JP3249308B2 - Label dyes and their precursors, methods for their synthesis, methods for using label dyes, and cocaine or methamphetamine detection devices using label dyes - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to merocyanine or methamphetamine derivative-labeled dyes useful for immunoassays and other biochemical experiments, precursor compounds thereof, methods for synthesizing them, and methods for using the dyes. The present invention relates to a detection device for detecting cocaine and methamphetamine by an immunoassay.

[0002]

2. Description of the Related Art First, as an example of the prior art, a case where methamphetamine (MA) is to be detected will be described. As a labeling dye in which MA and a fluorescent substance are chemically bonded, a compound in which a dansyl group is bonded to MA has been proposed as described in Japanese Patent Application Laid-Open No. 3-223673. 330n of dansylated MA (hereinafter DNS-MA)
It is known that fluorescence of 525 nm is emitted by excitation of m, and the fluorescence intensity is changed by binding to an anti-MA antibody. When DNS-MA is bound to an anti-MA antibody in advance, and MA is added thereto, DNS-MA is detached from the antibody as MA binds to the antibody, and the fluorescence intensity at 525 nm changes. By measuring the change in intensity, MA can be detected with high sensitivity.

[0003]

However, there is no precedent for detecting cocaine (CC), and the conventional technique cannot detect CC. Therefore, C
In order to detect C, development of a labeled dye that can be detected by an immunoassay has been required. In addition, since many substances having fluorescence near 525 nm exist naturally, when these impurities are mixed, the above-described measurement method using DNS-MA requires MA
Was difficult to detect.

[0004] In order to solve the above-mentioned problems, the present invention provides a labeled dye capable of detecting CC or methamphetamine contained in a substance to be measured, a precursor compound thereof, a method for synthesizing the same, and a method for using the dye. With the goal. It is another object of the present invention to provide a cocaine / methamphetamine detection device using a labeling dye.

[0005]

In order to achieve the above object, the labeling dye of the present invention comprises a merocyanine derivative represented by the above formula (Formula 1).

Next, the propyl halide derivative of the present invention is a precursor of a labeled dye comprising the merocyanine derivative represented by the above formula (Formula 1), and is represented by the above formula (Formula 2). Next, the first method for producing a labeled dye of the present invention comprises dissolving a propyl halide derivative represented by the above formula (Formula 2) in an organic solvent, and adding norcocaine represented by the above formula (Formula 3) thereto. It has a configuration of heating.

[0007] manufacturing method of the dye precursors of the present invention then, under
Trimethyl indolenium salt represented by the serial formula (Formula 4) and
And dimethylaminobenzaldehyde represented by the following formula (Formula 5) was dissolved in an organic solvent, those having a structure that synthesize propyl halide derivative represented by the above formula (Formula 2) by heating.

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[0008] The second labeling dye of the present invention comprises a methamphetamine derivative represented by the above formula (Formula 6).
Next, the methanesulfonic acid derivative of the present invention is represented by the above formula (Formula 7) and is a precursor of the second labeled dye of the present invention.

[0009] Next hydroxy derivatives of the present invention is represented by the following formula (Formula 8), which is a precursor of a second labeling dye of the present invention. Then acetyl derivative of the present invention is represented by the following formula (Formula 9), which is a precursor of a second labeling dye of the present invention.

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[0010] The following method for producing a second labeling dye of the present invention causes a substitution reaction of methane sulfonic acid derivative and methamphetamine represented by the Formula (7) in an acidic solvent, wherein
A methamphetamine derivative represented by the formula (Formula 6) is synthesized.
It has a configuration of

[0011] Next, the method of using the dye of the present invention is to determine the fluorescence intensity of the dye-derived fluorescence emitted from a buffer solution containing a labeled dye comprising a merocyanine derivative represented by the above formula (Formula 1) and an anti-cocaine antibody, or Intensity change between the fluorescence intensity of the fluorescence derived from the dye emitted from the buffer solution containing the labeled dye represented by the above formula (Formula 6) and the anti-methamphetamine antibody, and the fluorescence intensity when the substance to be measured is added to the buffer solution Is measured, and cocaine or methamphetamine contained in the substance to be measured is detected.

Next, the cocaine / methamphetamine detection apparatus of the present invention reacts an anti-methamphetamine antibody or an anti-cocaine antibody with a sample solution obtained by dissolving a sample in a dye solution containing an antigen-labeled dye, and changes the fluorescence intensity of the sample solution A detector for measuring the presence or absence or the concentration of methamphetamine, cocaine or a derivative thereof in a sample liquid, wherein the labeling dye is a compound of the formula (1) or (6)
A container for storing at least one dye selected from the labeling dyes shown above, and an antibody solution containing the dye solution and the antibody, an elution device for eluting the sample, and the sample solution, A reaction container for reacting the antibody solution and the antibody solution, supply means for supplying the antibody solution and the sample solution to the reaction container, and a fluorescence detector for detecting the fluorescence intensity of the liquid to be measured in the reaction container And at least the following.

In the above construction, the supply means is preferably a syringe pump and a valve. Further, in the above configuration, it is preferable that the reaction vessel is a rectangular cell having an inlet and an air vent hole at an upper portion and a discharge nozzle at a lower portion.

Further, in the above configuration, the inside of the reaction vessel is
It is preferable to provide a stirring unit driven by a magnetic force for stirring the sample solution, the dye solution, and the antibody solution. Further, in the above configuration, it is preferable to provide a constant temperature device using a Peltier element in the reaction vessel.

[0015] In the above structure, it is preferable that the storage device and the supply means are provided at least inside the air-conditioning type constant temperature device.

