JP2014136675A - Metal ion fluorescence probe - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a compound useful as a fluorescence probe for metal ion measurement.SOLUTION: This invention relates to a compound represented by the formula (I) or salt thereof, wherein R1 and R2 independently represent a 2-quinolyl group which may be substituted or a 8-quinolyl group which may be substituted, and m and n independently represent 0, 1 or 2, with m+n=1, 2, 3 or 4, provided that 1,4-bis(quinoline-2-ylmethyl)-1,4-diazacycloheptane is excluded.

Description

  The present invention relates to a compound useful as a fluorescent probe for metal ions, particularly iron ions, mercury ions or cadmium ions. The present invention also relates to a method for measuring a metal ion concentration using the above compound.

  Conventionally, atomic absorption analyzers and inductively coupled plasma (ICP) emission analyzers have been widely used to measure metals contained in trace amounts in samples, but these are both large and very expensive. In addition, a certain skill is required to operate these devices.

  Furthermore, a method is known in which a plurality of metals contained in a sample are simultaneously measured by separating a plurality of metals by capillary electrophoresis and measuring ultraviolet absorption. Although this method can measure without using an expensive apparatus, many coexisting chemical substances in the sample also absorb ultraviolet light, which may interfere with the measurement.

  On the other hand, the fluorometric method has the advantages that it has higher sensitivity than the above-described absorption detection method, has an inexpensive apparatus, and is easy to handle including pretreatment. In addition, there are significant advances in measuring instruments such as fluorescence microscopes and confocal laser scanning fluorescence microscopes. As one of the methods for quantifying trace metals and tracking the dynamic behavior of metal ions in vivo, It is widely used (Non-Patent Document 1).

  Metals, particularly heavy metals represented by mercury, cadmium and the like, have been widely used industrially and industrially for a long time because of their many advantages such as electrical / thermal conductivity and color development characteristics. These heavy metals, when taken into the body as ions, have been confirmed to accumulate in the body, inhibit protein function, run away, and cause organ damage to the digestive system and nervous system. There are strict usage control restrictions. However, effluents from water sources and soils before the use regulations and environmental management were enacted can still cause health damage, and a certain amount of these heavy metals exist in nature as well. From the viewpoint of environment and health management, it is urgent to establish an analytical method that enables simple and rapid detection of harmful heavy metal ions.

  In addition, there are many metals involved in the maintenance of biological functions such as metabolism and synthesis of physiologically active substances in the living body. Among them, iron is the most essential metal element. Therefore, detection and quantification of iron ions are extremely important because they can be applied to medical fields such as blood and urine analysis. So far, color reaction using hexacyanoiron ions and colorimetric analysis of divalent iron ions using 1,10-phenanthroline are well known, but none of them has high detection sensitivity.

  Recently, several fluorescent probes (sometimes referred to as “metal ion fluorescent sensors”) for quantifying metal ions such as heavy metals and iron have been reported (Non-Patent Documents 2 and 3).

  On the other hand, the present inventors have reported that N, N, N ′, N′-tetrakis (2-quinolylmethyl) ethylenediamine (TQEN) and the like that can be produced at low cost are useful as metal ion fluorescent sensors. Is a fluorescent sensor that specifically detects zinc ions (Patent Document 1).

JP 2005-194244 A

Yoshinori Katagani, Bunseki, 2008, 4, 158-162. Jeong, Y. and Yoon, Inorganica Chimica Acta, 2012, 381, 2-14. Hyman, L. M. and Franz, K. J., Coordination Chemistry Reviews, 2012, 256, 2333-2356.

  Many of the metal ion fluorescent probes reported so far (especially fluorescent probes for the measurement of iron ion, mercury ion or cadmium ion) all have a complex chemical structure, and the synthesis is multistep. It was necessary, and a problem was left in practical use.

  An object of the present invention is to provide a metal ion that can be synthesized easily and accurately and with high sensitivity without using an expensive apparatus, and capable of measuring metal ions such as heavy metals and iron easily and accurately. The object is to provide a fluorescent probe for measurement.

  As a result of intensive studies under such circumstances, the present inventor has obtained the following formula (I):

[Where
R ¹ and R ² each independently represent an optionally substituted 2-quinolyl group or an optionally substituted 8-quinolyl group;
m and n each independently represent 0, 1 or 2, and m + n = 1, 2, 3 or 4. ]
Or a salt thereof (hereinafter sometimes referred to as compound (I)) is selectively selected from a specific metal ion (particularly trivalent iron ion, mercury ion or cadmium ion) in an aqueous solvent. It was found for the first time that it exhibits a complex-forming ability and that the compound is a selective and highly sensitive fluorescent probe for trivalent iron ions, mercury ions or cadmium ions, and the present invention has been completed.

That is, the present invention is as follows.
[1] Formula (I):

[Where
R ¹ and R ² each independently represent an optionally substituted 2-quinolyl group or an optionally substituted 8-quinolyl group;
m and n each independently represent 0, 1 or 2, and m + n = 1, 2, 3 or 4. ]
Or a salt thereof (excluding 1,4-bis (quinolin-2-ylmethyl) -1,4-diazacycloheptane),
[2] The compound according to the above [1] or a salt thereof, wherein R ¹ and R ² are each independently an optionally substituted 2-quinolyl group,
[3] R ¹ and R ² are each independently a 2-quinolyl group optionally substituted by a C _1-6 alkoxy group, and m and n are each independently 1 or 2, A compound or a salt thereof according to the above [1], wherein m + n = 2 or 3;
[4] Formula (I):

[Where
R ¹ and R ² each independently represent an optionally substituted 2-quinolyl group or an optionally substituted 8-quinolyl group;
m and n each independently represent 0, 1 or 2, and m + n = 1, 2, 3 or 4. ]
A fluorescent probe for measuring a metal ion containing a compound represented by the formula:
[5] The fluorescent probe according to the above [4], wherein R ¹ and R ² are each independently an optionally substituted 2-quinolyl group,
[6] R ¹ and R ² are each independently a 2-quinolyl group optionally substituted by a C _1-6 alkoxy group, and m and n are each independently 1 or 2, And the fluorescent probe according to the above [4], wherein m + n = 2 or 3;
[7] The fluorescent probe according to any one of [4] to [6], wherein the metal ion is an iron ion, a mercury ion, or a cadmium ion,
[8] Formula (I):

