JP2014218456A - Novel hydrogen halide salts - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide novel substances excellent in water solubility and available as luminescent substrates in firefly bioluminescent systems.SOLUTION: There are provided hydrogen halide salts of compounds represented by the specified general formula (I) or the specified general formula (II).

Description

  The present invention relates to a hydrohalide salt, particularly a hydrohalide salt that is excellent in water solubility and can be used as a luminescent substrate in a firefly bioluminescence system.

Among the bioluminescent systems, the firefly luminescent system is known as a system excellent in luminous efficiency. In the firefly luminescence system, the luminescent substrate firefly luciferin is converted to excited oxyluciferin in the presence of the luminescent enzyme firefly luciferase, adenosine triphosphate (ATP) and magnesium ion (Mg ²⁺ ). When the oxyluciferin is deactivated to the ground state, yellow-green fluorescence having a wavelength of about 560 nm is emitted.

  Recently, compounds that realize various emission wavelengths have been synthesized as analogs of the luminescent substrate of such a firefly luminescent system. For example, Patent Document 1 below discloses a luciferin derivative in which the phenolic hydroxyl group of firefly luciferin is substituted with a secondary or tertiary amino group. Patent Documents 2 and 3 listed below disclose a luciferase luminescent substrate having a molecular structure similar to firefly luciferin.

  Among these firefly luciferin analogs, a long-wavelength light, in particular, a luminescent substrate that emits red light having a wavelength of 650 nm or more has a high transmissivity in the living body. For example, a firefly luciferin analog that emits long-wavelength light is commercially available from Wako Pure Chemical Industries, Ltd. under the trade name “Akalumine”.

JP 2007-91695 A JP 2009-184932 A International Publication No. 2013/027770

  In addition, the present inventors have further studied, a compound having a molecular structure similar to firefly luciferin and having two aromatic rings in the molecular structure functions as a luminescent substrate in the firefly bioluminescence system, It has been found to emit long wavelength light.

  However, although the above-mentioned luminescent substrate analogues of the firefly luminescence system can realize various emission wavelengths, they are low in water solubility, and are particularly prominent in luminescent substrates that emit light having a wavelength of 650 nm or more, which is useful for visualization in the deep part of the living body. is there. In general, in the administration to the living body of a laboratory animal such as a mouse or a rat, the luminescent substrate needs to have a solubility of about 10 to 15 mg / ml. Solubility in water was about 0.1 mg / ml, and there was a problem in practicality.

  Accordingly, an object of the present invention is to solve the above-mentioned problems of the prior art and to provide a novel substance that is excellent in water solubility and can be used as a luminescent substrate in a firefly bioluminescent system.

  As a result of intensive studies to achieve the above object, the present inventors have chlorinated a water-insoluble luminescent substrate having a specific molecular structure and functioning as a luminescent substrate in a firefly bioluminescent system with hydrogen halide. Thus, the present inventors have found that the water solubility is greatly improved while maintaining the luminous ability in the firefly bioluminescence system, and have completed the present invention.

［式中、Ｒ¹及びＲ²はそれぞれ独立して水素又は炭素数１〜３のアルキル基であり、Ｒ³はそれぞれ独立して炭素数１〜８のアルキル基又は炭素数２〜８のアルケニル基であり、ｎは０〜３の整数であり、ｍは０〜４の整数である］、又は、
下記一般式（II）：

［式中、Ｒ⁴及びＲ⁵はそれぞれ独立して水素又は炭素数１〜３のアルキル基であり、Ｒ⁶及びＲ⁷はそれぞれ独立して炭素数１〜８のアルキル基又は炭素数２〜８のアルケニル基であり、ｐ及びｑはそれぞれ独立して０〜４の整数である］で表される化合物のハロゲン化水素塩が提供され、該ハロゲン化水素塩は、水溶性に優れ、ホタル生物発光系における発光基質として機能する。 That is, according to the present invention, the following general formula (I):

[Wherein, R ¹ and R ² are each independently hydrogen or an alkyl group having 1 to 3 carbon atoms, and R ³ is each independently an alkyl group having 1 to 8 carbon atoms or an alkenyl group having 2 to 8 carbon atoms. A group, n is an integer of 0-3, m is an integer of 0-4], or
The following general formula (II):

[Wherein, R ⁴ and R ⁵ are each independently hydrogen or an alkyl group having 1 to 3 carbon atoms, and R ⁶ and R ⁷ are each independently an alkyl group having 1 to 8 carbon atoms or 2 to 2 carbon atoms. 8 is an alkenyl group, and p and q are each independently an integer of 0 to 4.], and the hydrogen halide salt has excellent water solubility, It functions as a luminescent substrate in a bioluminescent system.