[0016]

The labeling dye of the present invention represented by the above formula (Chemical Formula 1) (hereinafter abbreviated as CCIMC) is used in the long wavelength region (59).
(Around 0 nm). And CCIMC is anti-CC
Has affinity for the antibody, but its value is CC
Not as high as in the case. Therefore, in the absence of CC, CCIMC binds to the anti-CC antibody and changes in fluorescence intensity, but when CC is added thereto, CCIMC bound to the antibody replaces CC with high affinity, The fluorescence intensity approaches the initial fluorescence intensity in accordance with the amount thus obtained.
CC can be detected by measuring the change in the fluorescence intensity. Therefore, the dye consisting of CCIMC of the present invention is
It is possible to detect CC with high sensitivity.

Further, the propyl halide derivative represented by the chemical formula (Chemical Formula 2) of the present invention has an excellent halogen as a leaving group, so that the first dye of the present invention, CCIMC, is synthesized. Can provide useful precursors (intermediates).

Further, according to the first method for producing a labeled dye of the present invention, a propyl halide derivative represented by the above formula (Chemical Formula 2) is dissolved in an organic solvent, and the solution is dissolved in the organic solvent. Can be synthesized by adding norcocaine and heating, thereby providing a novel method for synthesizing CCIMC.

Further, according to the method for producing a dye precursor of the present invention, the trimethylindolenium salt represented by the above formula (Formula 4) and the dimethylaminobenzaldehyde represented by the above formula (Formula 5) are dissolved in an organic solvent. Then, by heating, the propyl halide derivative represented by the above formula (Formula 2) can be synthesized, and a novel method for synthesizing the propyl halide derivative can be provided.

The labeled dye of the present invention represented by the above formula (Formula 6) (hereinafter abbreviated as MAIMC) has fluorescence in a long wavelength region (around 590 nm). And although MAIMC has affinity for anti-MA antibodies, its value is not as high as in MA. Therefore, in the absence of MA, MAIMC binds to the anti-MA antibody and changes in fluorescence intensity. However, when MA is added thereto, MAIMC bound to the antibody replaces MA with high affinity, The fluorescence intensity approaches the initial fluorescence intensity in accordance with the amount thus obtained. MA can be detected by measuring the change in the fluorescence intensity.
Absorbs light centered on a wavelength of 0 nm and produces a long wavelength region (590 n
m) Since it is a dye having fluorescence in the ultraviolet region, it is hardly affected by impurities having absorption in the ultraviolet region and light scattering by a remarkable insoluble component in the ultraviolet region, so that it is easy to detect MA with high sensitivity. A dye can be provided.

Further, the methanesulfonyl derivative represented by the above formula (Formula 7) has a methanesulfonyl group which is excellent as a leaving group.
A precursor (intermediate) useful for synthesizing AIMC can be provided.

Further, the hydroxy derivative represented by the above formula (Formula 8) has a hydroxyl group and is therefore highly reactive with acid chlorides. Therefore, it reacts quickly with methanesulfonic anhydride to synthesize the methanesulfonyl derivative.

Further, the acetyl derivative represented by the above formula (Formula 9) can synthesize a dye skeleton at a high yield in order to remove the inhibitory effect of the reaction due to the hydroxyl group. That is, the reaction of N-hydroxypropyl-2,3,3-trimethylindolenine with pN, N-dimethylbenzaldehyde seems to be the shortest route to synthesize the hydroxy derivative. N-hydroxypropyl
Inhibited by the hydroxyl group of -2,3,3-trimethylindolenine and practically does not progress. Therefore, a dye skeleton, that is, an acetyl derivative represented by the above formula (Chem. 9) is synthesized while protecting the hydroxyl group on trimethylindolenin with an acetyl group, and then the acetyl group is removed by hydrolysis. Can be synthesized in yield.

According to the second method for producing a labeled dye of the present invention, the compound can be synthesized by subjecting a methanesulfonic acid derivative represented by the above formula (Formula 7) to a methamphetamine substitution reaction in an acidic solvent. , MAIMC can be provided.

The method of using the labeling dye of the present invention is as follows.
Buffer solution containing CIMC and anti-CC antibody, or MAIM
It is possible to detect cocaine or methamphetamine with high sensitivity by measuring the intensity change between the fluorescence intensity of a buffer solution containing C and an anti-methamphetamine antibody and the fluorescence intensity when a substance to be measured is added to the buffer solution. It becomes possible.
That is, since the labeling dye of the present invention is a labeling dye having fluorescence in a long wavelength region, it is possible to provide a detection method which is hardly affected by impurities and can detect CC with high sensitivity.
By using a mixture of CIMC and MAIMC, it is possible to detect a drug contained in the liquid to be measured, and at the same time, to specify whether the detected drug is CC or MA.

Next, the apparatus for detecting MA, CC or a derivative thereof according to the present invention provides an anti-MA antibody or an anti-CC antibody to a sample solution obtained by dissolving a sample in a dye solution containing an antigen-labeled dye.
MA, CC or a derivative thereof for measuring the presence or absence or the concentration of MA, CC or a derivative thereof in a sample solution from a change in the fluorescence intensity of the sample solution by reacting an antibody, wherein the labeled dye is At least one selected from the labeling dyes represented by the formulas (1) and (6).
Containers for storing the dye solution and the antibody solution containing the dye solution, an elution device for eluting the sample, and a reaction container for reacting the sample solution and the antibody solution. Supply means for supplying the antibody solution and the sample solution to the reaction vessel,
By providing at least a fluorescence detector for detecting the fluorescence intensity of the liquid to be measured in the reaction container, a detection device capable of quickly detecting MA, CC or a derivative thereof in a sample liquid is achieved. it can.

According to a preferred embodiment of the present invention in which the supply means is a syringe pump and a valve, the liquid can be easily supplied from the storage container to the reaction container, and can be easily detected.

