JP2006306752A - Chemiluminescent compound and marker comprising the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new chemiluminescent compound having high light emission efficiency and able to change wavelength of fluorescence by solely substituting a substituent, and a marker comprising the same. <P>SOLUTION: The chemiluminescent compound is expressed by general formula [I] wherein one of R<SP>1</SP>to R<SP>7</SP>expresses a chemiluminescent group such as a phthal carbazide group or the like and the others express each H or a group which does not inhibit luminescence of the compound; R<SP>8</SP>and R<SP>9</SP>each expresses F or an alkoxy. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a chemiluminescent compound and a labeling agent comprising the same.

  Chemiluminescent compounds are widely used as labeling agents in various fields including immunoassay. Many of them are a combination of a chemiluminescent substance having good performance such as a lucigenin derivative and a fluorescent dye such as rhodamine via a spacer (Patent Documents 1 to 13). However, these known chemiluminescent materials have problems such as unsatisfactory luminous efficiency, low stability, and difficulty in synthesis. In addition, with known labeling agents, it is difficult to change the fluorescence emission wavelength greatly only by changing the substituent. On the other hand, a fluorescent dye having a boron dipyrromethene skeleton is known (Patent Document 14). However, a chemiluminescent compound in which boron dipyrromethene fluorescent dye is bound to other chemiluminescent groups is not known.

International Publication WO98 / 54574 Japanese Unexamined Patent Publication No. 9-5238 Japanese Patent Laid-Open No. 10-81659 JP 2004-51490 A JP 2004-26665 A JP2003-183641 JP2003-137824 JP 2002-302483 A JP 2001-281257 A Japanese Patent Laid-Open No. 2001-115155 JP 2001-81455 A JP 2000-245499 A JP 2000-178550 A U.S. Pat.No. 5,723,218

  An object of the present invention is to provide a novel chemiluminescent compound having a high luminous efficiency and capable of changing the fluorescence emission wavelength greatly only by changing a substituent, and a labeling agent comprising the same.

  As a result of diligent research, the inventors of the present application have high luminous efficiency by combining a chemiluminescent group with a fluorescent dye having a boron dipyrromethene skeleton, and only changing the substituent bonded to the boron dipyrromethene skeleton. The present inventors have found that the fluorescence emission wavelength can be changed greatly and completed the present invention.

  That is, the present invention provides a chemiluminescent compound having a structure represented by the following general formula [I].

In the formula, R ¹ to R ⁷ , independently of each other, at least one of them is a chemiluminescent group, and the others are hydrogen or any group that does not inhibit light emission of the compound, R ⁸ and R ⁹ is independently a fluorine or alkoxy group.

  The present invention also provides a labeling agent comprising the chemiluminescent compound of the present invention. Furthermore, the present invention provides a combination of labeling agents comprising a plurality of types of the above-mentioned chemiluminescent compounds of the present invention having different emission wavelength adjusting groups from each other and thereby different emission wavelengths. Furthermore, the present invention provides a method for measuring a labeled substance, which comprises subjecting a substance labeled with the labeling agent of the present invention to a reaction, and measuring the substance by causing the labeling agent to emit light after the reaction. Furthermore, the present invention uses a plurality of chemiluminescent compounds of the present invention having different emission wavelength adjusting groups and thereby different emission wavelengths, as labeling agents, and a plurality of labels respectively labeled with the plurality of labeling agents. Provided is a method for simultaneously measuring a plurality of labeling substances, which comprises subjecting a kind of substance to a reaction and, after the reaction, causing each of the labeling agents to emit light and simultaneously measuring the plurality of types of the substance.

  INDUSTRIAL APPLICABILITY According to the present invention, a novel chemiluminescent compound that has high luminous efficiency and can greatly change the fluorescence emission wavelength simply by changing a substituent and a labeling agent comprising the same are provided. The boron dipyrromethene skeleton employed in the chemiluminescent compound of the present invention has high luminous efficiency.In particular, in a preferred embodiment of the present invention, the chemiluminescent group is directly bonded to the boron dipyrromethene skeleton without a spacer. Luminous efficiency can be increased. Further, in the chemiluminescent compound of the present invention, by changing the substituent on the boron dipyrromethene skeleton, preferably a single substituent, the fluorescence emission wavelength is greatly changed even if other structures are the same. Can do. Since introduction of an arbitrary substituent on the boron dipyrromethene skeleton can be easily performed by a conventional method, a plurality of chemiluminescent compounds having different fluorescence emission wavelengths can be easily synthesized according to the present invention. And since these may have the same structure other than the modified substituent, in particular the chemiluminescent group that chemiluminescents by the reaction, these chemiluminescent substances should emit light optimally under the same conditions. Can do. Therefore, by labeling a plurality of types of substances related to a single reaction system with different chemiluminescent compounds of the present invention, each of the plurality of types of substances can be measured simultaneously. Thereby, for example, in immunoassay and the like, it is possible to measure a plurality of different kinds of test substances at the same time. Therefore, the present invention provides a new measurement technique.

As described above, the chemiluminescent compound of the present invention has a structure represented by the above general formula [I]. In the general formula [I], R ¹ to R ⁷ are independently of each other, at least one of them is a chemiluminescent group, and the others are hydrogen or any group that does not inhibit the light emission of the compound, R ⁸ and R ⁹ are fluorine or an alkoxy group.

  The chemiluminescent group is a group having a structure that emits light by a chemical reaction. In the chemiluminescent compound of the present invention, the boron dipyrromethene moiety emits fluorescence by the light emitted from the chemiluminescent group, and this fluorescence is measured. Therefore, the chemiluminescent group does not necessarily need to emit visible light, and any one that emits light that can excite the boron dipyrromethene moiety to emit fluorescence can be used. Can do. As such a chemiluminescent group, any one can be used as long as it is used as a labeling agent in this field, and various ones are known or well known. Preferred examples include phthalcarbazide derivatives, dioxetane derivatives, lophine derivatives, acridine derivatives, indole derivatives, oxalic acid derivatives, diphenoyl derivatives or luciferin derivatives, all of which are well known in the art. These chemiluminescent groups are each represented by the following general formula.

In the above general formulas, R is independently of each other hydrogen, alkyl group, aryl group, halogen, alkoxy, arylalkyl, cycloalkyl, acyl, formyl, cyano, alkenyl, heteroaryl, mono- or di-alkyl. A substituent such as amino, and one R in each general formula is bonded to the boron dipyrromethene skeleton directly or via a spacer. In addition, carbon number of the alkyl group in the group containing an alkyl group or an alkyl group part is about 1-10 normally. In the present specification and claims, the term “alkyl group” includes both a linear alkyl group and a branched alkyl group. The same applies to the alkoxyl group. In the phthalcarbazide derivative described above, any of the four Rs is —NR ^a R ^b (wherein R ^a R ^b is independently hydrogen or an alkyl group) is a luminol derivative.

  Of the various chemiluminescent groups described above, a phthalhydrazide derivative that has a high emission quantum yield and can easily synthesize raw material phthalic acid that can be coupled to a boron dipyrromethene skeleton is preferable.

  In addition, although it is preferable to normally use one or a plurality of the above-mentioned chemiluminescent groups in each compound, a plurality of types may be combined. In addition, the chemiluminescent group can be bonded to the boron dipyrromethene skeleton through a spacer such as an alkylene group, but by bringing the chemiluminescent group as close as possible to the boron dipyrromethene skeleton, the fluorescence emitted from the boron dipyrromethene moiety can be reduced. In order to increase the strength, the chemiluminescent group is preferably bonded directly to the boron dipyrromethene skeleton without a spacer. By selecting the boron dipyrromethene skeleton as the fluorescent dye, the chemiluminescent group can be easily directly linked.