  The hydrohalide salt of the present invention is preferably a hydrochloride or hydrobromide of the compound represented by the general formula (I) or the general formula (II). In this case, the water solubility of the hydrogen halide salt is particularly excellent.

In another preferable example of the hydrogen halide salt of the present invention, R ¹ and R ² in the general formula (I), and R ⁴ and R ⁵ in the general formula (II) are 1 to 1 carbon atoms. 3 alkyl groups. Here, R ¹ and R ² in the general formula (I) and R ⁴ and R ⁵ in the general formula (II) are more preferably methyl groups. In this case, the luminous efficiency of the hydrogen halide salt is particularly excellent.

で表される化合物の塩酸塩又は臭化水素酸塩が特に好ましい。この場合、ハロゲン化水素塩の水溶性、発光効率、生体内深部の可視化、入手容易性が特に優れる。 Among the hydrogen halide salts of the present invention, the hydrogen halide salt of the compound represented by the general formula (I) is preferable, and the following chemical formula (III):

The hydrochloride or hydrobromide of the compound represented by the formula is particularly preferred. In this case, the water solubility, luminous efficiency, visualization of the deep part of the living body, and availability are particularly excellent.

  According to the present invention, a novel hydrogen halide salt that is excellent in water solubility and can be used as a luminescent substrate in a firefly bioluminescence system can be provided.

The IR spectrum of Aka Lumine is shown. Fig. 2 shows an IR spectrum of a red luminescence. 2 shows the UV spectrum of Acaluminescence. 2 shows the UV spectrum of acalumine hydrochloride. The luminescence intensity of Acalumine, Acalumine hydrochloride and Acalumine hydrobromide is shown. The emission spectra of acalumine, acalumine hydrochloride and acalumine hydrobromide are shown.

［式中、Ｘ¹はハロゲン元素であり、Ｒ¹及びＲ²はそれぞれ独立して水素又は炭素数１〜３のアルキル基であり、Ｒ³はそれぞれ独立して炭素数１〜８のアルキル基又は炭素数２〜８のアルケニル基であり、ｎは０〜３の整数であり、ｍは０〜４の整数であり、ａは０を超え且つ２以下である］、又は、
下記一般式（V）：

［式中、Ｘ²はハロゲン元素であり、Ｒ⁴及びＲ⁵はそれぞれ独立して水素又は炭素数１〜３のアルキル基であり、Ｒ⁶及びＲ⁷はそれぞれ独立して炭素数１〜８のアルキル基又は炭素数２〜８のアルケニル基であり、ｐ及びｑはそれぞれ独立して０〜４の整数であり、ｂは０を超え且つ２以下である］で表される。本発明のハロゲン化水素塩は、ハロゲン化水素が付加しているため、水溶性に優れ、また、分子構造がホタルルシフェリンと類似しているため、ホタル生物発光系における発光基質として利用できる。 The present invention is described in detail below. The hydrogen halide salt of the present invention is a hydrogen halide salt of a compound represented by the above general formula (I) or general formula (II), preferably the following general formula (IV):

[Wherein, X ¹ is a halogen element, R ¹ and R ² are each independently hydrogen or an alkyl group having 1 to 3 carbon atoms, and R ³ is each independently an alkyl group having 1 to 8 carbon atoms. Or an alkenyl group having 2 to 8 carbon atoms, n is an integer of 0 to 3, m is an integer of 0 to 4, and a is more than 0 and 2 or less], or
The following general formula (V):

[Wherein, X ² represents a halogen element, R ⁴ and R ⁵ each independently represent hydrogen or an alkyl group having 1 to 3 carbon atoms, and R ⁶ and R ⁷ each independently represent 1 to 8 carbon atoms. Or an alkenyl group having 2 to 8 carbon atoms, p and q are each independently an integer of 0 to 4, and b is more than 0 and 2 or less. The hydrogen halide salt of the present invention has excellent water solubility because of the addition of hydrogen halide, and has a molecular structure similar to that of firefly luciferin, so that it can be used as a luminescent substrate in a firefly bioluminescence system.