According to a preferred embodiment of the present invention, wherein the reaction vessel is a square cell having an inlet and an air vent hole at an upper portion and a discharge nozzle at a lower portion, the supply means and the discharge nozzle are provided. By controlling the opening and closing, it is possible to easily measure continuously different samples.

Further, according to a preferred embodiment of the present invention in which the reaction vessel is provided with a stirring means driven by magnetic force for stirring the sample liquid, the dye liquid and the antibody liquid, the sample liquid can be reacted quickly. And the time required for processing is reduced.

According to a preferred embodiment of the present invention in which the reaction vessel is provided with a thermostat using a Peltier element, detection can be performed under a constant temperature environment. Further, according to a preferred embodiment of the present invention in which the storage device and the supply means are provided at least inside the air-conditioning type constant temperature device, it becomes possible to detect CC or the like under any temperature environment.

[0031]

Embodiments of the present invention will be described below with reference to the drawings. The trimethylindolenium salt represented by the chemical formula (Formula 4) is, for example, chloroiodopropane and about
Mixing with 0.5 to 2 molar ratio of trimethylindolenine,
It can be obtained by heating at about 60-100 ° C. for about 1-5 hours.

The propyl halide derivative represented by the chemical formula (Chemical Formula 2) may be, for example, a solution of a trimethylindolenium salt represented by the chemical formula (Chemical Formula 4) in an organic solvent,
For example, an equimolar amount of dimethylaminobenzaldehyde represented by the above formula (Formula 3) is added to an ethanol solution,
By heating to 60 to 100 ° C., dimethylaminobenzaldehyde serves as a base, and can be obtained in a short time without adding pyridine, which was conventionally required as a base.

Further, CC represented by the chemical formula (Formula 1)
For example, IMC is prepared by adding a 2 to 10-fold molar amount of norcocaine to a solution of an iodopropyl derivative represented by the above chemical formula (Formula 2) in an organic solvent, for example, a chloroform solution, and adding about 60 to
It can be obtained by heating at 〜100 ° C. for about 5 to 12 hours.

The acetyl derivative represented by the above formula (Chemical formula 9) is equimolar to a solution of an acetyl-substituted trimethylindolenium salt represented by the formula (Chemical formula 4) in a basic organic solvent such as a pyridine solution. Of dimethylaminobenzaldehyde and heating to about 110 to 150 ° C. in a short time.

The hydroxy derivative represented by the above formula (Chemical formula 8) can be obtained by dissolving, for example, an acetyl derivative represented by the above formula (Chemical formula 9) in an organic solvent such as methanol, and a several-fold amount of an alkali such as sodium hydroxide. The reaction is terminated by mixing the aqueous solutions of the above and stirring at a normal temperature and a normal pressure for 1 to 2 hours. The resulting mixture is obtained by adjusting the pH and removing the solvent.

The methanesulfonic acid derivative represented by the above formula (Chemical formula 7) can be prepared by adding a methanesulfone derivative having a molar ratio of 2 to 20 to an organic solvent solution of the hydroxy derivative represented by the above formula (Chemical formula 8), for example, a chloroform solution. It can be obtained by adding an acid anhydride and stirring at room temperature or a low temperature of about 4 ° C. under normal pressure for 1 to 10 days.

The dye represented by the above general formula (Chemical formula 6) is prepared by adding a 2 to 20 molar ratio of MA to an acetic acid solution of a methanesulfonic acid derivative represented by the above general formula (Chemical formula 7),
It can be obtained by heating at 80 ° C. for several hours.

FIG. 1 is a block diagram showing the configuration of the detection device of the present invention. In FIG. 1, all components are placed in an air-conditioned room 17 provided with an air-conditioned unit 18.
It is kept at ° C. The containers 1, 2, and 3 store an eluate, an antibody solution, and a dye solution, respectively. The container 1 is connected with a valve 4 and a pump 7, the container 2 is connected with a valve 5 and a pump 8, and the container 3 is connected with a valve 6 and a pump 9, respectively. The container 1 is connected to a sample elution device 21 via a valve 4 and a valve 22. A liquid reservoir 23 is provided below the sample elution device 21. The liquid reservoir 23 is connected to the reaction vessel 11 in the fluorescence detector 14 through the switching valve 10.
It is connected to. In the reaction vessel 11, a magnetic stirrer bar 12 and a magnetic stirrer 13 are provided as stirring means. The reaction vessel 11 is maintained at 25 ° C. by a Peltier device 19. The reaction device 11, the sample elution device 21 and the liquid reservoir 23 are connected to the waste liquid reservoir 16 via a waste liquid valve 24 and a waste liquid pump 15, respectively.

The reaction vessel 11 is a so-called square cell having a rectangular cross section as shown in FIG. 2, and a switching valve 10 and a connection pipe 11c from the valve 5 and an air vent hole 11d are provided in an upper portion 11a. I have. In general, the shape of the reaction vessel is not limited to one having a rectangular cross section, and any shape other than a rectangular cross section such as a circular cross section can be used. However, the use of a material having a shape other than a rectangular cross section is not suitable because a light scattering phenomenon occurs when measuring the fluorescence intensity. In addition, when a liquid such as a sample liquid is injected, it is preferable to provide an air vent hole inside the reaction vessel 11 so that back pressure is not applied in order to accurately control the injection amount.