Since the boron dipyrromethene moiety emits fluorescence, the boron dipyrromethene skeleton emits fluorescence. Among R ¹ to R ⁷ , groups other than the chemiluminescent group are not particularly limited, and any one that does not inhibit the emission of hydrogen or a compound. It may be a group of

As one of the important features of the chemiluminescent compound of the present invention, by changing the substituent bonded to the boron dipyrromethene skeleton, the wavelength of fluorescence emission is greatly changed without changing other parts. There are things that can be done. In order to utilize this feature, it is preferable to have a light emission wavelength adjusting group as at least one of R ¹ to R ⁷ . Here, the “emission wavelength adjusting group” means a group in which the emission wavelength of fluorescence changes when this group is bonded as compared to the case where this group is not bonded. The emission wavelength adjusting group is a group in which the wavelength of fluorescence emitted from the boron dipyrromethene moiety is changed as compared with the fluorescence wavelength of a compound having no emission wavelength adjusting group by binding it to the boron dipyrromethene skeleton. The boron dipyrromethene skeleton is not particularly limited, and the fluorescence wavelength changes more or less by being affected by the substituent, and therefore the emission wavelength adjusting group may be an arbitrary group. However, it is preferable that the fluorescence wavelength changes relatively large by changing a single group, and it is particularly preferable that the fluorescence wavelength changes on the long wavelength side compared to the case where the emission wavelength adjusting group is not present. Preferred examples of the emission wavelength adjusting group include an alkyl group, an alkoxyl group, an aryl group, a heteroaryl group, and derivatives thereof. Here, as for carbon number of the alkyl part in an alkyl group or an alkoxyl group, 1-10 are preferable. The aryl group is preferably a phenyl group and a naphthalene group, and the heteroaryl group is preferably a group in which one or more carbon atoms in the phenyl group and naphthalene group are substituted with nitrogen, oxygen and / or sulfur. . In addition, as a derivative of an alkyl group or an alkoxyl group, a hydrogen atom bonded to one or a plurality of carbon atoms constituting these groups is halogen, cyano group, alkoxyl group, amino group, hydroxyl group, mono- or di- -The thing substituted by groups, such as an alkylamino group, a carbonyl group, a carboxyl group, a thiol group, a disulfide group, is preferable, As for carbon number of the alkyl part in these substituents, 1-10 are preferable, and 1-6 are further. preferable. A group in which one or more carbon atoms constituting the alkyl group or alkoxyl group are substituted with at least one of oxygen, sulfur and nitrogen is also preferable as the derivative of the alkyl group or alkoxyl group. In addition, as the derivative of the aryl group or heteroaryl group, a hydrogen atom bonded to one or more carbon atoms constituting these groups is halogen, cyano group, alkoxyl group, amino group, hydroxyl group, mono- Or the thing substituted by groups, such as a di-alkylamino group, a carbonyl group, a carboxyl group, a thiol group, and a disulfide group, is preferable, As for carbon number of the alkyl part in these substituents, 1-10 are preferable, 1-6 Is more preferable.

  As the emission wavelength adjusting group, a substituent such as benzocrown, N-arylazacrown, amide group, etc., whose electronic structure is changed by molecular recognition or chemical reaction and whose emission wavelength is changed can also be used.

Since the fluorescence wavelength of the boron dipyrromethene fluorescent dye is greatly influenced by the type of substituents bonded to R ¹ and R ⁷ in the general formula [I], the above emission wavelength adjusting group is R ¹ and / or R ⁷ is preferred. In addition, a plurality of emission wavelength adjusting groups can be bonded, and different types of emission wavelength adjusting groups can be bonded to one boron dipyrromethene skeleton. However, when multiple types of chemiluminescent labeling agents are used for simultaneous measurement of multiple types of test substances, which will be described in detail later, it is convenient that each labeling agent is subjected to a chemiluminescent reaction under the same conditions. Therefore, it is preferable that the chemiluminescent groups of the respective labeling agents are the same.

It is preferable that at least one of R ¹ to R ^{7 in} the general formula [I] has a binding group. Here, the binding group means a group that can be used for binding to another compound. Since chemiluminescent labeling agents are often bound to biologically related substances such as proteins, polypeptides, and sugars, amino groups, hydroxyl groups, and carboxyls that are convenient for binding to these biologically related substances can be used as binding groups. Preferred examples include groups, sulfonic acid groups, thiol groups, disulfide groups, isocyanate groups, thioisocyanate groups, succinimidyl ester groups, pentafluorophenyl ester groups, maleimide groups, and the like. In addition, since these groups should just be used for a coupling | bonding with another compound, arbitrary groups (for example, aminoalkyl groups etc.) containing at least any one of these groups are also utilized as a binding group. be able to. Note that only one binding group is sufficient, and it is usually preferable to specify one binding site in order to specify one binding site, but two or more binding groups may be included. Moreover, it is also possible to combine the above-mentioned amino group, hydroxyl group, carboxyl group, sulfonic acid group, etc. with a part of the bulky group, which will be described later, so that the binding group and bulky group can be used together.

In order to increase the synthesis yield, it is preferable that at least one of R ¹ to R ⁷ in the general formula [I] has a bulky group. Since the chemiluminescent compound of the present invention has high flatness of the boron dipyrromethene skeleton portion and the chemiluminescent group often has high flatness, the compound is often flat as a whole. In such a case, planar compounds are stacked and formed between π bonds of each aromatic ring, and each molecule is π-π bonded (π-π stacking) to cause aggregation, resulting in a decrease in yield. There is. In order to avoid such a situation and prevent the synthesis yield from decreasing, it has a bulky group that prevents π-π stacking as at least one of R ¹ to R ^{7 in} the general formula [I]. preferable. Here, the bulky group is a group that is thicker in the thickness direction (a direction perpendicular to the plane with the boron dipyrromethene skeleton as a plane) than the linear alkyl group, and preferably has 10 or more carbon atoms, Further, a branched alkyl group having 10 to 20 carbon atoms or a branched alkoxyl group is preferable.

Furthermore, a group for adjusting solubility may be included as at least one of R ¹ to R ⁷ as necessary. Since the chemiluminescent labeling agent is often used in an aqueous system, when it is desired to increase the hydrophilicity of the chemiluminescent compound and improve the solubility in the aqueous system, at least one of R ¹ to R ⁷ is used. As an alternative, a hydrophilic group may be included. Examples of such a hydrophilic group include a hydroxyl group, an amino group, a carbonyl group, a carboxyl group, an alkyl group or an alkoxyl group (one having 1 to 6 carbon atoms) containing one or more hydrophilic groups such as an ether bond, and the like. However, the present invention is not limited to these examples. Further, such a hydrophilic group may be used in combination with the above-described bulky group, binding group, emission wavelength adjusting group and the like.

In the general formula [I], R ⁸ and R ⁹ are each independently a fluorine or alkoxyl group, and the alkoxyl group preferably has 1 to 6 carbon atoms.

  Preferable examples of the chemiluminescent compound of the present invention include compounds having a structure represented by the following general formula [II].

(Wherein, R ^1a is an alkyl group, alkoxyl group, phenyl group or substituted phenyl group (the substituent is 1 to 5 alkyl groups, alkoxyl group or mono- or di-alkylamino group), and R ^3a is hydrogen. Or an alkyl group, R ^5a is a branched alkoxyl group). The number of carbon atoms in the alkyl group or alkyl moiety in R ^1a and R ^3a is 1 to 10 is preferable, the carbon number of R ^5a is preferably 10 to 20. In general formula [II], the phthalcarbazide derivative portion corresponds to a chemiluminescent group, R ^{1a corresponds} to a light emission wavelength adjusting group, R ^{3a corresponds} to hydrogen or an arbitrary substituent, and R ^5a corresponds to a bulky group.

  The chemiluminescent compound of the present invention can be produced by bonding a chemiluminescent group to a boron dipyrromethene skeleton to which the above-described substituent is bonded as required. Since the boron dipyrromethene skeleton and the chemiluminescent group are known and the production method thereof is also known, the chemiluminescent compound of the present invention can be easily produced by ordinary chemical synthesis, and several specific examples thereof are available. Examples are described in detail in the examples below.

  The chemiluminescent compound of the present invention can be used as a chemiluminescent labeling agent. The chemiluminescent labeling agent itself is well known, and the chemiluminescent compound of the present invention can also be used in the same manner as conventional chemiluminescent labeling agents. That is, the labeling substance is labeled by binding the chemiluminescent compound of the present invention to the labeling substance to be labeled. When the chemiluminescent compound of the present invention has the above-described binding group, the chemiluminescent compound of the present invention is bonded to the labeling substance via the above-mentioned binding group by a conventional method. When it does not have a binding group, an intermediate having a binding group introduced can be prepared by a conventional method, and this can be bound to a labeling substance.