[Where
R ¹ and R ² each independently represent an optionally substituted 2-quinolyl group or an optionally substituted 8-quinolyl group;
m and n each independently represent 0, 1 or 2, and m + n = 1, 2, 3 or 4. ]
Or a metal complex formed from a salt thereof and a metal ion (however, from 1,4-bis (quinolin-2-ylmethyl) -1,4-diazacycloheptane and an iron ion or a manganese ion) Excluding complexes formed),
[9] The metal complex according to the above [8], wherein R ¹ and R ² are each independently an optionally substituted 2-quinolyl group,
[10] R ¹ and R ² are each independently a 2-quinolyl group optionally substituted by a C _1-6 alkoxy group, and m and n are each independently 1 or 2, And the metal complex according to the above [8], wherein m + n = 2 or 3;
[11] The metal complex according to any one of [8] to [10], wherein the metal ion is an iron ion, a mercury ion, or a cadmium ion,
[12] A method for measuring metal ions, including the following steps:
(1) Formula (I):

[Where
R ¹ and R ² each independently represent an optionally substituted 2-quinolyl group or an optionally substituted 8-quinolyl group;
m and n each independently represent 0, 1 or 2, and m + n = 1, 2, 3 or 4. ]
A step of reacting the compound represented by the above or a salt thereof with a metal ion, and (2) a step of measuring the fluorescence intensity of the metal complex formed in the above step,
[13] The method of the above-mentioned [12], wherein R ¹ and R ² are each independently an optionally substituted 2-quinolyl group,
[14] R ¹ and R ² are each independently a 2-quinolyl group optionally substituted by a C _1-6 alkoxy group, and m and n are each independently 1 or 2, The method according to [12] above, wherein m + n = 2 or 3;
[15] The method according to any one of [12] to [14] above, wherein the metal ion is an iron ion, a mercury ion, or a cadmium ion,
Etc.

  According to the present invention, there is provided a fluorescent probe for metal measurement that has a selective complex formation ability with a specific metal ion, particularly trivalent iron ion, mercury ion, and cadmium ion, and can be synthesized inexpensively and easily. Can do. In addition, the fluorescent probe according to the present invention can easily perform functional group conversion, and can easily adjust the type and detection sensitivity of metal ions detectable by the functional group conversion. Furthermore, according to the present invention, a highly sensitive measurement method of metal ions using the fluorescent probe can also be provided.

It is the figure which showed the fluorescence response of the compound (I-1a) in the presence of various metal ions (2 equivalents). It is the figure which showed the fluorescence response of the compound (I-1b) in the presence of various metal ions (2 equivalents). It is the figure which showed the fluorescence response of the compound (I-1d) in the presence of various metal ions (2 equivalents). It is the figure which showed the result of having titrated the compound (I-1a) with the mercury ion in acetonitrile, and tracking the change by the ultraviolet visible absorption spectrum. It is the figure which titrated the compound (I-1a) with acetonitrile in acetonitrile, and showed the change of the fluorescence intensity in wavelength 408nm in the addition amount of mercury ion. It is the figure which showed the result of having titrated the compound (I-1a) with trivalent iron ion in acetonitrile, and tracking the change by the ultraviolet-visible absorption spectrum. It is the figure which titrated the compound (I-1a) with trivalent iron ion in acetonitrile, and showed the change of the fluorescence intensity in wavelength 411nm in the addition amount of a trivalent iron ion. It is the figure which showed the result of having titrated the compound (I-1d) with the cadmium ion in acetonitrile, and tracking the change by the ultraviolet visible absorption spectrum. It is the figure which titrated the compound (I-1d) with cadmium ion in acetonitrile, and showed the change of the fluorescence intensity in wavelength 405nm in the addition amount of cadmium ion.

  Hereinafter, the present invention will be described in detail.

(Definition)

  In the present specification, “measurement” includes both quantification and detection.

In the present specification, the metal to be measured using the metal measuring fluorescent probe is a metal to be measured for the presence or absence or the concentration thereof in the sample.
Examples of the metal include alkali metals, alkaline earth metals, transition metals, and other metals.

  More specifically, examples of the alkali metal include lithium, sodium, potassium, rubidium, cesium, and francium.

  Examples of the alkaline earth metal include beryllium, magnesium, calcium, strontium, barium, and radium.

  Examples of the transition metal include scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, yttrium, zirconium, niobium, molybdenum, technetium, rubidium, radon, palladium, silver, hafnium, tantalum, and tungsten. , Rhenium, osmium, iridium, platinum or gold.

  Furthermore, examples of other metals include aluminum, zinc, gallium, germanium, arsenic, selenium, cadmium, indium, tin, antimony, tellurium, mercury, thallium, lead, bismuth, and polonium.

  A particularly preferable metal for performing measurement using the fluorescent probe for metal measurement of the present invention is iron, mercury or cadmium.

A sample for measuring a metal using the fluorescent probe for measuring a metal according to the present invention is a sample that may contain the metal, and is intended to measure the sample.
Metals take various forms such as simple substances, ions and compounds, and exist in a free state or in a state bound to a carrier (carrier), and are contained in various objects. The metal in the sample to be measured using the fluorescent probe for use is not particularly limited, and any metal can be used as long as it can be measured directly or by processing.

  The fluorescent probe for measuring metal according to the present invention is characterized in that metal can be measured simply, accurately, and with high sensitivity. Therefore, biological samples, meat, vegetables, grains, fruits, marine products, processed foods, beverages, beverages It is particularly effective for measuring metals contained in samples that may contain trace amounts of metals such as water, well water, river water, lake water, seawater, soil, air, or pharmaceuticals.

  For example, biological samples include human or animal blood, serum, plasma, urine, stool, cerebrospinal fluid, saliva, sweat, tears, ascites, amniotic fluid, brain and other organs, hair, skin, nails, muscles, nerves, etc. Examples include tissues and cells.