FIG. 3A is a front view showing a cell holder on which the Peltier element 19 of the fluorescence detector 14 of the detection apparatus according to the present embodiment is mounted, and FIG. 3B is a side view thereof. FIG.
In (a), 11c is a liquid inlet, 11b is a waste liquid port,
32 is an aluminum fin. In FIG. 3B, the rectangular reactor 11 is fixed to one surface of an aluminum block 31, and a thermostat 33 provided with a Peltier element 19 is provided near the reactor. The Peltier device 19 is fixed to the other surface of the block 31. further,
Aluminum fins 3 are provided on the surface opposite to the Peltier element 19.
2 are installed. Thus, the Peltier device 19 is connected to the reactor 11 via the block 31 having a large heat capacity.
By mounting the above, a sudden change in temperature when a sample solution or the like is injected into the reaction vessel 11 or when the Peltier element 19 generates heat is reduced. Further, since the entire apparatus is provided inside the air-conditioning type constant temperature chamber 17, stable detection is performed regardless of temperature conditions such as a cold area or a warm area.

As shown in FIG. 4, the sample elution device 21
Piston 41, O-ring 42, eluate inlet / outlet 4
3 and a filter mounting portion 44. In addition, the computer 7, the pumps 7, 8, 9 and the valves 4, 5,
6, 10, 22, 24 and magnetic stirrer 1
3 is controlled, the data from the fluorescence detector 14 is fetched, and the data is processed. Therefore, a detection operation can be easily performed even by a person unfamiliar with the handling of the apparatus without special knowledge.

Since the method for detecting CC or MA using the labeled dye of the present invention measures the change in the fluorescence intensity of CCIMC or MAIMC, the wavelength of the fluorescence to be measured is preferably 550 to 640 nm, which is strong. Preferred, especially 590n
The fluorescence of m is preferred because it is the strongest. In addition, as described above, the measurement using such a long wavelength has little effect even if impurities are mixed, and has a high sensitivity and a high sensitivity.
Alternatively, it is preferable because MA can be detected. Also, the excitation light
The use of 550 nm is optimal in terms of fluorescence intensity, but the excitation wavelength far from the half-plane fluorescence wavelength is superior in terms of stray light. Therefore, in practice, it is preferable to excite at around 530 nm.

Hereinafter, the present invention will be described in more detail with reference to specific examples. In the following experiments, all the measurements of liquid chromatography (hereinafter, HPLC) were performed under the following conditions.

Apparatus: Nippon Millipore Limited, 600 system Detector: Otsuka Electronics, MCPD3600 Column: Nippon Millipore Limited, C18 (15μ, 100
0A, 7.9 mm × 30 cm) Solvent: methanol / hydrochloric acid (0.01 N) = 3/2 Flow rate: 2.0 ml / min Quantification: Quantification by area of absorption peak at 550 nm wavelength (Example 1) (Synthesis of trimethylindolenium salt ) Trimethylindolenine (10 g, MW = 159.2, 0.063 mol) and chloroiodopropane (50 g, MW = 204.4, 0.24 mol) were mixed using 30 ml of benzene, and heated at 80 ° C. for 6 hours under a nitrogen stream. Benzene evaporated during the reaction time. The reaction was cooled and washed repeatedly with diethyl ether to give a pale red powder. The product was identified by NMR as 1- (3-iodopropyl) -2,3,3-trimethylindolenium chloride (MW = 363.5). The yield was 20 g (87%).

Table 1 shows the chemical shifts of ¹ H-NMR in deuterated chloroform and the assignment of each peak.

[0046]

[Table 1]

Example 2 (Synthesis of Iodopropyl Derivative) 4.4 g (12.10 mmol) of trimethylindolenium salt was dissolved in 200 ml of ethanol, and 1.81 g (12.10 mmol) of dimethylaminobenzaldehyde was added thereto. Refluxed for 2 hours. The reaction was cooled to room temperature, and the precipitated solid was filtered out and washed with ether. Recrystallized from ethanol, 6.3g (MW = 49
4.7, 100%).

Table 2 shows chemical shifts of ¹ H-NMR in deuterated chloroform and assignment of each peak.

[0049]

[Table 2]

(Example 3) (Synthesis of norcocaine) 1 g (3.29 mmol) of cocaine was dissolved in a mixed solvent of 55 ml of acetonitrile and 110 ml of water, and 1 ml of acetic acid was added thereto to obtain a weakly acidic solution. .
To this solution, 42 ml of an aqueous solution of 1.06 g (7.55 mmol) of potassium permanganate was added dropwise over 8 hours. After the addition, the mixture was stirred at room temperature overnight. The resulting precipitate was removed by filtration,
Potassium carbonate was added to the filtrate to make a weakly basic solution. This solution was extracted three times with diethyl ether, and anhydrous sodium sulfate was added to the organic layer, followed by drying. After ether was distilled off under reduced pressure, silica gel thin layer chromatography (developing solvent:
Purified using ammonia-saturated chloroform), 536 mg
(MW = 289.4, 56%) norcocaine was obtained.

Table 3 shows the chemical shifts of ¹ H-NMR in deuterated chloroform and the assignment of each peak.

[0052]

[Table 3]

Example 4 (Synthesis of CCIMC) 100 mg (0.20 mmol) of an iodopropyl derivative and 292 mg (1.01 mmol) of norcocaine were mixed for 30 m.
The mixture was dissolved in 1 l of chloroform and refluxed for 8 hours under a nitrogen stream. After cooling the reaction solution to room temperature, it was added to about 1000 ml of ether with stirring. The resulting solid was filtered, washed with ether and 125 mg (MW = 655.5, 94%) of C
CIMC was obtained.

Table 4 shows the chemical shifts of ¹ H-NMR in deuterated chloroform and the assignment of each peak.

[0055]

[Table 4]

(Example 5) (Measurement of change in fluorescence intensity of CCIMC) 1487. CCIMC (final concentration 7.09 × 10 ⁻⁶ M) solution in PBS (phosphate buffer).
Take 5 μl, stir at 25 ° C for 1 minute, and excite at 530 nm
Was used to measure the fluorescence intensity at 590 nm. Its fluorescence intensity was 110.4. To this solution, 114.3 μl of an anti-CC antibody adjusted to a final concentration of 1 × 10 ⁻⁶ M was added, and the mixture was stirred at 25 ° C. for 1 minute. After measuring the fluorescence intensity under the same conditions as above, the fluorescence intensity was increased to 165.5.