  The mode of use of the chemiluminescent labeling agent is exactly the same as in the past. After labeling the labeling substance as described above, the labeled substance (referred to as “labeling substance” in this specification) is subjected to the reaction, and if necessary After performing a washing step or the like, the label is chemiluminescent and the luminescence is measured. For example, in the case of immunoassay in which chemiluminescent labeling agents are widely used, the labeling agent can be used as follows. For example, when immunoassay of a target antigen in a test sample is performed by the sandwich method, a primary antibody against the target antigen is bound to a solid phase, and this is reacted with the test sample, so that the target antigen in the test sample is immobilized on the solid phase. Conjugated to the primary antibody. After washing, a second antibody labeled with a chemiluminescent labeling agent is reacted. After washing, the chemiluminescent labeling agent of the second antibody bound to the target antigen is chemiluminescent by a chemical reaction according to a conventional method, and luminescence is measured. In the present specification and claims, “measurement” includes any of detection, quantification, and semi-quantification. Chemiluminescence of the labeling agent can be performed by performing a chemical reaction according to the chemiluminescent group in each chemiluminescent labeling agent by a conventional method. For example, when a phthalcarbazide derivative, which is a preferred example of a chemiluminescent group, is used as a chemiluminescent group, as is well known, it is reacted with hydrogen peroxide in the presence of potassium hexacyanoiron (III) (potassium ferricyanide) as a catalyst. Thus, the chemiluminescent group can emit light. The boron dipyrromethene skeleton is excited by light from the chemiluminescent group, and fluorescence is emitted from the boron dipyrromethene skeleton, and this fluorescence emission is measured.

  One of the advantageous features of the present invention is that the fluorescence wavelength can be greatly changed to be discernable by changing the emission wavelength adjusting group. Bonding a substituent on the boron dipyrromethene skeleton can be easily performed by ordinary chemical synthesis. Accordingly, a plurality of types of chemiluminescent compounds of the present invention having different fluorescence emission wavelengths can be easily produced. By using a plurality of types of chemiluminescent compounds of the present invention as the chemiluminescent labeling agents that are different to the extent that the wavelength of the fluorescence emission can be identified, it becomes possible to label a plurality of types of substances with different chemiluminescent labeling agents, As a result, it becomes possible to simultaneously measure a plurality of different test substances. For example, when multiple types of chemiluminescent labeling agents are used for the immunoassay by the sandwich method described above, different types of secondary antibodies can be labeled with different labeling agents so that the fluorescence wavelength can be identified. By discriminating and measuring luminescence from, multiple types of antigens can be immunoassayed simultaneously. The plural kinds of labeling agents to be used are different from each other so that the respective wavelengths of the fluorescence emission can be discriminated (that is, the emission wavelength ranges do not overlap at all or hardly overlap), and one of them is the emission wavelength adjustment. It may have no group. That is, the present invention has the emission wavelength adjusting groups different from each other (however, only one kind of chemiluminescent compound may not have the emission wavelength adjusting group), and the emission wavelengths are thereby different. Using the types of chemiluminescent compounds of the present invention as labeling agents, a plurality of types of substances labeled with the plurality of labeling agents are used for the reaction, and after the reaction, each of the labeling agents is allowed to emit light, thereby The present invention also provides a method for simultaneously measuring a plurality of labeling substances including simultaneous measurement of substances, and also provides a combination of a plurality of labeling agents used in this simultaneous measuring method.

  Conventionally, various chemiluminescent labeling agents having different fluorescence emission wavelengths are known. However, even when trying to measure a plurality of types of test substances simultaneously by using them in combination, a reaction that causes each chemiluminescent labeling agent to chemiluminescent Since the reaction conditions themselves and optimum reaction conditions are different, there is a problem that the measurement sensitivity of any labeling agent is lowered or the measurement becomes complicated. On the other hand, the chemiluminescent labeling agent of the present invention can use a plurality of types of chemiluminescent labeling agents in which the chemiluminescent group and the skeleton portion of the fluorescent dye are the same, and only the substituents on the boron dipyrromethene skeleton are different. Therefore, it is possible to label multiple types of substances with multiple types of labeling agents with the same chemiluminescent group luminescence reaction and optimal conditions, so that each labeling agent is highly sensitive to the same reaction. It is possible to measure simultaneously. Thereby, for example, in immunoassay and the like, it is possible to measure a plurality of different kinds of test substances at the same time. Therefore, the present invention provides a new measurement technique.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

Production of KCB-lime A chemiluminescent compound of the present invention (named “KCB-lime”) having the structure represented by the following formula 13 was produced.

  The chemical synthesis scheme is as follows.

  Hereinafter, each step will be described in detail.

(1) Synthesis of 3,5-dimethyl-1H-pyrrole-2-carbaldehyde

  Dissolve 2,4-dimethylpyrrole (30 g, 315 mmol, 1 eq) and dimethylformamide (DMF) (53.1 mL, 690 mmol, 2.2 eq) in hexane (600 mL) under an Ar stream, And stirred. Phosphoryl chloride (105.8 g, 690 mmol, 2.2 eq) was added dropwise over 1.5 hours under ice cooling. The reaction system was filtered, and the residual solid was washed with 5 N NaOH aqueous solution. The obtained liquid was cooled to 0 ° C., and the precipitated solid was filtered off to obtain the target compound (2) (28.8 g, yield; 74.2%) as a yellow solid.

TLC; R _f = 0.1 (eluent: chloroform)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 9.50 (s, 1 H), 9.46 (s, 1 H), 5.86 (s, 1 H), 2.32 (s, 3 H), 2.30 (s, 3 H).

(2) Synthesis of 4-methoxy-1,5-dihydro-pyrrol-2-one

  A 500 mL eggplant flask was charged with 10% aqueous ammonia (300 mL) and heated to 65 ° C. 4-Chloro-3-methoxy-2 (E) -butenonic acid methyl ester (75.8 g, 461 mmol) was added dropwise from the dropping funnel over 1.5 hours. After completion of dropping, the mixture was stirred for 4 hours. After completion of the reaction, the mixture was allowed to cool, methylene chloride was added, and the organic phase was extracted. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The obtained residue was dissolved in toluene, heated and gradually cooled. The obtained crystals were filtered off to obtain the target compound (4) (32.6 g, yield: 62.7%) as a white needle-like solid.

TLC: Rf = 0.2 (eluent: chloroform / ethyl acetate = 4/1, v / v)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 5.88 (s, 1 H), 5.08 (s, 2 H), 3.93 (s, 2 H), 3.82 (s, 1 H).

(3) Synthesis of 4- (2-butyl-octyloxy) -1,5-dihydro-pyrrol-2-one

In a 200 mL three-necked flask, compound (4) (30.0 g, 265 mmol, 1 eq), 1,2-butyl-1-octanol (118 mL, 530 mmol, 2 eq) and methanesulfonic acid (2.4 mL, 33 mmol, 0.13 eq) was added, and the mixture was stirred at 80 ° C for 15 minutes. Next, the mixture was stirred at 80 ° C under reduced pressure for 24 hours. After completion of the reaction, the reaction mixture was allowed to cool, and the precipitated solid was filtered off, and a saturated aqueous NaHCO ₃ solution and methylene chloride were added to the filtrate to extract the organic phase. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The obtained residue was separated and purified by silica gel chromatography (eluent: chloroform / ethyl acetate = 2/1) to obtain the target compound (5) (47.5 g, yield; 67.0%) as a white solid.

TLC: Rf = 0.1 (eluent: chloroform / ethyl acetate = 2/1, v / v)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 5.65 (s, 1 H), 5.03 (s, 1 H), 3.93 (s, 2 H), 3.82 (d, J = 4 Hz, 1 H), 1.74-1.65 (m, 1 H), 1.34- 1.28 (m, 16 H), 0.94-0.89 (m, 6 H).