  In the measurement of a metal in a sample performed using the metal measurement fluorescent probe of the present invention, the sample mixed with and brought into contact with the metal measurement fluorescent probe is preferably a liquid. On the other hand, when the sample containing metal is not liquid, pretreatment such as extraction or solubilization may be performed according to a known method so that the metal is contained in the liquid.

  In the present specification, a metal measuring fluorescent probe is capable of forming a complex by contacting with a metal in a sample to be measured to coordinate with the metal. Specifically, the fluorescent probe for metal measurement has the formula (I):

[Where
R ¹ and R ² each independently represent an optionally substituted 2-quinolyl group or an optionally substituted 8-quinolyl group;
m and n each independently represent 0, 1 or 2, and m + n = 1, 2, 3 or 4. ]
Or a salt thereof.

In the present specification, “optionally substituted” means that it may have one or more substituents unless otherwise specified. As the “substituent”, ( 1) halogen atom, (2) alkoxy group, (3) alkoxyalkoxy group, (4) alkylenedioxy group, (5) nitro group, (6) cyano group, (7) hydroxy group, (8) amino group, (9) mercapto group, (10) carbamoyl group, (11) alkyl group, (12) carboxy group, (13) cycloalkyl group, (14) cycloalkenyl group, (15) alkenyl group, (16) alkynyl group, (17) aryl group, (18) aryloxy group, (19) aralkyl group, (20) aralkyloxy group, (21) alkoxy-carbonyl group, (22) aralkyloxy-carbonyl group, (23) a Kill-carbonyl group, (24) aryl-carbonyl group, (25) succimidyloxycarbonyl group, (26) alkyl-carbonyloxy group, (27) aryl-carbonyloxy group, (28) aryloxy-carbonyl group, (29) alkoxy-carbonyl-alkoxy group, (30) aryloxy-carbonyl-alkyloxy group, (31) sulfo group or ester thereof, (32) phosphono group or ester thereof, (33) alkylsulfonyl group, (34) Arylsulfonyl group, (35) formyl group, (36) azide group, (37) alkylthio group, (38) arylthio group, (39) alkylsulfonoxy group, (40) arylsulfonoxy group, (41) alkyl Sulfonate, (42) aryl sulfonate, (43) mono-young Kuwaji -C _1-6 alkylamino - carbonyl group, (44) heterocyclic group, (45) tri-substituted silyl group, (46) but such protected amino group, is limited to Absent. Among them, halogen, nitro, cyano, C _1-6 alkoxy, C _1-6 alkoxy-C _1-6 alkoxy, methylenedioxy, C _1-6 alkyl, C _1-6 alkoxy-carbonyl, benzyloxycarbonyl, carboxy, Acetyl, benzoyl, formyl, carbamoyl, benzyl, phenyl, phenoxy, naphthyl, piperidyl, pyrrolidinyl, thienyl, furyl, pyridyl, pyrrolyl, indolyl, quinolyl, azide, trimethylsilyl, triethylsilyl, di-C _1-6 alkylamino, mono- C _1-6 alkylamino, acetylamino, benzyloxycarbonylamino, tert-butoxycarbonylamino are preferred. When a plurality of substituents are present, each substituent may be the same or different.

The above substituent may be further substituted with the above substituent. The number of substituents is not particularly limited as long as it is a substitutable number, but is preferably 1 to 5, more preferably 1 to 3. When a plurality of substituents are present, each substituent may be the same or different. The substituent may further include a C _1-6 alkyl group, a C _2-6 alkenyl group, a C _2-6 alkynyl group, a C _3-8 cycloalkyl group, a C _1-6 alkoxy group, a C _3-8 cycloalkenyl group, It may be substituted with a C _6-14 aryl group, C _7-14 aralkyl group, heterocyclic group, halogen atom, hydroxy group, carboxy group, amino group, carbamoyl group, cyano group, nitro group, oxo group or the like. The number of substituents is not particularly limited as long as it is a substitutable number, but is preferably 1 to 5, more preferably 1 to 3. When a plurality of substituents are present, each substituent may be the same or different.

  In the present specification, the “halogen atom” means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

In the present specification, the “alkyl group” means a linear or branched monovalent alkyl group having 1 or more carbon atoms. When there is no particular limitation on the carbon number range, C _{1- 12} alkyl group, preferably C _1-8 alkyl group, more preferably C _1-6 alkyl group. In addition, alkyls of other substituents having an alkyl moiety (for example, alkoxy groups, alkylthio groups, alkylamino groups, dialkylamino groups, alkylsulfonyl groups, alkyl-carbonyl groups, alkylsulfonyloxy groups, alkylsulfonate groups, etc.) The same applies to the part.

In the present specification, the “alkenyl group” means a linear or branched monovalent alkenyl group having 2 or more carbon atoms, and the carbon number range is not particularly limited, but preferably C _2-6 alkenyl. It is a group.

In the present specification, the “C _2-6 alkenyl group” means a linear or branched alkenyl group having 2 to 6 carbon atoms, such as ethenyl, 1-propenyl, 2-propenyl, 2-methyl. -1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1 -Hexenyl, 3-hexenyl, 5-hexenyl and the like. Among these, a C _2-4 alkenyl group is particularly preferable.

In the present specification, the “alkynyl group” means a linear or branched monovalent alkynyl group having 2 or more carbon atoms, and the carbon number range is not particularly limited, but preferably a C _2-6 alkynyl group. It is a group.

In the present specification, the “C _2-6 alkynyl group” means a linear or branched alkynyl group having 2 to 6 carbon atoms, such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl. 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl and the like. Among these, a C _2-4 alkynyl group is preferable.

In the present specification, examples of the “cycloalkyl group” include cyclic alkyl groups having 3 to 8 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like. Among these, a C _3-6 cycloalkyl group is preferable.