Next, 150 μl of a CC aqueous solution having a final concentration of 1.9 × 10 ⁻⁴ M was added to this solution, and the excitation wavelength was 530 nm.
Was used to measure the fluorescence intensity at 590 nm. The fluorescence intensity was 113.9. That is, it is understood that the fluorescence enhancement due to the binding of the antibody and CCIMC was almost completely inhibited by the addition of CC.

When the final concentration of CC is 7.71 × 10 ⁻⁵ M, 1.94 × 10
^-5 M, 7.84 × 10 ^-6 M, 1.97 × 10 ^-6 M, 7.96 × 10 ^-7 M, 1.99 ×
For 10 ^-7 M, 7.93 × 10 ^-8 M, 1.84 × 10 ^-8 M, and 6.21 × 10 ^-9 M, the fluorescence intensity was measured in the same manner as described above. The respective fluorescence intensities were 116.6 (7.71 × 10 ⁻⁵ M), 12
5.1 (1.94 × 10 ^-5 M), 135.2 (7.84 × 10 ^-6 M), 149.8 (1.97 ×
10 ^-6 M), 156.5 (7.96 x 10 ^-7 M), 161.1 (1.99 x 10 ^-7 M),
162.8 (7.93 x 10 ^-8 M), 163.7 (1.84 x 10 ^-8 M), 165.1
(6.21 × 10 ⁻⁹ M).

FIG. 5 is a CC detection curve obtained in one embodiment of the present invention. In this embodiment, such a characteristic curve could be obtained. (Example 6) (Synthesis of acetyl derivative) Synthesis of propyl 3-bromoacetate (3-Bromopropylacetate, BPA) 102.520 g of bromopropanol (BPO) (MW = 139.00, 0.737)
Acetic anhydride (Acetic unhydrid)
e, AcU) 91.364 g (MW = 102.99, 0.8871 mole) of benzene 5
0 ml of the solution was gradually added to avoid heat generation, and the mixture was stirred at room temperature overnight. Gas-liquid chromatography (GLC) confirmed that all the bromopropanol had reacted. The reaction solution was washed five times with pure water to hydrolyze unreacted acetic anhydride and remove most of acetic acid.
After drying the benzene layer with magnesium sulfate all day and night,
Benzene was evaporated. Distill to 66mmHg, 11
A fraction at 3-115 ° C was collected. Alkylation of 2,3,3-trimethylindolenine (TMI) 43.9 g of TMI (MW = 159.23, 0.2758 mole) and 50 g of BPA (MW = 181.
3, 0.2758 mole) was mixed using 30 ml of benzene and heated at 130 ° C. for 6 hours under a nitrogen stream. Benzene evaporated during the reaction time. Upon cooling the reaction was tar-like. The reaction product was repeatedly washed with diethyl ether to obtain a pale red powder. The product was analyzed by NMR for 1-
It was identified as (3-acetylpropyl) -2,3,3-trimethylindolenium bromide (APTMI, MW = 340.26). The yield was 81 g (0.2380 mole, 86.3%). Here, APTMI seems to have a strong bond with unreacted TMI, and it is difficult to purify the tar-like reactant. When handling in large quantities, small quantities are transferred to a mortar and refined by grinding in ether. Further, in a state where the crude purification has been completed and the solid has been formed, the solid is dispersed in ether in a flask and subjected to ultrasonic waves for 0.5 to 2 hours for purification. The purity at this point is sufficient for synthesis. For further purification, such as for analysis, it was carried out by dissolving in a small amount of acetone and diluting it with benzene. Synthesis of acetyl derivative APTMI (9.9906 g, MW = 340.263, 0.0294 mole) was dissolved in 75 ml of ethanol with gentle heating to obtain solution 1. 4.5994 g of p-dimethylaminobenzaldehyde (MW = 14
9.19, 0.0308 mole) was dissolved in 50 ml of ethanol with gentle heating to obtain solution 2. When solutions 1 and 2 were mixed, red-violet coloring was immediately observed at room temperature. After refluxing for 2 hours, the mixture was cooled at room temperature overnight to obtain a purple solid precipitate. This was filtered and washed with benzene. The solid component was 9.7609 g. What was obtained from the result of NMR could be identified as the acetyl derivative of the formula (Formula 9). The filtrate became a brown candy-like product when the solvent was distilled off. At this time, care must be taken because bumping easily occurs due to high viscosity. It is presumed that the cause of the substance which should be a solid in such a candy state is water, and the substance was solid when vacuum dried at 60 ° C. in the presence of diphosphorus pentoxide. Then, when dissolved in 50 ml of hot ethanol and allowed to stand at −20 ° C. overnight, purple crystals appeared. After filtration in the same manner as above, the resultant was washed with benzene. It turned out that the purity of the product can be easily increased by these conditions. The yield obtained by this operation was 4.16 g. The molecular weight of the acetyl derivative was 471.44, and the mass obtained by the two operations was 13.92 g in total, that is, 0.0295 mole, and the yield was 100%.

Table 5 shows ¹ H-NM in deuterated chloroform.
The chemical shift of R and the assignment of each peak are shown.

