbital and ethylene urea in tetrahydrofuran were injected into the solution of Compound 6, and the fluorescence intensities of these solutions were measured in the same manner as in Application Example 1. It was measured under the conditions. The results are shown in FIGS. 4 and 5 as a relationship between the relative value of Im or Iex and the concentration of the added analyte molecule.

As is clear from these results, the fluorescence intensity of compound 6 increases in proportion to the concentration of the analyte molecule, and therefore, the concentration of the target molecule in the test solution can be analyzed from the fluorescence intensity. .

<< Application Example 3 >> About 4 ml of cyclohexane was placed in a 5 ml volumetric flask, and 10 μl of a 1 mM solution of compound 6 in tetrahydrofuran was injected into the flask.
Was formed. Inject 10 μl of 25 mM tetrahydrofuran or a mixed solution of tetrahydrofuran and methanol of various molecules having a ureide bond or a salt thereof into this solution, and add exactly 5 ml of cyclohexane.
Diluted. The fluorescence intensities of these solutions were examined under the same measurement conditions as in Application Example 1. Table 3 shows the results.

The results in Table 3 show that Compound 6 is an excellent probe for the analysis of molecules having a ureido bond, as shown in Application Example 1.

[0069]

[Table 3]

<< Application Example 4 >> 20 μl of a 1 mM solution of compound 6 in tetrahydrofuran was injected into 10 ml of cyclohexane. 10 ml of this solution and an aqueous solution containing a molecule having various ureido bonds at a concentration of 1 mM (pH adjusted to 6.5 using a 0.05 M phosphate buffer)
Was placed in a 25 ml flask with a stopper and vigorously shaken for 5 minutes. After standing for 1 minute, the fluorescence intensity of the upper layer (cyclohexane layer) was measured under the same conditions as in Application Example 1. Table 4 shows the results.

As is clear from the results shown in Table 4, the compound 6 according to the present invention is an excellent fluorescent probe capable of analyzing a target molecule in an aqueous solution sample with high sensitivity. In addition, as a result of performing similar tests using aqueous solution samples containing various concentrations of analyte molecules, a positive proportional relationship between the concentration and the fluorescence intensity was established for each analyte molecule shown in Table 4, and this It was confirmed that the quantitative analysis of each sample molecule can be performed using the relationship.

[0072]

[Table 4]

<< Preparation Example 2 >> According to the flowchart shown in FIG. 6, a fluorescent compound 9 according to the present invention suitable for analysis of a molecule having a ureido bond was prepared.

Preparation of Compound 8 : 0.85 g (2.4 mmol) of Compound 3 and 0.36 g (3.6 mmol) of triethylamine prepared in the same manner as in Preparation Example 1 were dissolved in 100 ml of anhydrous tetrahydrofuran. 0.44 g (3.6 mmol) of 1-pentanoyl chloride was added to this solution.
l) in anhydrous tetrahydrofuran (100 ml)
The mixture was added dropwise with stirring at room temperature under a nitrogen atmosphere. After the solution was further stirred for 2 hours, volatile components were distilled off using a rotary evaporator. The solid residue was washed with a 0.05M aqueous sodium hydroxide solution, followed by water. Further, the solid residue was dispersed in 200 ml of a 0.05 M aqueous hydrochloric acid solution, and insoluble components were removed by filtration. After neutralizing this solution with sodium hydrogen carbonate, the resulting precipitate was extracted with 100 ml of chloroform. Chloroform was distilled off under reduced pressure, and the solid residue was purified by silica gel column chromatography using a mixed solution of chloroform and tetrahydrofuran as a developing solvent to obtain 0.34 g of pure compound 8 (0.79 mmol,
32%): white powder, mp 141-143 ° C .; ¹ HNMR (CDC
l ₃ ) δ = 0.95 (t, 3H, J = 7H _Z ), 1.42 (m, 2H), 1.72 (m, 2H), 2.44
_{(t, 2H, 7H Z)} , 4.44 (s, 2H), 6.28 (d, 1H, J = 8H Z), 7.49 (t, 1H, J
= 8H _Z ), 7.61 (t, 1H, J = 8H _Z ), 7.71 (d, 1H, J = 8H _Z ), 7.73 (t, 2H,
J = 8H _Z ), 7.95 (d, 1H, J = 8H _Z ), 8.00 (d, 1H, J = 8H _Z ), 8.09 (s, 1
H), 8.13 (d, 2H , J = 8H Z), 8.45 (s, 1H), 8.46 (s, 1H), 9.14 (s, 1
H) calculated for _{.C 23 H 24 N 6 O 3} : C, 63.88; H, 5.
59; N, 19.45%. Found: C, 63.80; H, 5.65; N, 1
9.27%.

Preparation of compound 9 : 0.32 g of compound 8
(0.74 mmol), and 0.2 of triethylamine
2 g (2.2 mmol) of anhydrous tetrahydrofuran 40
Dissolved in ml. To this solution was added 30 ml of a solution of 0.32 g (1.0 mmol) of Compound 5 prepared in the same manner as in Preparation Example 1 in anhydrous tetrahydrofuran, and the mixture was stirred at room temperature under a nitrogen atmosphere for 3 hours. The volatile components were distilled off under reduced pressure, and the solid residue was washed with a 0.05 M aqueous sodium hydroxide solution, a 0.05 M aqueous hydrochloric acid solution, and subsequently with water. This solid was purified by silica gel column chromatography using a mixed solution of chloroform and tetrahydrofuran as a developing solvent to obtain 0.10 g (0.14 mmol, 20%) of pure compound 9: white powder, mp 165-167 ° C; IR (KBr) ν3297,1667,16
^{51,1586cm -1; 2 HNMR (THF-} d8) δ = 0.90 (t, 3H, J = 7H Z), 1.33
(m, 2H), 1.66 (m, 2H), 2.22 (m, 2H), 2.35 (t, 2H, J = 7H _Z ), 2.50
(t, 2H, J = 7H Z), 3.41 (t, 2H, J = 8H Z), 7.53-7.71 (m, 3H), 7.84
-8.13 (m, 14H), 8.38 (d, 2H, J = 9H Z), 8.47 (s, 1H), 9.11 (s, 1
H), 9.21 (s, 1H), 9.28 (s, 1H), 9.51 (s, 1H). Calculated value (C ₄₃ H
₃₈ N ₆ O ₄ ): C, 73.49; H, 5.45; N, 11.96
%. Found: C, 73.55; H, 5.50; N, 11.87%.