(4) 4- (2-Butyl-octyloxy) -5- (3,5-dimethyl-1H-pyrrol-2-ylmethylene)
Synthesis of 1,5-dihydro-pyrrol-2-one

  Compound (5) (16.0 g, 60 mmol, 1 eq), compound (2) (7.4 g, 60 mmol, 1 eq), DMSO (192 mL) and 2N NaOH aq. (172 mL) were placed in a 500 mL three-necked flask. added. This was stirred at 60 ° C for 20 hours under Ar flow. After completion of the reaction, the mixture was allowed to cool, and the precipitated solid was filtered off and washed with water. The obtained solid (brown) was dissolved in ethyl acetate, and water was added to extract the organic phase. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure to obtain the target compound (6) (15.5 g, yield; 69.3%) as a brown solid.

TLC: Rf = 0.1 (eluent: chloroform)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 10.80 (s, 1 H), 10.30 (s, 1 H), 6.34 (s, 1 H), 5.81 (s, 1 H), 5.05 (s, 1 H), 3.54 (d, J = 5 Hz, 2 H), 2.39 (s, 3 H), 2.16 (s, 1 H), 1.85-1.83 (m, 1 H), 1.39-1.28 (m, 16 H), 0.91-0.86 (m, 6 H).

(5) Synthesis of trifluoro-methanesulfonic acid-4- (2-butyl-octyloxy) -5- (3,5-dimethyl-1H-pyrrol-2-ylmethylene) -5H-pyrrol-2-yl ester

Under Ar, Compound (6) (12.7 g, 34.2 mmol, 1 eq) and methylene chloride (100 mL) were placed in a 300 mL three-necked eggplant flask and cooled to 0 ° C. Trifluoromethanesulfonic anhydride (14.5 g, 51.3 mmol, 1.5 eq) dissolved in methylene chloride (100 ml) was added dropwise from the dropping funnel over 3 hours. The mixture was then stirred at room temperature for 2 days. After completion of the reaction, sat. NaHCO ₃ aq. And ethyl acetate were added to the reaction system, and the organic phase was extracted. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The obtained residue was separated and purified by silica gel chromatography (eluent: hexane / ethyl acetate = 40/1) to obtain the target compound (7) (5.1 g, yield: 29.7%) as a reddish brown oil. It became a yellow solid when stored frozen.

TLC: Rf = 0.2 (eluent: hexane / ethyl acetate = 40/1, v / v)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 10.70 (s, 1 H), 7.03 (s, 1 H), 5.87 (s, 1 H), 5.37 (s, 1 H), 3.87 (d, J = 6 Hz, 2 H), 2.30 (s, 3 H), 2.20 (s, 1 H), 1.83-1.81 (m, 1 H), 1.39-1.24 (m, 16 H), 0.90-0.86 (m, 6 H).

(6) Synthesis of 5-bromo-2-methyl-isoindole-1,3-dione

  Add toluene (30 mL) and methylamine solution (8.2 mL, 88.9 mmol, 1.5 eq) to a 100 mL two-necked flask, add 4-bromophthalic anhydride (13.5 g, 59.2 mmol, 1 eq), and add Dean-Stark. At reflux for 7 hours. It became a yellowish green transparent solution. After allowing to cool, suction filtration was performed, and the resulting white solid was vacuum dried. The target compound (9) (10.1 g, yield; 71.0%) was obtained as a white solid.

TLC; R _f = 0.5 (eluent: chloroform)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 7.98 (s, 1 H), 7.85 (d, J = 8 Hz, 1 H), 7.70 (d, 1 H), 3.18 (s, 3 H).

(7) Synthesis of 2-methyl-5- (4,4,5,5-tetramethyl- [1,3,2] dioxaborolan-2-yl) -isoindole-1,3-dione

4-Bromomethylphthalimide (3.0 g, 12.5 mmol, 1 eq) was placed in a 100 mL eggplant flask and dissolved in DMSO (75 mL). Bis (pinacolato) diboron (3.3 g, 13 mmol, 1.05 eq), PdCl ₂ (dppf) (0.3 g, 0.38 mmol, 0.03 eq) and KOAc (3.7 g, 37.5 mmol, 3 eq) were added to it, and under Ar The mixture was stirred at 80 ° C for 27 hours. A blackish brown solution was obtained. After completion of the reaction, the mixture was allowed to cool, and toluene and water were added to extract the organic layer. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The obtained residue was separated and purified by silica gel chromatography (eluent: chloroform) to obtain the target compound (10) (948 mg, yield: 26.3%) as a white solid.

TLC; R _f = 0.2 (eluent: chloroform)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 8.23 (s, 1 H), 8.14 (d, J = 7 Hz, 1 H), 7.83 (d, J = 7 Hz, 1 H), 3.18 (s, 3 H), 1.37 (s, 12 H).

(8) 5- [4- (2-Butyl-octyloxy) -5- (3,5-dimethyl-1H-pyrrol-2-ylmethylene) -5H-pyrrol-2-yl] -2-methyl-isoindole- Synthesis of 1,3-dione

Dissolve dipyrromethene triflate (7) (100 mg, 0.2 mmol, 1 eq) in 1,4-dioxane (50 ml) in a 300 mL three-necked flask, then (10) (86.1 mg, 0.3 mmol, 1.5 eq) , K ₂ CO ₃ (220 mg, 1.6 mmol, 8 eq) and Pd (pph ₃ ) ₄ (116 mg, 0.1 mmol, 0.5 eq) were added. The mixture was stirred at 85 ° C for 1 day under Ar flow. After completion of the reaction, the mixture was allowed to cool, and chloroform and water were added to extract the organic layer. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The obtained residue was separated and purified by alumina chromatography (eluent: methylene chloride / ethyl acetate = 40/1) to obtain the target compound (11) (20 mg, yield; 19.4%) as a reddish brown solid.

TLC; R _f = 0.2 (eluent: methylene chloride / ethyl acetate = 40/1, v / v)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 8.36 (s, 1 H), 8.30 (d, J = 5 Hz, 1 H), 7.89 (s, 1 H), 7.88 (d, J = 5 Hz, 1 H), 7.20 (s, 1 H) , 6.12 (s, 1 H), 5.93 (s, 1 H), 4.97 (d, J = 2 Hz, 2 H), 3.22 (s, 3 H), 2.44 (s, 3 H), 2.27 (s, 3 H), 1.87-1.80 (m, 1 H), 1.49-1.20 (m, 16 H), 0.95-0.78 (m, 6 H).

(9) Synthesis of boron dipyrromethene derivatives

(11) (20 mg, 0.04 mmol, 1 eq) was dissolved in toluene (15 mL) in a 50 mL eggplant flask. Et ₃ N (0.5 mL) and BF ₃ • Et ₂ O (0.75 mL) were then added and refluxed for 1 hour. After completion of the reaction, the mixture was allowed to cool, and a saturated aqueous NaHCO ₃ solution was added to extract the organic layer. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The obtained residue was separated and purified by silica gel chromatography (eluent: toluene / ethyl acetate = 19/1) to obtain a red solid. The obtained solid was further purified by GPC to obtain the target compound (12) (4 mg, yield; 19.4%) as a red solid.

TLC; R _f = 0.2 (eluent: toluene / ethyl acetate = 19/1, v / v)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 8.36 (dd, J = 2, 8 Hz, 1 H), 8.23 (s, 1 H), 7.90 (dd, J = 2, 8 Hz, 1 H), 7.28 (s, 1 H), 6.11 (s , 1 H), 6.04 (s, 1 H), 4.00 (d, J = 6 Hz 2 H), 3.21 (s, 3 H), 2.49 (s, 3 H), 2.29 (s, 3 H), 1.89 -1.85 (m, 1 H), 1.42-1.25 (m, 16 H), 0.95-0.87 (m, 6 H).

(10) Synthesis of KCB-lime

  (12) (8.67 mg, 0.02 mmol, 1 eq) was placed in a 50 mL eggplant flask and dissolved in ethanol (4 mL). Hydrazine monohydrate (40 μL, 0.82 mmol, 57 eq) was added and refluxed for 30 minutes. After completion of the reaction, the mixture was allowed to cool, and ether and 1N HCl aq were added to extract the organic layer. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The target compound (13) (12.5 mg, yield; 147%) was obtained as a red solid.