[0061]

[Table 5]

Example 7 (Synthesis of hydroxy derivative) Acetyl derivative 7.6582 g (1
6.24 mmole) was dissolved in 500 ml of methanol. While stirring the solution, 3 ml (30 mmole) of 10N sodium hydroxide was added, and the mixture was allowed to stand at room temperature. The solution, which was initially dark purple, almost decolorized after a few minutes. A check by liquid chromatography confirmed that about 25% of the acetyl derivatives were unreacted after 0.5 hour, but all acetyl derivatives were reacted after 1 hour. Total 2 just in case
After stirring for hours, 5.1019 g (84.96 mmole) of acetic acid was added. The decolorized solution immediately regained its original color.
The methanol and acetic acid were removed by evaporation. Chloroform was added to the almost dry product and undissolved sodium acetate was filtered. The filtrate was evaporated again and the remaining dark purple paste was ultrasonically washed with diethyl ether to give a purple solid. The final product was vacuum dried (60 ° C.) in the presence of diphosphorus pentoxide. The yield is 5.
It was 7413 g (MW = 429.5, 13.37 mmole, 82.3%).

Comparison of the infrared absorption spectra (KBr tablet method) of the acetyl derivative and the hydroxy derivative, which are the raw materials in this reaction, revealed that the peak derived from the acetyl group (1700, 1240 cm ⁻¹ ) observed in the acetyl derivative was completely obtained. Had disappeared. As a result, this reaction product was determined to be the target HPIMC.

Example 8 (Synthesis of methanesulfonic acid derivative) Hydroxy derivative
1.000 g (MW = 429.5, 2.33 mmole) and 2.5377 g of methanesulfonic anhydride (MW = 174.2, 14.57 mmole) were dissolved in 15 ml of chloroform and stirred at room temperature for 13 days. 3 with 3% saline
And the chloroform layer was dried over magnesium sulfate. Twenty days later, magnesium sulfate was filtered, and the solvent was distilled off, followed by washing with diethyl ether. Yield 0.6853 g (MW = 507.4
9, 1.35mmole, 58.0%). When the starting hydroxy derivative was compared with the product of this reaction by HPLC, the retention time of HPLC was about 11 minutes, and that of this reaction product was about 17 minutes. It was also found at the same time that the reaction product contained no starting material.

Example 9 (Synthesis of MAIMC) Methanesulfonic acid derivative 55.1 mg
(MW = 507.49, 0.109mmole) and 133.8mg of methamphetamine
(MW = 149.24, 0.897 mmole) was dissolved in 235.7 mg of acetic acid, and HPLC
The mixture was stirred at 80 ° C. while monitoring the progress of the reaction. After the reaction for 2 hours, the retention time of the methanesulfonic acid derivative as a raw material was about 17 minutes, and the retention time was 10 minutes at 11 minutes.
% Of the reaction product was found. After the reaction for 17 hours, it was confirmed that all the methanesulfonic acid derivatives were almost quantitatively changed to the product at the position of 11 minutes. From the structure of the compounds of Examples 1 to 3, the fact that this reaction proceeded almost without side reactions, and the general reaction route of methanesulfonic acid ester and amine, the present reaction product has the structure shown in Chemical formula 1. It was determined to be MAIMC. The product is under ultrasound
After washing with diethyl ether three times, the product was confirmed to be pure by HPLC. From the result of HPLC monitoring, it can be said that the reaction yield was almost 100%.

(Example 10) (Measurement of change in fluorescence intensity of MAIMC) MAIMC (8.6
1600 µl of a solution of × 10 ⁻⁶ M) of PBS (phosphate buffer) was stirred at 25 ° C. for 1 minute, and the fluorescence intensity at 590 nm at an excitation wavelength of 530 nm was measured. Its fluorescence intensity was 32.0. To this solution was added 100 μl of an anti-MA antibody adjusted to a final concentration of 6.1 × 10 ⁻⁶ M, and the mixture was stirred at 25 ° C. for 1 minute. The fluorescence intensity was measured under the same conditions as above, and the fluorescence intensity was increased to 54.8. Next, MA solution was added to this solution to give a final concentration of 2.3 × 10 ⁻⁵ M.
/ PBS solution (100 μl) was added, and the fluorescence intensity at 660 nm was measured at an excitation wavelength of 600 nm. The fluorescence intensity was 31.0. That is, it is understood that the enhancement of fluorescence due to the binding of the antibody and MAIMC was almost completely inhibited by the addition of MA.
In addition, the same experiment was performed, and when the same amount of PBS was added instead of the MA / PBS solution, the fluorescence intensity was 53.0, and no inhibition of the fluorescence enhancement was observed. That is, it can be said that MA was detected by the immunological method using the labeled dye MAIMC of the present invention.

Example 11 In this example, a mixed dye of a labeled dye represented by the above formula (Chem. 1) and a labeled dye represented by the above formula (Chem. 6) was used by using the detection apparatus shown in FIG. The change in the fluorescence intensity associated with the binding of the solution, the anti-MA antibody and the anti-CC antibody was examined. First, 1.25 ml of the dye solution was injected into the syringe pump for eluate 7 from the eluate solution container 1 through the eluate valve 4. This dye solution is composed of a 6 × 10 ⁻⁷ M (mol / l) aqueous solution of the dye represented by the formula (Chem. 1) and a 6 × 10 ⁻⁷ M aqueous solution of the dye represented by the formula (Chem. 6). The mixture adjusted by volume was used. After standing for a few seconds, the valve 22 for the sample elution device was switched to collect the eluate in the reservoir 23. At this time, the reason why the eluate is temporarily stored in the liquid reservoir 23 is to remove bubbles contained in the eluate. Thereafter, 1.25 ml of the eluate was aspirated into the syringe pump 9 for dye solution through the switching valve 10 and the valve 6 for dye solution. After that, the switching valve 10 is switched, and the angular cell 11 of the fluorescence detector 14 (manufactured by JASCO, FP-821) is supplied from the syringe pump 9 for the dye solution.
Was injected with 1.10 ml of eluate. The corner cell 11 could be injected quantitatively because the upper part was an open system and no back pressure was applied. While stirring the solution by rotating the magnetic stir bar 12, the excitation wavelength is 600 nm and the fluorescence wavelength is 6
The fluorescence intensity of the eluate was measured at 60 nm and the intensity was 6.
44. Thereafter, the antibody liquid is passed from the antibody liquid container 2 through the antibody liquid valve 5 into the syringe liquid pump 8 for 1 ×.
0.08 ml of 10 ^-5 M anti-MA antibody solution was aspirated. The antibody solution valve 5 is switched, and 1 × 10 ⁻⁵ M anti-MA antibody solution 0.08 ml is injected into the square cell 11 of the fluorescence detector 14 (FP-821 manufactured by JASCO Corporation) from the antibody solution syringe pump 8. did. When the fluorescence intensity was measured under the same conditions as above, the fluorescence intensity was increased to 6.55. The same measurement was performed several times while increasing the amount of the injected antibody.