In the present specification, the “aryl group” means a monocyclic or polycyclic (condensed) hydrocarbon group exhibiting aromaticity, and specifically includes, for example, phenyl, 1-naphthyl, 2-naphthyl. , C _6-22 aryl groups such as biphenylyl, terphenyl, diphenylnaphthyl, 2-anthryl, phenanthryl and the like. Among them, a C _6-14 aryl group is preferable, and a C _6-10 aryl group is particularly preferable. In addition, the aryl part of other substituents having an aryl part (for example, an aryloxy group, an aryl-carbonyl group, an aryl-carbonyloxy group, an arylsulfonyl group, an arylthio group, an arylsulfonoxy group, an arylsulfonate group, etc.) The same applies to.

In the present specification, examples of the “C _6-10 aryl group” include phenyl, 1-naphthyl, and 2-naphthyl, and phenyl is particularly preferable.

  Examples of the “heterocycle (group)” in the present specification include an aromatic heterocyclic group and a non-aromatic heterocyclic group.

  In the present specification, the “aromatic heterocyclic group” means an aromaticity containing 1 to 4 heteroatoms selected from an oxygen atom, a sulfur atom and a nitrogen atom in addition to a carbon atom as a ring constituent atom. Means a monocyclic or polycyclic (fused) heterocyclic group shown.

  In the present specification, examples of the “monocyclic aromatic heterocyclic group” include furyl, thienyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl (1, 2,4-oxadiazolyl, 1,3,4-oxadiazolyl), thiadiazolyl (1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl), triazolyl (1,2,4-triazolyl, 1,2,3- Triazolyl), tetrazolyl, triazinyl and the like. Among them, a 5- or 6-membered monocyclic aromatic heterocyclic group is preferable, and pyridyl is particularly preferable.

  In the present specification, the “polycyclic (fused) aromatic heterocyclic group” means that the monocyclic aromatic heterocyclic group is a monocyclic aromatic ring (preferably a benzene ring or a monocyclic aromatic group). Heterocycle), such as quinolyl, isoquinolyl, quinazolyl, quinoxalyl, benzofuranyl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzimidazolyl, benzotriazolyl , Indolyl, indazolyl, pyrrolopyridyl, pyrazolopyridyl, imidazopyridyl, thienopyridyl, pyrrolopyrazinyl, pyrazolopyrazinyl, imidazopyrazinyl, thienopyrazinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, imidazopyrimidinyl, thienopyrimidinyl, etc. .

  Examples of the “non-aromatic heterocyclic group” in the present specification include, for example, 4 to 7 members containing 1 to 4 heteroatoms selected from an oxygen atom, a sulfur atom and a nitrogen atom in addition to a carbon atom as a ring constituent atom (Preferably 5 or 6 membered) monocyclic non-aromatic heterocyclic group or condensed non-aromatic heterocyclic group. Examples of the condensed non-aromatic heterocyclic group include a ring corresponding to the 4- to 7-membered monocyclic non-aromatic heterocyclic group, and a 5- or 6-membered aromatic containing 1 or 2 nitrogen atoms. 1 or 2 rings selected from a heterocyclic ring (eg, pyrrole, imidazole, pyrazole, pyrazine, pyridine, pyrimidine), a 5-membered aromatic heterocyclic ring containing 1 sulfur atom (eg, thiophene) and a benzene ring Examples thereof include a group derived from a condensed ring and a group obtained by partial saturation of the group.

  Preferred examples of the non-aromatic heterocyclic group include azetidinyl, pyrrolidinyl, piperidyl, morpholinyl, thiomorpholinyl, piperazinyl, hexamethyleneiminyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, oxazolinyl, thiazolinyl, imidazolinyl, dioxolanyl, dioxolanyl, , Monocyclic non-aromatic heterocyclic groups such as pyranyl, tetrahydropyranyl, thiopyranyl, tetrahydrothiopyranyl, tetrahydrofuryl, pyrazolidinyl, pyrazolinyl, tetrahydropyrimidinyl, dihydrotriazolyl, tetrahydrotriazolyl, dihydroindolyl, dihydro Isoindolyl, dihydrobenzofuranyl, dihydrobenzodioxinyl, dihydrobenzodioxepinyl, tetrahydrobenzo Ranil, chromenyl, dihydrochloride Mesnil, dihydroquinolinyl, tetrahydroquinolinyl, dihydroisoquinolinyl, tetrahydroisoquinolinyl, and the like condensed non-aromatic heterocyclic group such as dihydrophthalazinyl the like.

In the present specification, “aralkyl” means a group in which an “alkyl group” is substituted with an “aryl group”, and preferably “C _7-14 aralkyl”. The same applies to the aralkyl moiety of other substituents having an aralkyl moiety (for example, an aralkyloxy group).

In the present specification, “C _7-14 aralkyl” means a group in which “C _1-4 alkyl group” is replaced by “C _6-10 aryl group”, and examples thereof include benzyl, 1-phenylethyl, 2 -Phenylethyl, (naphthyl-1-yl) methyl, (naphthyl-2-yl) methyl, 1- (naphthyl-1-yl) ethyl, 1- (naphthyl-2-yl) ethyl, 2- (naphthyl-1) -Yl) ethyl, 2- (naphthyl-2-yl) ethyl, biphenylylmethyl and the like.

In the present specification, the “mono- or di-C _1-6 alkylamino-carbonyl group” means a carbonyl group on a mono- or di-substituted amino group with a linear or branched alkyl group having 1 to 6 carbon atoms. Means a bonded group, for example, methylaminocarbonyl group, ethylaminocarbonyl group, propylaminocarbonyl group, isopropylaminocarbonyl group, dimethylaminocarbonyl group, diethylaminocarbonyl group, dipropylaminocarbonyl group, diisopropylaminocarbonyl group, etc. Can be mentioned.

In the present specification, the “tri-substituted silyl group” means a silyl group substituted by three identical or different substituents (eg, C _1-6 alkyl group, C _6-10 aryl group), As the group, a trimethylsilyl group, a triethylsilyl group, a triisopropylsilyl group, a tert-butyldimethylsilyl group and the like are preferable.