<< Application Example 5 >> Preparation Example 1 using Compound 9
The response to a molecule having ureido binding was examined by the test method described in (1). The results are shown in Table 5.

From Table 5, it is clear that Compound 9 changes the fluorescence intensity greatly in response to the target molecule sharply and is an excellent analytical fluorescent probe.

[0078]

[Table 5]

<< Preparation Example 3 >> A fluorescent compound of the present invention suitable for analysis of a molecule having a carboxyl group was prepared according to the flowchart of FIG.

Preparation of compound 10 : The compound 3 was prepared in the same manner as in the preparation of compound 3 except that terephthaloyl dichloride was used instead of isophthaloyl dichloride in the preparation method of compound 3 shown in Preparation Example 1. 10 3.
60 g (95% yield) were obtained: white powder, ¹ H NMR (THF-d
8) δ = 5.07 (s, 4H), 6.20 (d, 2H, J = 8H Z), 7.38 (t, 2H, J = 8H Z),
7.62 (d, 2H, J = 8HZ), 8.04 (s, 4H), 9.30 (s, 2H).

Preparation of Compound 11 0.40 g (1.1 mmol) of Compound 10 and Compound 5
Using 0.96 g (3.1 mmol), a crude product of compound 11 was obtained according to the synthesis method of compound 6. This was recrystallized from tetrahydrofuran to give pure compound 11
0.65 g (0.73 mmol, 67%) was obtained: white powder, IR (KBr) ν3306,1667,1586 cm ⁻¹ ; ¹ HNMR (DMF = d7) δ =
2.26 (m, 4H), 2.75 (t, 4H, J = 7H Z), 3.51 (t, 4H, J = 8H Z), 7.90
(t, 2H, J = 8H Z), 8.01-8.33 (m, 24H), 8.52 (d, 2H, J = 9H Z), 10.
43 (s, 2H), 10.65 (s, 2H), ( as _{C 58 H 44 N 6 O 4} ) Calculated: C, 78.36; H, 4.99 ; N, 9.45%. Measured value: C, 7
8.54; H, 5.06; N, 9.36%.

<< Application Example 6 >> 1 ml of chloroform and 3 ml of cyclohexane were placed in a 5 ml volumetric flask, and 10 μl of a 1 mM solution of compound 11 in N, N-dimethylformamide was injected. 10 μl of methanol solution containing various carboxyl-containing molecules in this solution
And diluted to exactly 5 ml with cyclohexane.
The fluorescence of these solutions was measured under the same conditions as described in Application Example 1. However, excimer fluorescence (Iex) is 500
Measured in nm. Table 6 shows the results.

As is clear from Table 6, the compound 11
It changes fluorescence in response to a molecule having a carboxyl group and functions as an excellent fluorescent probe for analysis.

[0084]

[Table 6]

[Brief description of the drawings]

FIG. 1 shows a reaction scheme for synthesizing preferred examples of the fluorescent compound of the present invention.

FIG. 2 shows the fluorescence mechanism of the fluorescent compound of the present invention.

FIG. 3 shows a change in fluorescence spectrum when the compound of the present invention captures barbital and ethylene urea.

FIG. 4 is a graph showing the relationship between pyrene monomer fluorescence intensity and barbital concentration when the fluorescent compound of the present invention is used.

FIG. 5 is a graph showing the relationship between pyrene excimer fluorescence intensity and ethylene urea concentration when the fluorescent compound of the present invention is used.

FIG. 6 shows a reaction scheme for synthesizing another preferred example of the fluorescent compound of the present invention.

FIG. 7 shows a reaction scheme for synthesizing still another preferred example of the fluorescent compound of the present invention.

Continuation of front page (56) References Am. Chem. Soc. , Vol. 112, p. 9408-9410 (1990) (58) Fields investigated (Int. Cl. ⁷ , DB name) C07D 213/00-213/75 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. A fluorescent compound represented by the following general formula [1]. Embedded image (However, in the formula, _two or more of X ₁ , X ₂ or X ₃ are an atomic group in which the same fluorescent molecule is bonded, and the following [Chemical Formula 2], [Chemical Formula 3], [Chemical Formula 4] , [Chem. 5], [Chem. 6],
Among X ₁ , X ₂ and X ₃ , those which do not bind to a fluorescent molecule are atoms or atomic groups which do not impair the fluorescence of the compound, and are selected from [Chemical formula 7] and [Chemical formula 8].
, Alkyl ether, alkyl ester, alkyl ester
And a hydrogen atom . ) Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image (Where Y is an atomic group connecting the (pyrid-2-yl) aminocarbonyl group of [Chemical Formula 1] and the fluorescent molecule,
It is selected from chain hydrocarbons which may contain an ether group, an ester group, a carbonyl group or an amide group. ) 2. A fluorescent probe for analyzing a molecule having a ureide bond, a molecule having a carboxyl group, or a salt of the molecule, comprising the fluorescent compound according to claim 1.