TLC; R _f = 0.1 (eluent: toluene / ethyl acetate = 1/1, v / v)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 8.64 (s, 1 H), 8.57 (s, 1 H), 8.30 (d, J = 8 Hz, 1 H), 7.26 (s, 1 H), 6.17 (s, 1 H), 6.10 (s, 1 H), 4.00 (d, J = 5 Hz, 2 H), 2.51 (s, 3 H), 2.30 (s, 3 H), 1.87-1.83 (m, 1 H), 1.62-1.28 (m, 16 H), 1.05-0.85 (m, 6 H).

(7 ') Synthesis of 4-boronate methylphthalimide

4-Bromomethylphthalimide (879 mg, 3.7 mmol, 1 eq) was placed in a 100 mL eggplant flask and dissolved in DMSO (45 mL). Add bis (neopentylglycolate) diboron (867 mg, 3.8 mmol, 1.05 eq), PdCl ₂ (dppf) (89.8 mg, 0.11 mmol, 0.03 eq) and KOAc (1.08 g, 11 mmol, 3 eq). The mixture was stirred at 80 ° C for 7 hours under Ar. A blackish brown solution was obtained. After completion of the reaction, the mixture was allowed to cool, and toluene and water were added to extract the organic layer. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The obtained residue was separated and purified by silica gel chromatography (eluent: chloroform) to obtain the target compound (10 ′) (614 mg, yield: 61.4%) as a white solid.

TLC; R _f = 0.2 (eluent: chloroform)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 8.27 (s, 1 H), 8.13 (d, J = 7 Hz, 1 H), 7.80 (d, J = 7 Hz, 1 H), 3.81 (s, 4 H), 3.18 (s, 3 H) , 1.04 (s, 6 H).

(8 ') 5- [4- (2-Butyl-octyloxy) -5- (3,5-dimethyl-1H-pyrrol-2-ylmethylene) -5H-pyrrol-2-yl] -2-methyl-isoindole Synthesis of 1,3-dione

Dissolve dipyrromethene triflate (7) (126 mg, 0.25 mmol, 1 eq) in 1,4-dioxane (50 mL) in a 300 mL three-necked flask, then (10 ') (100 mg, 0.37 mmol, 1.5 eq ), K ₂ CO ₃ (276 mg, 2.0 mmol, 8 eq) and Pd (pph ₃ ) ₄ (144 mg, 0.13 mmol, 0.5 eq) were added. The mixture was stirred at 85 ° C for 1 day under Ar flow. After completion of the reaction, the mixture was allowed to cool, and chloroform and water were added to extract the organic layer. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The obtained residue was separated and purified by silica gel chromatography (eluent: methylene chloride / ethyl acetate = 40/1) to obtain the target compound (11) (61 mg, yield; 47.2%) as a reddish brown solid.

TLC; R _f = 0.2 (eluent: methylene chloride / ethyl acetate = 40/1, v / v)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 8.36 (s, 1 H), 8.30 (d, J = 5 Hz, 1 H), 7.89 (s, 1 H), 7.88 (d, J = 5 Hz, 1 H), 7.20 (s, 1 H) , 6.12 (s, 1 H), 5.93 (s, 1 H), 4.97 (d, J = 2 Hz 2 H), 3.22 (s, 3 H), 2.44 (s, 3 H), 2.27 (s, 3 H), 1.87-1.80 (m, 1 H), 1.49-1.20 (m, 16 H), 0.95-0.78 (m, 6 H).

Production of KCB-orange A chemiluminescent compound of the present invention (named “KCB-orange”) having the structure represented by the following formula 22 was produced.

  The chemical synthesis scheme is as follows.

  Hereinafter, each step will be described in detail.

(1) Synthesis of 2- (4-methoxy-phenyl) -pyrrole-1-carboxylic acid tert-butyl ester

In a 300 mL three-necked flask, N-tert-butoxycarbonyl-2-bromopyrrole (7.5 g, 30.5 mmol, 1 eq), 4-methoxyphenylboronic acid (4.6 g, 30.5 mmol, 1 eq), Pd (PPh ₃ ) ₄ (347 mg, 0.3 mmol, 0.01 eq) was added, followed by ultrasonically degassed 2 N Na ₂ CO ₃ aq (18 mL, 36.6 mmol, 1.2 eq). Toluene (150 mL) and methanol (30 ml) were added thereto, vacuum degassed, and the mixture was stirred at 80 ° C. for 20 hours under Ar flow. After the reaction was completed, water was added and the organic phase was extracted. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The obtained residue was separated and purified by silica gel chromatography (eluent: hexane / ethyl acetate = 29/1) to obtain the target compound (15) (4.3 g, yield; 51.8%) as a colorless transparent oil. .

TLC: Rf = 0.2 (eluent: hexane / ethyl acetate = 29/1, v / v)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 7.32 (dd, J = 3, 2 Hz, 1 H), 7.27 (d, J = 9 Hz, 2 H), 6.90 (d, J = 9 Hz, 2 H), 6.20 (dd, J = 3, 3 Hz, 1 H), 6.13 (dd, J = 3, 2 Hz, 1 H), 3.83 (s, 3 H), 1.39 (s, 9 H).

(2) Synthesis of 2- (4-methoxy-phenyl) -1H-pyrrole

  Compound (15) (4.3 g, 15.7 mmol, 1 eq) was placed in a 200 mL two-necked flask and dissolved in THF (70 mL). Then 28% NaOMe methanol solution (15 mL) was added and stirred at room temperature for 3 hours. After the reaction was completed, toluene and water were added to extract the organic layer. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The target compound (16) (2.64 g, yield; 97.8%) was obtained as a brownish white solid.

TLC: Rf = 0.1 (eluent: hexane / ethyl acetate = 29/1, v / v)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 8.30 (s, 1 H), 7.38 (d, J = 9 Hz, 2 H), 6.90 (d, J = 9 Hz, 2 H), 6.80 (d, J = 2 Hz, 1 H), 6.40 ( s, 1 H), 6.28 (d, J = 3 Hz, 1 H), 3.81 (s, 3 H).

(3) Synthesis of 5- (4-methoxy-phenyl) -1H-pyrrole-2-carbaldehyde

Compound (16) (2.6 g, 15 mmol, 1 eq) dissolved in methylene chloride (30 mL) was added to a 100 mL three-necked flask under N ₂ flow, and DMF (2.6 mL, 30 mmol, 2 eq) was added. . POCl ₃ (4.6 g, 30 mmol, 2 eq) was then added dropwise in a 0 ° C. ice bath. It changed from a brown clear solution to a black-green clear solution. After the addition, 5 N NaOH aq (30 mL) was added to hydrolyze, and methylene chloride and water were added to extract the organic layer. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The obtained residue was separated and purified by silica gel chromatography (eluent; methylene chloride / ethyl acetate = 40/1) to obtain the target compound (17) (2.56 g, yield; 84.8%) as a pink solid.

TLC: Rf = 0.2 (eluent: methylene chloride / ethyl acetate = 40/1, v / v)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 10.10 (s, 1 H), 9.47 (s, 1 H), 7.60 (d, J = 9 Hz, 2 H), 7.02 (d, J = 4 Hz, 1 H), 6.95 (d, J = 9 Hz, 2 H), 6.50 (d, J = 9 Hz, 1 H), 3.84 (s, 3 H).

(4) Synthesis of 4- (2-butyl-octyloxy) -5- [5- (4-methoxy-phenyl) -1H-pyrrol-2-ylmethylene] -1,5-dihydro-pyrrol-2-one

  Compound (17) (284 mg, 1.4 mmol, 1 eq) was dissolved in DMSO (4.5 mL) under a stream of Ar in a 50 mL three-necked flask, and compound (5) (750 mg, 2.8 mmol, 2 eq), 2 N NaOH aq (4.5 mL) was added. The mixture was stirred for 48 hours at 60 ° C under Ar flow. After completion of the reaction, water was added and suction filtration was performed. The obtained filtrate was dissolved in ethyl acetate and water was added to extract the organic layer. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The obtained residue was separated and purified by silica gel chromatography (eluent; chloroform / ethyl acetate = 19/1) to obtain the target compound (18) (153 mg, yield; 24.3%) as a yellow solid.