In the present specification, the “protected amino group” means an amino group protected by the same or different one or two “protecting groups”. As the “protecting group”, for example, a protecting group for an amino group described in Protective Groups in Organic Synthesis, published by John Wiley and Sons (1980) can be used, and a C _1-6 alkyl group, a C _2-6 alkenyl group can be used. C _6-10 aryl group, C _7-14 aralkyl group, formyl group, C _1-6 alkyl-carbonyl group, C _1-6 alkoxy-carbonyl group, C _2-6 alkenyloxy-carbonyl group, C _6-10 Aryl-carbonyl group, C _7-14 aralkyl-carbonyl group, C _6-10 aryloxy-carbonyl group, C _7-14 aralkyloxy-carbonyl group, C _6-10 arylsulfonyl group, benzhydryl group, trityl group, tri-C Protecting groups such as _1-6 alkylsilyl group, 9-fluorenylmethyloxycarbonyl group, phthaloyl group and the like can be mentioned. The protecting group may be substituted with a halogen atom, a C _1-6 alkyl group, a C _1-6 alkoxy group or a nitro group. Specific examples of the protective group for the amino group include acetyl, trifluoroacetyl, pivaloyl, tert-butoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, benzhydryl, Examples include trityl, phthaloyl, allyloxycarbonyl, p-toluenesulfonyl, o-nitrobenzenesulfonyl, trimethylsilylethoxycarbonyl and the like.

(Compound of the present invention)
The compound of the present invention has the following formula (I):

[Where
R ¹ and R ² each independently represent an optionally substituted 2-quinolyl group or an optionally substituted 8-quinolyl group;
m and n each independently represent 0, 1 or 2, and m + n = 1, 2, 3 or 4. ]
Or a salt thereof (compound (I)).

  Hereinafter, each group of the compound (I) of the present invention will be described.

R ¹ and R ² each independently represent a 2- or 8-quinolyl group which may be substituted with the above-described substituent, and when having a plurality of substituents, they may be the same or different. Good. By selecting substituents on R ¹ and R ² , it is possible to control the excitation and fluorescence wavelength, metal ion selectivity, fluorescence intensity and the like of the compound (I) of the present invention.

The substituent on R ¹ and R ² is preferably a hydrogen atom, an alkoxy group (eg, C _1-6 alkoxy group), an amino group (eg, amino group, di-C _1-6 ) which may be protected. Alkylamino group), cyclic amino group (eg, aziridinyl group, azetidinyl group, pyrrolidinyl group, piperidyl group, etc.), carboxy group, alkoxycarbonyl group and the like. Of these, a hydrogen atom or a C _1-6 alkoxy group (eg, methoxy group) is particularly preferable.

R ¹ and R ² are preferably the same group from the viewpoint of ease of synthesis. As the compound (I), the following compounds are suitable.

[Compound (IA)]
Compound (I) or a salt thereof, wherein R ¹ and R ² are both a 2-quinolyl group or an 8-quinolyl group; and m is 1 and n is 2.

[Compound (IB)]
Compound (I) or a salt thereof, wherein R ¹ and R ² are both 2-quinolyl group or 8-quinolyl group; and m and n are both 1.

[Compound (IC)]
R ¹ and ^{R 2} is 8-quinolyl group substituted by 2-quinolyl group or a _{C 1-6} alkoxy group both substituted with _{C 1-6} alkoxy group; and m is 1, and n is 2, Compound (I) or a salt thereof.

[Compound (ID)]
R ¹ and ^{R 2} is 8-quinolyl group substituted by 2-quinolyl group or a _{C 1-6} alkoxy group both substituted with _{C 1-6} alkoxy group; and m and n are both 1, Compound (I) or a salt thereof.

  Particularly preferable compounds are the following compounds.

The compound (I) of the present invention can exist as an acid addition salt, and can also exist as a base addition salt depending on the type of substituent. Examples of the acid addition salt include mineral acid salts such as hydrochloride, sulfate, and nitrate, or organic acid salts such as methanesulfonate, p-toluenesulfonate, oxalate, citrate, and tartrate. Can be mentioned. A base addition salt is formed when it has a substituent such as a carboxyl group, for example, a metal salt such as a sodium salt, potassium salt, calcium salt, magnesium salt, an ammonium salt, or an organic amine salt such as a triethylamine salt. Can be mentioned. In addition to these, a salt with an amino acid such as glycine may be formed. The compound of the present invention or a salt thereof may exist as a hydrate or a solvate, and any of these substances is included in the compound (I) of the present invention. Compound (I) may also be labeled with an isotope (eg, ³ H, ¹⁴ C, etc.). Further, compound (I) may be a deuterium converter.

(Synthesis of Compound (I) of the Present Invention)
Although it does not specifically limit as a manufacturing method of compound (I) of this invention, For example, it can synthesize | combine through the following reactions.

  The raw material compounds can be easily obtained as commercial products unless otherwise stated, or can be produced according to a method known per se.

[Production Method 1]
The compound (I) of the present invention (specifically, the compound (I-1)) can be produced, for example, from a commercially available product or a known material in a literature in one step as shown below.

[In the formula, R ³ represents a substituent which may be present, and X represents a leaving group (eg, halogen atom, alkylsulfonoxy group (eg, trifluoromethanesulfonyloxy group, methanesulfonyl group)). Roxy group, etc.), arylsulfonyloxy group (eg, toluenesulfonoxy group, benzenesulfonyloxy group, etc.), and other symbols are as defined above.]

This step is a reaction for producing compound (I-1) by quinolylmethylation by reacting two amino groups constituting the ring of compound (1) with compound (2). As the compound (1), an acid addition salt thereof (for example, hydrochloride etc.) can also be used.
This step is performed in the presence of a base in a solvent that does not affect the reaction.

Examples of the base include alkali metal hydroxides such as lithium hydroxide, sodium hydroxide, and potassium hydroxide; alkaline earth metals such as magnesium hydroxide and calcium hydroxide; alkali metals such as sodium carbonate and potassium carbonate Alkali metal hydrogen carbonates such as sodium hydrogen carbonate and potassium hydrogen carbonate; trimethylamine, triethylamine, diisopropylethylamine, pyridine, picoline, N-methylpyrrolidine, N-methylmorpholine, N, N-dimethylaniline, 1,5-diazabicyclo [4; .3.0] -5-nonene, 1,4-diazabicyclo [2.2.2] octane, 1,8-diazabicyclo [5.4.0] -7-undecene (DBU), and organic such as tetramethylguanidine Bases, etc., among which potassium carbonate, triethyla Emissions, diisopropylethylamine and the like are preferable.
The amount of the base to be used is generally 2 to 8 equivalents relative to 1 equivalent of compound (1).