After the completion of each measurement, the following washing operation was performed. The rotation of the magnetic stirrer bar 12 was stopped, the waste liquid pump 15 was operated, and the measurement liquid present in the square cell 11 was discarded into the waste liquid container 16 through the waste liquid port 11b shown in FIG. The dye solution 1.5 is supplied from the dye solution container 3 to the dye solution syringe type pump 9 through the dye solution valve 6.
ml was aspirated. The dye liquid valve 6 is switched, and the dye cell syringe type pump 9 is used to switch the square cell 11 of the fluorescence detector 14.
And the waste liquid pump 15 was operated again, and the dye liquid present in the square cell 11 was discarded into the waste liquid container 16. By repeating this operation two or three times, the inside of the square cell 11 and the inside of the tube were washed.

Next, the same measurement as described above was performed using a 1 × 10 ⁻⁵ M anti-CC antibody solution at an excitation wavelength of 530 nm and a fluorescence wavelength of 5 μm.
The measurement was performed at 90 nm, and the change in fluorescence intensity associated with the binding between the dye solution and the anti-CC antibody was measured. After completion of each measurement, the same washing as described above was performed.

Next, an anti-MA antibody and an anti-CC antibody were injected into the mixed dye solution by the same operation as described above, and an excitation wavelength of 600
After measuring the fluorescence intensity of the eluate at a wavelength of 660 nm and a fluorescence wavelength of 660 nm, a MA solution of a known concentration was temporarily injected into the eluate, and the change in the fluorescence intensity was measured.
After completion of each measurement, the same washing as described above was performed.

Further, the same measurement as described above was performed at the excitation wavelength 53.
0 nm, at a fluorescence wavelength of 590 nm, to which a CC solution of a known concentration was temporarily injected, and a change in fluorescence intensity was measured.
A characteristic curve was obtained. After completion of each measurement, the same washing as described above was performed.

In the actual work of detecting methamphetamine or the like, the collected filter is set in the filter mounting section 44 of the elution device 21, and the sample is eluted from the filter.
The presence or absence and the concentration of methamphetamine and the like in the eluate can be determined from the fluorescence intensity of the eluate with reference to the characteristic curve obtained in this example.

When excitation light of a plurality of wavelengths is irradiated, the emitted fluorescence is spectrally separated by a spectroscope built in the fluorescence detector, and then the fluorescence intensity is measured at the same time.

[0074]

As described above, the propyl halide derivative represented by the chemical formula (Formula 2) of the present invention easily reacts with dimethylaminobenzaldehyde to form a basic skeleton of a dye. Therefore, it is a useful precursor when synthesizing the CCIMC represented by the chemical formula (Formula 1). In addition, CCIMC comprising the merocyanine derivative represented by the chemical formula (Formula 1) can provide a labeled dye capable of detecting CC with high sensitivity. Further, the novel synthesis method of the propyl halide derivative and CCIMC of the present invention has made it possible to produce CCIMC which is a labeling dye.

Further, the acetyl derivative represented by the above formula (Chemical Formula 9) of the present invention can be easily deprotected in an alkaline aqueous solution, and is a useful precursor for synthesizing the hydroxy derivative of the chemical formula (Chemical Formula 8). Be a body. The hydroxy derivative represented by the above formula (Formula 8) can easily react with an acid anhydride and introduce an excellent leaving group in a high yield. Therefore, it is a useful precursor when synthesizing the methanesulfonic acid derivative represented by the above formula (Formula 7). Since the methanesulfonic acid derivative represented by the formula (Formula 7) has a methanesulfonyl group which is an excellent leaving group, it is a useful precursor for synthesizing the MAIMC represented by the formula (Formula 6). Be a body.

The MAIMC of the present invention represented by the above formula (Formula 6) is hardly affected by impurities, has high sensitivity,
A labeled dye capable of detecting A can be provided. In addition, the novel method for synthesizing MAIMC of the present invention has made it possible to produce MAIMC which is a labeling dye. In addition, according to the method of using the labeled dye of the present invention, since the immunoassay uses CCIMC or MAIMC, CC or MA can be detected with high sensitivity without being affected by impurities.