TLC: Rf = 0.2 (eluent: chloroform / ethyl acetate = 19/1, v / v)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 11.4 (s, 1 H), 10.5 (s, 1 H), 7.90 (d, J = 9 Hz, 2 H), 6.94 (d, J = 9 Hz, 2 H), 6.52 (s, 2 H) , 6.34 (s, 1 H), 5.09 (s, 1 H), 3.92 (d, J = 5 Hz, 2 H), 3.86 (s, 3 H), 1.87-1.83 (m, 1 H), 1.42- 1.28 (m, 16 H), 0.95-0.83 (m, 6 H).

(5) Trifluoro-methanesulfonic acid 4- (2-butyl-octyloxy) -5- [5- (4-methoxy-phenyl) -1H-pyrrol-2-ylmethylene] -5H-pyrrol-2-yl ester Composition

Under Ar, compound (18) (153 mg, 0.34 mmol, 1 eq) was dissolved in methylene chloride (15 mL) in a 50 mL three-necked flask and cooled to 0 ° C. Trifluoromethanesulfonic acid (677 mg, 2.4 mmol, 2 eq) dissolved in methylene chloride (5 mL) was added dropwise from the dropping funnel over 10 minutes. The mixture was then stirred at room temperature for 2 hours. After completion of the reaction, saturated NaHCO ₃ aqueous solution and ethyl acetate were added to the reaction system, and the organic layer was extracted. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The obtained residue was separated and purified by silica gel chromatography (eluent; hexane / methylene chloride = 9/1) to obtain the target compound (19) (35 mg, yield; 17.7%) as a yellow solid.

TLC: Rf = 0.2 (eluent: hexane / methylene chloride = 9/1, v / v)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 10.5 (s, 1 H), 7.50 (d, J = 9 Hz, 1 H), 7.23 (d, J = 9 Hz, 2 H), 6.96 (s, 1 H), 6.90 (d, J = 9 Hz, 2 H), 6.55 (s, 1 H), 5.30 (d, J = 5 Hz, 1 H), 3.82 (d, J = 6 Hz, 2 H), 3.79 (s, 3 H), 1.78- 1.72 (m, 1 H), 1.23-1.03 (m, 16 H), 0.85-0.72 (m, 6 H).

(6) 5- {4- (2-Butyl-octyloxy) -5- [5- (4-methoxy-phenyl) -1H-pyrrol-2-ylmethylene] -5H-pyrrol-2-yl} -2-methyl Of 2-isoindole-1,3-dione

Dissolve compound (19) (35 mg, 0.06 mmol, 1 eq) in 1,4-dioxane (20 mL) in a 300 mL three-necked flask, then (10 ') (24.6 mg, 0.09 mmol, 1.5 eq), K ₂ CO ₃ (66.3 mg, 0.48 mmol, 8 eq) and Pd (pph ₃ ) ₄ (34.7 mg, 0.03 mmol, 0.5 eq) were added. The mixture was stirred at 85 ° C for 1 day under Ar flow. After completion of the reaction, the mixture was allowed to cool, and chloroform and water were added to extract the organic layer. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The obtained residue was separated and purified by silica gel chromatography (eluent: methylene chloride / ethyl acetate = 80/1) to obtain the target compound (20) (10 mg, yield: 28.1%) as a reddish brown solid.

TLC; R _f = 0.5 (eluent: methylene chloride / ethyl acetate = 80/1, v / v)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ8.23 (s, 1 H), 8.20 (d, J = 8 Hz, 1 H), 7.80 (d, J = 8 Hz, 1 H), 7.41 (d, J = 9 Hz, 2 H), 7.03 (s, 1 H), 7.01 (d, J = 9 Hz, 2 H), 6.66 (s, 1 H), 6.09 (s, 1 H), 3.95 (d, J = 6 Hz, 2 H), 3.92 (s, 3 H), 3.18 (s, 3 H), 1.88-1.80 (m, 1 H), 1.50-1.15 (m, 16 H), 0.95-0.80 (m, 6 H).

(7) Synthesis of boron dipyrromethene derivatives

(20) (10 mg, 0.017 mmol, 1 eq) was dissolved in toluene (15 mL) in a 50 mL eggplant flask. Et ₃ N (0.5 mL) and BF ₃ • Et ₂ O (0.75 mL) were then added and refluxed for 1 hour. After completion of the reaction, the mixture was allowed to cool, and sat.NaHCO ₃ aq was added to extract the organic layer. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The obtained residue was separated and purified by silica gel chromatography (eluent: toluene / ethyl acetate = 49/1) and then further purified by GPC to obtain the target compound (21) (6 mg, yield; 55.5%) as red. Obtained as a purple solid.

TLC; R _f = 0.2 (eluent: toluene / ethyl acetate = 49/1, v / v)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 8.20 (dd, J = 2, 8 Hz, 1 H), 8.05 (d, J = 2 Hz 1 H), 7.80 (d, J = 8 Hz, 1 H), 7.33 (s, 1 H), 7.24 (d, J = 9 Hz, 2 H), 7.23 (s, 1 H), 6.89 (d, J = 9 Hz 2 H), 6.87 (s, 1 H), 5.96 (s, 1 H), 4.00 ( d, J = 6 Hz, 2 H), 3.83 (s, 3 H), 3.17 (s, 1H), 1.86-1.82 (m, 1 H), 1.33-0.93 (m, 16 H), 0.90-0.81 ( m, 6 H).

(8) Synthesis of KCB-orange

(21) (2.5 mg, 0.004 mmol, 1 eq) was taken into a 50 mL eggplant flask and dissolved in ethanol (4 mL). Hydrazine monohydrate (40 μL, 0.82 mmol, 57 eq) was added and refluxed for 30 minutes. After completion of the reaction, the mixture was allowed to cool, and diethyl ether and 1N HCl aq were added to extract the organic layer.
The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The target compound (22) (2.6 mg, yield; 104%) was obtained as a red solid.

TLC; R _f = 0.1 (eluent: toluene / ethyl acetate = 1/1, v / v)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 8.50 (d, J = 8 Hz, 1 H), 8.48 (s, 1 H), 8.24 (d, J = 8 Hz, 1 H), 7.34 (s, 1 H), 7.32 (s, 1 H) , 7.31 (d, J = 9 Hz, 2 H), 6.90 (d, J = 9 Hz 2 H), 6.88 (s, 1 H), 6.09 (s, 1 H), 4.03 (d, J = 6 Hz , 2 H), 3.83 (s, 3 H), 3.17 (s, 1H), 1.90-1.82 (m, 1 H), 1.40-1.11 (m, 16 H), 0.98-0.85 (m, 6 H).

Production of KCB-apple A chemiluminescent compound of the present invention (named “KCB-apple”) having a structure represented by the following formula 30 was produced.

  The chemical synthesis scheme is as follows.

  Hereinafter, each step will be described in detail.

(1) Synthesis of 2- (4-dimethylamino) -pyrrole-1-carboxylic acid tert-butyl ester

In a 300 mL three-necked flask, N-tert-butoxycarbonyl-2-bromopyrrole (6.67 g, 27 mmol, 1 eq), 4-dimethylaminoboronic acid (4.47 g, 27 mmol, 1 eq), Pd (PPh ₃ ) ₄ (312 mg, 0.3 mmol, 0.01 eq) was added followed by ultrasonically degassed 2 N Na ₂ CO ₃ aq (20 mL, 32.4 mmol, 1.2 eq). Toluene (150 mL) and methanol (30 mL) were added thereto, vacuum degassed, and the mixture was stirred at 80 ° C. for 22 hours under Ar flow. After the reaction was completed, water was added and the organic phase was extracted. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The obtained residue was separated and purified by silica gel chromatography (eluent: hexane / ethyl acetate = 29/1) to obtain the target compound (23) (3.39 g, yield; 44.0%) as a light gray solid.