Moreover, this process can also accelerate | stimulate reaction by performing in presence of potassium iodide.
The usage-amount of potassium iodide is 2-8 equivalent normally with respect to 1 equivalent of compound (1).

  Examples of the solvent include nitriles such as acetonitrile and propionitrile; halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, and trichloroethylene; dioxane, tetrahydrofuran (THF), diethyl ether, tert-butyl methyl ether, and diisopropyl. Ethers, ethers such as ethylene glycol-dimethyl ether (DME), diethylene glycol dimethyl ether (diglyme); amides such as dimethylformamide (DMF); hydrocarbons such as hexane, benzene and toluene, among which acetonitrile is Particularly preferred.

The reaction temperature is generally −78 ° C. to 120 ° C., preferably room temperature to 110 ° C.
The reaction time is usually 0.5 to 96 hours.

  The compound (I) of the present invention is useful as a fluorescent probe for metal ions, particularly trivalent iron ions, mercury ions, cadmium ions and the like. That is, Compound (I) does not have a property of emitting strong fluorescence per se, but when it captures a specific metal ion to form a metal complex, it emits strong fluorescence. Compound (I) is particularly characterized in that trivalent iron ions, mercury ions and cadmium ions can be specifically captured, and complex formation is extremely rapid. Compound (I) is characterized by almost no background fluorescence, and the formed metal complex emits stable and strong fluorescence that is hardly affected by pH in the neutral region (region of pH 6 to 8). Have. Therefore, Compound (I) is extremely useful as a fluorescent probe for measuring metal ions in living cells and living tissues under physiological conditions.

  The method for using the fluorescent probe for measuring metal ions of the present invention is not particularly limited, and can be used in the same manner as conventionally known fluorescent probes. Usually, the compound (I) is added to an aqueous medium such as physiological saline or a buffer, or a mixture of an aqueous medium such as ethanol, acetone, acetonitrile, ethylene glycol, dimethyl sulfoxide, dimethylformamide and an aqueous medium. And the fluorescence spectrum may be measured by adding this solution to an appropriate buffer containing cells and tissues. Moreover, you may use the fluorescent probe of this invention in the form of a composition combining with an appropriate additive. For example, it can be combined with additives such as a buffer, a solubilizing agent and a pH adjuster.

The present invention will be described more specifically with reference to the following examples and test examples. However, the present invention is not limited thereby, and may be changed without departing from the scope of the present invention.
The reaction was monitored by thin layer chromatography using Merck 60 F254 silica gel plates (thickness 0.25 mm).
¹ H and ¹³ C-NMR spectra were measured using Varian Gemini 2000 and deuterated chloroform or deuterated methanol as a solvent. Data for ¹ H-NMR are chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet, dd = double doublet, dt = It is shown as double triplet, brs = broad singlet), coupling constant (Hz), integration and allocation.
Elemental analysis was performed using J-Science Micro Corder JM10.
Jasco FP-6300 was used for the fluorescence measurement of the prepared complex of the compound (I) of the present invention and a metal ion.
“Room temperature” in the following examples usually indicates about 10 ° C. to about 30 ° C. The ratio shown in the mixed solvent is a volume ratio unless otherwise specified. Unless otherwise indicated, “%” indicates “% by weight”.

  In the following examples, the piperazine or homopiperazine used as the compound (1) was a commercially available product (manufactured by Wako Pure Chemical Industries, Ltd.). Further, 2-chloromethylquinoline hydrochloride or 6-methoxy-2-chloromethylquinoline used as compound (2) is a commercially available product (manufactured by Tokyo Chemical Industry Co., Ltd.) or a method known per se (Tetrahedron 2004, 60, 11057-11065) or a method analogous thereto.

Example 1
Synthesis of 1,4-bis (quinolin-2-ylmethyl) piperazine (compound (I-1a))

To a solution of 2-chloromethylquinoline hydrochloride (993 mg, 4.64 mmol) in acetonitrile (dried with CaSO ₄ ) (70 mL), piperazine (200 mg, 2.32 mmol), potassium carbonate (2.60 g, 18. 8 mmol) and potassium iodide (385 mg, 2.32 mmol) were added. The reaction was heated to reflux at 110 ° C. for 2 days. After the solvent was distilled off, acetonitrile was added and filtered. After the solvent was further distilled off, chloroform was added and further filtered, the solvent was distilled off, and a yellow solid and orthorhombic colorless crystals (774 mg (2. 10 mmol): yield 91%).
¹ H NMR (CDCl ₃ , 300 MHz): δ (ppm) 8.12 (d, J = 8.9 Hz, 2H), 8.06 (d, J = 8.9 Hz, 2H), 7.79 (d, J = 8.2 Hz, 2H), 7.68-7.63 (m, 4H), 7.51 (d, J = 7.6 Hz2H), 3.86 (s, 4H), 2.63 (s, 8H).
¹³ C NMR (CDCl _3, 75.5 MHz): δ (ppm) 159.8 _, 147.8 _, 136.6 _, 129.6 _, 129.3 _, 127.8 _, 127.6 _, 126.4, 121. 4, 65.7, 54.0.