Next, the apparatus for detecting MA, CC or a derivative thereof according to the present invention provides an anti-MA antibody or an anti-CC antibody to a sample solution obtained by dissolving a sample in a dye solution containing an antigen-labeled dye.
MA, CC or a derivative thereof for measuring the presence or absence or the concentration of MA, CC or a derivative thereof in a sample solution from a change in the fluorescence intensity of the sample solution by reacting an antibody, wherein the labeled dye is At least one selected from the labeling dyes represented by the formulas (1) and (6).
Containers for storing the dye solution and the antibody solution containing the dye solution, an elution device for eluting the sample, and a reaction container for reacting the sample solution and the antibody solution. Supply means for supplying the antibody solution and the sample solution to the reaction vessel,
By providing at least a fluorescence detector for detecting the fluorescence intensity of the liquid to be measured in the reaction container, a detection device capable of quickly detecting MA, CC or a derivative thereof in a sample liquid is achieved. it can. When the supply means is a syringe pump and a valve, the liquid can be easily supplied from the storage container to the reaction container, and can be easily detected. Further, when the reaction vessel is a rectangular cell having an inlet and an air vent hole at an upper part and a discharge nozzle at a lower part, the opening / closing of the supply means and the discharge nozzle is controlled, so that the reaction vessel is continuously different. Sample can be easily measured. Further, if a stirring means driven by magnetic force for stirring the sample liquid, the dye liquid and the antibody liquid is provided in the reaction vessel, the sample liquid can be reacted quickly, and the time required for the processing is reduced. If the reaction vessel is provided with a thermostat using a Peltier element, detection can be performed under a constant temperature environment. Further, if at least a storage device and a supply means are provided inside the air-conditioning type constant temperature device, it becomes possible to detect CC or the like under any temperature environment.

[Brief description of the drawings]

FIG. 1 is a block diagram showing a cocaine / methamphetamine detection device according to one embodiment of the present invention.

FIG. 2 is a perspective view of a reaction device mounted on a fluorescence detector of the detection device according to one embodiment of the present invention.

FIG. 3 (a) is a front view showing a configuration of a reaction apparatus equipped with a Peltier element of a fluorescence detector of a detection apparatus according to an embodiment of the present invention, and FIG. 3 (b) is a side view thereof.

FIG. 4 is a perspective view of a sample elution device of the detection device according to one embodiment of the present invention.

FIG. 5 is a CC detection curve diagram obtained by using the detection device according to one embodiment of the present invention.

[Explanation of symbols]

 1, 2, 3: Storage containers 4, 5, 6: Valves 7, 8, 9: Pump 10: Switching valve 11: Reaction container 11a: Upper part of the reactor 11b: Injection port 11c: Waste liquid port 12: Magnetic stir bar 13: Magnetic stirrer 14: Fluorescence detector 15: Waste liquid pump 16: Waste liquid reservoir 17: Air-conditioning type constant temperature chamber 18: Air-conditioning type constant temperature device 19: Peltier device 20: Computer 21: Sample elution device 22: Valve for sample elution device 23 : Liquid reservoir 24: Waste liquid valve 31: Aluminum block 32: Aluminum fin 33: Constant temperature device 41: Piston 42: O-ring 43: Injection / discharge port 44: Filter mounting part

──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Tadayasu Mitsumata 1006, Kazuma, Kadoma, Osaka Prefecture Inside Matsushita Electric Industrial Co., Ltd. (56) References JP-A-3-50558 (JP, A) JP-A-55 -88047 (JP, A) Ukr. Khim. Zh. , (1986), 52 (5), p. 509-13 (58) Field surveyed (Int. Cl. ⁷ , DB name) C07D 209/14 C07D 451/06 C09B 23/00 G01N 21/78 G01N 33/53 CA (STN) REGISTRY (STN)

Claims (11)

(57) [Claims] 1. A labeled dye comprising a merocyanine derivative represented by the following formula (Formula 1). Embedded image 2. A halogenated phosphor represented by the following formula (2):
The propyl derivative is dissolved in an organic solvent,
Norcocaine shown in 3) is added and heated, and the above formula is added.
A label for synthesizing a merocyanine derivative represented by (Chemical Formula 1)
Dye production method. Embedded image Embedded image 3. A methampheta represented by the following formula (6):
Labeled dye consisting of a min derivative. Embedded image 4. A methanesulfur represented by the following formula (7):
Displacement of sulfonic acid derivative and methamphetamine in acidic solvent
Methamphetamine represented by the above formula (Formula 6)
Derivative synthesis method. Embedded image 5. The method according to claim 1, wherein the labeled dye represented by the formula (1) is
Derived from dye emitted from buffer solution containing cocaine antibody
Or the label represented by the above formula (Formula 6)
Emitted from a buffer solution containing a dye and an anti-methamphetamine antibody
The fluorescence intensity of the dye derived from the dye
Measures the change in intensity from the fluorescence intensity when the target substance is added
And cocaine or methanephene contained in the substance to be measured.
How to use a dye to detect Tamine. 6. A dye containing an antigen-labeling dye.
Add the anti-methamphetamine antibody or
Reacts the anti-cocaine antibody and adjusts the fluorescence intensity of the sample solution.
From the change to methamphetamine and cocaine in the sample solution.
Or a method to determine the presence or concentration of
Detection device for tamfetamine, cocaine or their derivatives
Wherein the labeling dye is one of the formulas (1) and (2)
At least one selected from the labeling dyes represented by 6)
A dye, and an antibody containing the dye solution and the antibody.
A container for storing body fluids, and the sample
An elution device for elution, the sample solution and the antibody solution
A reaction vessel for reacting the antibody solution and the sump.
Supply means for supplying a liquid to the reaction vessel,
Fluorescence for detecting the fluorescence intensity of the liquid to be measured in the reaction vessel
And a methane detector at least comprising a detector.
An apparatus for detecting ethamine, cocaine or their derivatives. 7. The supply means is a syringe pump and a valve.
The detection device according to claim 6, wherein 8. The reaction vessel has an inlet and an air vent at the top.
Is a square cell with a hole and a discharge nozzle at the bottom.
The detection device according to claim 6. 9. A sample solution, a dye solution and
Stirring means driven by magnetic force for stirring the antibody liquid
The detection device according to claim 6, comprising: 10. A constant temperature using a Peltier device for a reaction vessel.
The detection device according to claim 6, further comprising a device. 11. An air-conditioning type constant temperature device for a storage device and a supply means.
7. The detection device according to claim 6, wherein the detection device is provided at least inside the device.
Place.