TLC: Rf = 0.2 (eluent: hexane / ethyl acetate = 29/1, v / v)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 7.29 (dd, J = 3, 3 Hz, 1 H), 7.23 (d, J = 9 Hz, 2 H), 6.71 (d, J = 9 Hz, 2 H), 6.19 (dd, J = 3, 3 Hz, 1 H), 6.11 (dd, J = 3, 3 Hz, 1 H), 2.96 (s, 6 H), 1.41 (s, 9 H).

(2) Synthesis of dimethyl- [4- (1H-pyrrol-2-yl) -phenyl] -amine

  Compound (23) (3.39 g, 11.8 mmol, 1 eq) was placed in a 100 mL two-necked flask and dissolved in THF (60 mL). Then 28% NaOMe methanol solution (11 mL) was added and stirred at room temperature for 1 hour. After the reaction was completed, toluene and water were added to extract the organic layer. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The target compound (24) (1.98 g, yield; 90.0%) was obtained as a silver-white solid.

TLC: Rf = 0.1 (eluent: hexane / ethyl acetate = 29/1, v / v)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 8.30 (s, 1 H), 7.35 (d, J = 9 Hz, 2 H), 6.80 (dd, J = 4,4 Hz, 1 H), 6.75 (dd, J = 9 Hz, 2 H), 6.35 (dd, J = 4,6 Hz, 1 H), 6.27 (dd, J = 4,6 Hz, 1 H), 2.96 (s, 6 H).

(3) Synthesis of 5- (4-dimethylamino-phenyl) -1H-pyrrole-2-carbaldehyde

Compound (24) (1.98 g, 10.6 mmol, 1 eq) in N ₂ stream was dissolved in methylene chloride (45 mL) in a 100 mL three-necked flask, and DMF (3.0 mL, 21.2 mmol, 2 eq) was added. . Then POCl ₃ (2.1 mL, 21.2 mmol, 2 eq) was added dropwise in a 0 ° C. ice bath and stirred for 2 hours. It changed from a dark brown clear solution to a blue-green clear solution. After the addition, 5 N NaOH aq (30 mL) was added to hydrolyze, and methylene chloride and water were added to extract the organic layer. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The obtained residue was separated and purified by alumina chromatography (eluent: chloroform) to obtain the target compound (25) (1.05 g, yield: 46.3%) as a greenish white solid.

TLC: Rf = 0.2 (eluent: chloroform)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 9.43 (s, 1 H), 9.33 (s, 1 H), 7.47 (d, J = 9 Hz, 2 H), 6.99 (dd, J = 4,6 Hz, 1 H), 6.75 (d, J = 9 Hz, 2 H), 6.50 (dd, J = 4,6 Hz, 1 H), 3.02 (s, 6 H).

(4) Synthesis of 4- (2-butyl-octyloxy) -5- [5- (4-dimethylamino-phenyl) -1H-pyrrol-2-ylmethylene] -1,5-dihydro-pyrrol-2-one

  Compound (25) (350 mg, 1.6 mmol, 1 eq) was dissolved in DMSO (5 mL) under a stream of Ar in a 50 mL three-necked flask, and compound (5) (655 mg, 2.5 mmol, 1.5 eq), 2 N NaOH aq (5 mL) was added. The mixture was stirred for 48 hours at 60 ° C under Ar flow. After completion of the reaction, water was added and suction filtration was performed. The obtained filtrate was dissolved in ethyl acetate and water was added to extract the organic layer. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The obtained residue was separated and purified by alumina chromatography (eluent: chloroform / ethyl acetate = 40/1) to obtain the target compound (26) (335 mg, yield; 44.3%) as an orange solid.

TLC: Rf = 0.2 (eluent: chloroform / ethyl acetate = 40/1, v / v)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 11.7 (s, 1 H), 10.5 (s, 1 H), 7.82 (d, J = 9 Hz, 2 H), 6.76 (d, J = 9 Hz, 2 H), 6.51 (dd, J = 3 , 3 Hz, 1 H), 6.46 (dd, J = 3,3 Hz, 1 H), 6.34 (s, 1 H), 5.12 (s, 1 H), 3.88 (d, J = 6 Hz, 2 H ), 3.00 (s, 6 H), 1.87-1.81 (m, 1 H), 1.40-1.30 (m, 16 H), 0.92-0.87 (m, 6 H).

(5) Trifluoro-methanesulfonic acid 4- (2-butyl-octyloxy) -5- [5- (4-dimethylamino-phenyl) -1H-pyrrol-2-ylmethylene] -5H-pyrrol-2-yl ester Synthesis of

Under Ar, Compound (26) (335 mg, 0.72 mmol, 1 eq) was dissolved in methylene chloride (15 mL) in a 50 mL three-necked flask and cooled to 0 ° C. Trifluoromethanesulfonic acid (406 mg, 1.44 mmol, 2 eq) dissolved in methylene chloride (5 mL) was added dropwise from the dropping funnel over 10 minutes. The mixture was then stirred at room temperature for 2 hours. After completion of the reaction, saturated NaHCO ₃ aqueous solution and ethyl acetate were added to the reaction system, and the organic layer was extracted. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The obtained residue was separated and purified by silica gel chromatography (eluent; hexane / chloroform = 6/1) to obtain the target compound (27) (163 mg, yield; 38.0%) as a red glossy solid.

TLC: Rf = 0.2 (eluent: hexane / chloroform = 6/1, v / v)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 11.4 (s, 1 H), 7.52 (d, J = 9 Hz, 1 H), 7.01 (s, 1 H), 6.78 (s, 1 H), 6.75 (d, J = 9 Hz, 2 H) , 6.52 (s, 1 H), 5.37 (s, 1 H), 3.88 (d, J = 6 Hz, 2 H), 3.02 (s, 6 H), 1.82-1.76 (m, 1 H), 1.40- 1.29 (m, 16 H), 0.94-0.87 (m, 6 H).

(6) 5- {4- (2-Butyl-octyloxy) -5- [5- (4-dimethylamino-phenyl) -1H-pyrrol-2-ylmethylene] -5H-pyrrol-2-yl} -2- Synthesis of methyl-isoindole-1,3-dione

Compound (27) (163 mg, 0.27 mmol, 1 eq) was dissolved in 1,4-dioxane (70 mL) in a 100 mL three-necked flask, then (10 ') (110 mg, 0.41 mmol, 1.5 eq), K ₂ CO ₃ (299 mg, 2.16 mmol, 8 eq) and Pd (pph ₃ ) ₄ (156 mg, 0.14 mmol, 0.5 eq) were added. The mixture was stirred at 85 ° C for 1 day under Ar flow. After completion of the reaction, the mixture was allowed to cool, and chloroform and water were added to extract the organic layer. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The obtained residue was separated and purified by alumina chromatography (eluent: methylene chloride / ethyl acetate = 40/1) to obtain the target compound (28) (41 mg, yield; 24.7%) as a black blue green solid. .

TLC; R _f = 0.3 (eluent: methylene chloride / ethyl acetate = 40/1, v / v)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 8.49 (s, 1 H), 8.32 (d, J = 8 Hz, 1 H), 7.86 (d, J = 9 Hz, 2 H), 7.84 (s, 1 H), 7.72 (s, 1 H) , 7.64 (d, J = 9 Hz, 2 H), 7.02 (s, 1 H), 6.89 (d, J = 8 Hz, 1 H), 6.14 (s, 1 H), 3.96 (d, J = 6 Hz, 2 H), 3.19 (s, 6H), 3.08 (s, 3 H), 1.88-1.80 (m, 1 H), 1.50-1.15 (m, 16 H), 0.93-0.89 (m, 6 H) .

(7) Synthesis of boron dipyrromethene derivatives

(28) (38 mg, 0.06 mmol, 1 eq) was dissolved in toluene (15 mL) in a 50 mL eggplant flask. Et ₃ N (0.5 mL) and BF ₃ • Et ₂ O (0.75 mL) were then added and refluxed for 1 hour. After completion of the reaction, the mixture was allowed to cool, and a saturated aqueous NaHCO ₃ solution was added to extract the organic layer. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The obtained residue was separated and purified by silica gel chromatography (eluent: toluene / ethyl acetate = 40/1) and then further purified by GPC to obtain the target compound (29) (16 mg, yield; 39.0%) as blue. Obtained as a green solid.