(Example 2)
Synthesis of 1,4-bis (quinolin-2-ylmethyl) -1,4-diazacycloheptane (compound (I-1b))

To a solution of 2-chloromethylquinoline hydrochloride (3.81 g, 17.8 mmol) in acetonitrile (dried with CaSO ₄ ) (70 mL), homopiperazine (1.00 g, 10.0 mmol), potassium carbonate (12 0.0 g, 86.8 mmol) and potassium iodide (3.42 g, 20.6 mmol) were added. The reaction was heated to reflux at 100 ° C. for 2 days. After the solvent was distilled off, acetonitrile was added and filtered. After the solvent was further distilled off, chloroform was added and further filtered, and the solvent was distilled off to obtain a black oil (3.31 g (8.65 mmol): Rate 87%).
¹ H NMR (CDCl ₃ , 300 MHz): δ (ppm) 8.24 (d, J = 8.2 Hz, 2H), 8.19 (d, J = 8.2 Hz, 2H), 7.77-7. 92 (m, 6H), 7.62 (dd, J = 7.0, 7.0 Hz, 2H), 4.13 (s, 4H), 3.00 (t, J = 6.0 Hz, 4H), 2.94 (s, 4H), 1.99 (quant., J = 6.0 Hz, 2H).
¹³ C NMR (CDCl _3, 75.5 MHz): δ (ppm) 160.5 _, 147.2 _, 136.0 _, 129.0 _, 128.7 _, 127.2 _, 127.1 _, 125.8, 120. 87, 65.0, 55.7, 54.7, 28.0.

(Example 3)
Synthesis of 1,4-bis (6-methoxyquinolin-2-ylmethyl) piperazine (compound (I-1c))

To a solution of 6-methoxy-2-chloromethylquinoline (968 mg, 4.66 mmol) in acetonitrile (dried with CaSO ₄ ) (70 mL), piperazine (200 mg, 2.32 mmol), potassium carbonate (1.84 g, 13 .3 mmol) and potassium iodide (802 mg, 4.83 mmol) were added. The reaction was heated to reflux at 100 ° C. for 2 days. After the solvent was distilled off, acetonitrile was added and filtered, and after the solvent was further distilled off, chloroform was added and further filtered, and the solvent was distilled off to obtain a yellow solid. Further, washing with acetonitrile gave a pale yellow powder (716 mg (1.67 mmol): yield 72%).
¹ H NMR (CDCl ₃ , 300 MHz): δ (ppm) 8.01 (d, J = 8.9 Hz, 2H), 7.95 (d, J = 8.9 Hz, 2H), 7.58 (d, J = 8.5 Hz, 2H), 7.33 (dd, J = 2.7, 9.0 Hz, 2H), 7.06 (d, J = 2.7 Hz, 2H), 3.92 (s, 6H) ), 3.81 (s, 4H), 2.61 (s, 8H).
¹³ C NMR (CDCl _3, 75.5 MHz): δ (ppm) 157.6 _, 157.1 _, 143.9 _, 135.4 _, 130.7 _, 128.5 _, 122.1 _, 121.8, 105. 5, 65.57, 56.0, 53.9.

(Example 4)
Synthesis of 1,4-bis (6-methoxyquinolin-2-ylmethyl) -1,4-diazacycloheptane (compound (I-1d))

To a solution of 6-methoxy-2-chloromethylquinoline (839 mg, 4.04 mmol) in acetonitrile (dried with CaSO ₄ ) (70 mL), homopiperazine (200 mg, 2.00 mmol), potassium carbonate (1.71 g, 12.4 mmol) and potassium iodide (689 mg, 4.15 mmol) were added. The reaction was heated to reflux at 110 ° C. for 4 days. After the solvent was distilled off, acetonitrile was added and filtered. After the solvent was further distilled off, chloroform was added and further filtered, and the solvent was distilled off to obtain a brown oil. The oil was washed with acetonitrile to obtain a pale yellow powder (495 mg (1.21 mmol): yield 60%).
¹ H NMR (CDCl ₃ , 300 MHz): δ (ppm) 8.00 (d, J = 8.4 Hz, 2H), 7.94 (d, J = 9.2 Hz, 2H), 7.64 (d, J = 8.4 Hz, 2H), 7.31 (dd, J = 2.8, 9.2 Hz, 2H), 7.05 (d, J = 2.8 Hz, 2H), 3.94 (s, 4H) ), 3.91 (s, 6H), 2.84 (dd, J = 5.8, 5.8 Hz, 4H), 2.79 (s, 4H), 1.85 (quint, J = 5.8 Hz) , 2H).
¹³ C NMR (CDCl _3, 75.5 MHz): δ (ppm) 157.9 _, 157.0 _, 143.2 _, 134.9 _, 130.1 _, 128.0 _, 121.5 _, 121.2, 105. 0, 64.9, 55.6, 55.5, 54.7, 28.0.

(Test Example 1)
When the compound (I-1a) was irradiated with light having a wavelength of 317 nm in acetonitrile, almost no fluorescence was emitted.
The fluorescence response of compound (I-1a) in acetonitrile was examined in the presence of various metal ions (2 equivalents).
As a result, compound (I-1a) emitted strong fluorescence in the presence of trivalent iron ions and mercury ions, but other metal ions had almost no effect on the fluorescence of compound (I-1a). (Figure 1).
According to FIG. 1, compound (I-1a) fluoresces only when trivalent iron ions and mercury ions are present, and does not complex with other metal ions, or fluoresces even when complexed. I knew that I would not.

(Test Example 2)
When the compound (I-1b) was irradiated with light having a wavelength of 317 nm in acetonitrile, almost no fluorescence was emitted.
The fluorescence response of compound (I-1b) in acetonitrile was examined in the presence of various metal ions (2 equivalents).
As a result, the compound (I-1b) emitted strong fluorescence in the presence of trivalent iron ions and mercury ions, and also emitted weak fluorescence in the presence of chromium ions, copper ions or lead ions. Other metal ions hardly affected the fluorescence of compound (I-1b) (FIG. 2).
According to FIG. 2, compound (I-1b) fluoresces only when trivalent iron ions and mercury ions are present, and does not complex with other metal ions, or fluoresces even when complexed. I knew that I would not.

(Test Example 3)
When the compound (I-1d) was irradiated with light having a wavelength of 338 nm in acetonitrile, the compound (I-1d) hardly emitted fluorescence.
The fluorescence response of the compound (I-1d) in acetonitrile was examined in the presence of various metal ions (2 equivalents).
As a result, compound (I-1d) emitted strong fluorescence in the presence of cadmium ions. Further, although weak fluorescence was emitted even in the presence of trivalent iron ions, mercury ions, or calcium ions, other metal ions hardly affected the fluorescence of compound (I-1d) (FIG. 3).
According to FIG. 3, it was found that the compound (I-1d) emits fluorescence only when cadmium ions are present, and does not complex with other metal ions or does not emit fluorescence even when complexed.