TLC; R _f = 0.2 (eluent: toluene / ethyl acetate = 40/1, v / v)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 8.33 (d, J = 8 Hz, 1 H), 7.87 (d, J = 9 Hz, 2 H), 7.86 (s, 1 H), 7.27 (s, 1 H), 7.07 (d, J = 4 Hz, 1 H), 6.72 (s, 1 H), 6.70 (d, J = 4 Hz, 1 H), 6.01 (s, 1 H), 4.00 (d, J = 6 Hz, 2 H), 3.20 ( s, 3 H), 3.02 (s, 6 H), 1.85-1.83 (m, 1 H), 1.44-1.25 (m, 16 H), 0.95-0.88 (m, 6 H).

(8) Synthesis of KCB-apple

  (29) (2.0 mg, 0.003 mmol, 1 eq) was taken into a 50 mL eggplant flask and dissolved in ethanol (4 mL). Hydrazine monohydrate (40 μL, 0.82 mmol, 57 eq) was added and refluxed for 30 minutes. After completion of the reaction, the mixture was allowed to cool, and diethyl ether and 1N HCl aq were added to extract the organic layer. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, the desiccant was filtered off, and the solvent was concentrated under reduced pressure. The target compound (30) (1.9 mg, yield; 95%) was obtained as a blue-green solid.

TLC; R _f = 0.1 (eluent: toluene / ethyl acetate = 1/1, v / v)

¹ H-NMR (300MHz, CDCl ₃ , TMS, rt)
δ 8.60 (d, J = 8 Hz, 1 H), 8.59 (s, 1 H), 7.90 (d, J = 9 Hz, 2 H), 7.31 (s, 1 H), 7.29 (s, 1 H) , 7.07 (d, J = 4 Hz, 1 H), 6.70 (d, J = 9 Hz 2 H), 6.15 (s, 1 H), 4.31 (s, 1 H), 4.03 (d, J = 6 Hz , 2 H), 3.01 (s, 6H), 2.28-2.00 (m, 1 H), 1.55-1.26 (m, 16 H), 0.99-0.83 (m, 6 H).

The light emission states of KCB-lime, KCB-orange and KCB-apple synthesized in Examples 1, 2, and 3 were visually observed. Add A solution (30% H ₂ O ₂ aqueous solution: 10% NaOH aqueous solution = 10: 1) and B solution (1 mmol / l potassium hexacyanoiron (III) potassium) to ethanol solution of KCB (lime / orange / apple). When well shaken, the yellow-green, orange, and red emission colors could be clearly distinguished by visual observation.

  Next, KCB-lime, KCB-orange, and KCB-apple were caused to emit light under the same conditions as described above, and an emission spectrum was measured. For the measurement, a double beam spectrophotometer (Hitachi U-2001) and a spectrofluorimeter (Hitachi F-4500) were used. In the emission spectrum measurement, the “luminescence” mode was selected in the spectrofluorometer (Hitachi F-4500). Table 1 below shows the equipment conditions for emission spectrum measurement. For KCB-apple, since the emission intensity was weak, we measured the sensitivity of the instrument conditions more than the other two.

  The measured emission spectrum is shown in FIG. As shown in FIG. 1, the emission wavelength peaks of KCB-lime were 545 nm, KCB-orange was 567 nm, and KCB-apple was 672 nm, which were clearly different from each other. Thus, the chemiluminescent compound of the present invention has a plurality of chemiluminescent properties that clearly differ in emission wavelength peak when emitted under the same conditions by simply changing the substituent bonded to the boron dipyrromethene skeleton. It has become clear that it is possible to produce compounds.

  At 672 nm, the peak of the emission wavelength of KCB-apple, almost no other two emissions are observed. In addition, at 545 nm and 567 nm, which are the emission wavelength peaks of KCB-lime and KCB-orange, part of the emission of other chemiluminescent compounds is also observed, but the ratio of the respective emission intensities becomes clear from FIG. Therefore, it is possible to calculate each concentration by measuring the emission intensity at each peak wavelength. Therefore, simultaneous measurement of a plurality of test substances is possible by labeling a plurality of different substances with these chemiluminescent compounds.

  Incidentally, the fluorescence spectrum after light emission of each chemiluminescent compound was measured and compared with the emission spectrum. The results are shown in Table 2 below.

  As shown in Table 2, the fluorescence spectrum after light emission almost coincides with the light emission spectrum. From this, it can be seen that the emission is a fluorescence emission of a boron dipyrromethene moiety.

It is a figure which shows the emission spectrum of each chemiluminescent compound synthesize | combined in the Example of this invention.

Claims (16)

A chemiluminescent compound having a structure represented by the following general formula [I].

In the formula, R ¹ to R ⁷ , independently of each other, at least one of them is a chemiluminescent group, and the others are hydrogen or any group that does not inhibit light emission of the compound, R ⁸ and R ⁹ is independently a fluorine or alkoxy group. The chemiluminescent compound according to claim 1, which has an emission wavelength adjusting group as at least one of R ¹ to R ^{7 in} the general formula [I].   The chemiluminescent compound according to claim 2, wherein the emission wavelength adjusting group is a group selected from the group consisting of an alkyl group, an alkoxyl group, an aryl group, a heteroaryl group, and derivatives thereof. The chemiluminescent compound according to claim 2 or 3, wherein R ¹ and / or R ⁷ is the emission wavelength adjusting group. 5. The compound according to claim 2, wherein R ¹ to R ⁷ in the general formula [I] have a binding group and / or a bulky group as the chemiluminescent group, the emission wavelength adjusting group, or a group other than hydrogen. The chemiluminescent compound of any one of Claims.   The binding group is an amino group, a hydroxyl group, a carboxyl group, a sulfonic acid group, a thiol group, a disulfide group, an isocyanate group, a thioisocyanate group, a succinimidyl ester group, a pentafluorophenyl ester group, a maleimide group, or at least one of these. 6. The chemiluminescent compound according to claim 5, which is selected from the group consisting of groups comprising   The chemiluminescent compound according to claim 5 or 6, wherein the bulky group is a branched alkyl group, a branched alkoxyl group or a group having them as a skeleton.   The chemiluminescent compound according to any one of claims 1 to 7, wherein the chemiluminescent group is a phthalcarbazide derivative, dioxetane derivative, lophine derivative, acridine derivative, indole derivative, oxalic acid derivative, diphenoyl derivative or luciferin derivative.   The chemiluminescent compound according to any one of claims 1 to 8, wherein the chemiluminescent group is directly connected to the boron dipyrromethene skeleton represented by the general formula [I] without a spacer. The chemiluminescent compound according to claim 1, which has a structure represented by the following general formula [II].

In the formula, R ^1a is an alkyl group, an alkoxyl group, a phenyl group, or a substituted phenyl group (the substituent is 1 to 5 alkyl groups, an alkoxyl group, or a mono- or di-alkylamino group), and R ^3a is hydrogen or The alkyl group, R ^5a is a branched alkoxyl group.   A labeling agent comprising the chemiluminescent compound according to any one of claims 1 to 10.   A plurality of kinds of the light emission wavelength adjusting groups different from each other (however, only one kind of chemiluminescent compound may not have the light emission wavelength adjusting group), and thereby the light emission wavelengths are different. 12. A combination of labeling agents comprising the chemiluminescent compound according to any one of 11 above.   The combination of labeling agents according to claim 12, wherein the plurality of labeling agents have the same chemiluminescent group.   A method for measuring a labeling substance, comprising: subjecting a substance labeled with the labeling agent according to claim 12 to a reaction, and after the reaction, causing the labeling agent to emit light and measuring the substance.   A plurality of kinds of the light emission wavelength adjusting groups different from each other (however, only one kind of chemiluminescent compound may not have the light emission wavelength adjusting group), and thereby the light emission wavelengths are different. 10. The chemiluminescent compound according to any one of 10 is used as a labeling agent, a plurality of types of substances labeled with the plurality of labeling agents are used for the reaction, and after the reaction, the labeling agent is caused to emit light, A method for simultaneously measuring a plurality of labeled substances, comprising simultaneously measuring a plurality of kinds of substances. The measurement method according to claim 15, wherein the plurality of labeling agents have the same chemiluminescent group.

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