(Test Example 4)
Compound (I-1a) was titrated with mercury ions, and the change was followed by fluorescence spectrum (FIG. 4). The change in fluorescence intensity with respect to the added amount of mercury ions at a wavelength of 408 nm was also traced (FIG. 5). As a result, it did not have fluorescence up to 1 equivalent of mercury ion, but emitted fluorescence when exceeding 1 equivalent, and the change became maximum at around 1.5 equivalent. That is, it was found that the first complex formed of mercury ion: compound (I-1a) = 1: 1 did not exhibit fluorescence, and the complex formed in an excess of mercury has fluorescence.

(Test Example 5)
The compound (I-1a) was titrated with trivalent iron ions, and the change was followed by the fluorescence spectrum (FIG. 6). The change in fluorescence intensity with respect to the added amount of trivalent iron ions at a wavelength of 411 nm was also traced (FIG. 7). As a result, although there is no fluorescence up to 0.5 equivalent of trivalent iron ion, it emits fluorescence when exceeding 0.5 equivalent, and the change shows an inflection point in the vicinity of 1 equivalent and the maximum in the vicinity of 1.5 equivalent. It became. That is, the initially formed complex of trivalent iron ion: compound (I-1a) = 0.5: 1 does not exhibit fluorescence, and trivalent iron ion: compound (I-1a) = 1: 1. This complex showed moderate fluorescence, and it was found that the complex formed with an excess of trivalent iron ions has higher fluorescence.

(Test Example 6)
Compound (I-1d) was titrated with cadmium ions, and the change was monitored by fluorescence spectrum (FIG. 8). Light of 338 nm was used for excitation. The change in fluorescence intensity with respect to the amount of cadmium ions added at a wavelength of 405 nm was also traced (FIG. 9). As a result, it was found that simply by introducing a methoxy group at the 6-position of the quinolyl group of R ¹ and R ² , the ability to form a complex with respect to cadmium ions is improved as compared with trivalent iron ions and mercury ions. Moreover, it turned out that the complex of cadmium ion: compound (I-1d) = 1: 1 shows fluorescence.

  ADVANTAGE OF THE INVENTION According to this invention, the fluorescent probe for a metal measurement which forms complex with a specific metal ion, especially trivalent iron ion, mercury ion, and cadmium ion, and has selective recognition ability can be provided. Since the fluorescent probe according to the present invention can be easily synthesized from an inexpensive raw material, functional group conversion can be easily performed, and the types and detection sensitivity of metal ions that can be detected by the functional group conversion can be easily adjusted. It also has the advantage of being able to. Furthermore, according to the present invention, a highly sensitive measurement method of metal ions using the fluorescent probe can also be provided.

Claims (15)

Formula (I):

[Where
R ¹ and R ² each independently represent an optionally substituted 2-quinolyl group or an optionally substituted 8-quinolyl group;
m and n each independently represent 0, 1 or 2, and m + n = 1, 2, 3 or 4. ]
Or a salt thereof (excluding 1,4-bis (quinolin-2-ylmethyl) -1,4-diazacycloheptane). The compound or a salt thereof according to claim ^1, wherein R ¹ and R ² are each independently a 2-quinolyl group which may be substituted. R ¹ and R ² are each independently a 2-quinolyl group optionally substituted by a C _1-6 alkoxy group, and m and n are each independently 1 or 2, and The compound or its salt of Claim 1 which is m + n = 2 or 3. Formula (I):

[Where
R ¹ and R ² each independently represent an optionally substituted 2-quinolyl group or an optionally substituted 8-quinolyl group;
m and n each independently represent 0, 1 or 2, and m + n = 1, 2, 3 or 4. ]
The fluorescent probe for a metal ion measurement containing the compound or its salt represented by these. The fluorescent probe according to claim 4, wherein R ¹ and R ² are each independently a 2-quinolyl group which may be substituted. R ¹ and R ² are each independently a 2-quinolyl group optionally substituted by a C _1-6 alkoxy group, and m and n are each independently 1 or 2, and The fluorescent probe according to claim 4, wherein m + n = 2 or 3.   The fluorescent probe according to any one of claims 4 to 6, wherein the metal ion is an iron ion, a mercury ion or a cadmium ion. Formula (I):

[Where
R ¹ and R ² each independently represent an optionally substituted 2-quinolyl group or an optionally substituted 8-quinolyl group;
m and n each independently represent 0, 1 or 2, and m + n = 1, 2, 3 or 4. ]
Or a metal complex formed from a salt thereof and a metal ion (however, from 1,4-bis (quinolin-2-ylmethyl) -1,4-diazacycloheptane and an iron ion or a manganese ion) Excluding complexes formed). The metal complex according to claim 8, wherein R ¹ and R ² are each independently an optionally substituted 2-quinolyl group. R ¹ and R ² are each independently a 2-quinolyl group optionally substituted by a C _1-6 alkoxy group, and m and n are each independently 1 or 2, and The metal complex according to claim 8, wherein m + n = 2 or 3.   The metal complex according to any one of claims 8 to 10, wherein the metal ion is an iron ion, a mercury ion or a cadmium ion. A method for measuring metal ions, including the following steps:
(1) Formula (I):

[Where
R ¹ and R ² each independently represent an optionally substituted 2-quinolyl group or an optionally substituted 8-quinolyl group;
m and n each independently represent 0, 1 or 2, and m + n = 1, 2, 3 or 4. ]
A step of reacting the compound represented by the above or a salt thereof with a metal ion, and (2) a step of measuring the fluorescence intensity of the metal complex formed in the above step. The method according to claim 12, wherein R ¹ and R ² are each independently an optionally substituted 2-quinolyl group. R ¹ and R ² are each independently a 2-quinolyl group optionally substituted by a C _1-6 alkoxy group, and m and n are each independently 1 or 2, and The method according to claim 12, wherein m + n = 2 or 3.   The method according to any one of claims 12 to 14, wherein the metal ions are iron ions, mercury ions or cadmium ions.

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