<Hydrogen halide salt of compound represented by general formula (I), hydrohalide salt represented by general formula (IV)>
In the general formula (IV), X ¹ is a halogen element, and examples of the halogen element include fluorine, chlorine, bromine, iodine, etc. Among these, chlorine and bromine are preferable from the viewpoint of water solubility. That is, as the hydrogen halide salt of the compound represented by the general formula (I) and the hydrogen halide salt represented by the general formula (IV), hydrochloride and hydrobromide are preferable.

In the general formulas (I) and (IV), R ¹ and R ² are hydrogen or an alkyl group having 1 to 3 carbon atoms, and R ¹ and R ² may be the same or different. Here, examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, and an i-propyl group. In addition, as R < ¹ > and R < ² >, a C1-C3 alkyl group is preferable from a viewpoint of luminous efficiency, and a methyl group is especially preferable.

In the general formulas (I) and (IV), R ³ is an alkyl group having 1 to 8 carbon atoms or an alkenyl group having 2 to 8 carbon atoms, and when a plurality of R ³ are present, each R ³ is the same or different. May be. Here, examples of the alkyl group include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, and an octyl group. Examples of the alkenyl group include a vinyl group, an allyl group, a butenyl group, a pentenyl group, and an octenyl group. Is mentioned.
Further, in the general formula (I) and (IV), R ³ is a substituent of the benzene ring, m indicating the number of the substituents is an integer of 0-4. In the present invention, from the viewpoint of ease of synthesis, it is preferable that m is 0, that is, no substituent R ³ is present on the benzene ring.

  In the general formulas (I) and (IV), n represents the number of repeating vinylene units (—CH═CH—) and is an integer of 0 to 3. Since the emission wavelength becomes longer as the number of n is larger, n is preferably 2 or 3 from the viewpoint of visualization of the deep part in the living body, and n is preferably 2 from the viewpoint of availability.

In the general formula (IV), a represents an average addition number of hydrogen halide (HX ¹ ), which is greater than 0 and not greater than 2, and preferably 1. In the hydrogen halide salt of the compound represented by the general formula (I) and the hydrogen halide salt represented by the general formula (IV), hydrogen halide can be added to two nitrogen atoms in the molecule.

The hydrogen halide salt of the compound represented by the above general formula (I) (particularly, the hydrogen halide salt represented by the above general formula (IV)) can be synthesized as follows. For example, as described in JP-A-2009-184932 (the above-mentioned Patent Document 2), an aldehyde such as 4-dimethylaminocinnamic aldehyde is used as a starting material and reacted with carbethoxymethylenetriphenylphosphorane to produce ethyl. An ester is obtained, and then the ethyl ester is converted to a carboxyl in an aqueous sodium hydroxide solution. On the other hand, a D-cysteine-S-trityl compound is reacted in hydrogen chloride and 1,4-dioxane solution to prepare a methyl ester form, and then the methyl ester form is reacted with the carboxyl form to produce an amide. Generate a body. Next, the amide form is cyclized with triphenylphosphine oxide and trifluoromethanesulfonic anhydride to form a heterocyclic ring to obtain a thiazoline form. Next, the methyl ester portion of the thiazoline body is hydrolyzed to obtain a carboxyl body having a thiazoline ring (that is, a compound represented by the above general formula (I)). Subsequently, a carboxylate having a thiazoline ring is reacted with hydrogen halide (hydrogen chloride, hydrogen bromide, etc.) to give a hydrogen halide salt of the compound represented by the general formula (I) (in particular, the general formula (IV ) Can be obtained. In addition, the desired hydrogen halide salt can be obtained by appropriately changing the starting material, introducing various substituents, or utilizing other synthetic routes. In the reaction of a carboxylate having a thiazoline ring and a hydrogen halide, the reaction temperature is not particularly limited, and the amount of hydrogen halide used can be changed according to the purpose. It is preferable to use an excess amount relative to the carboxylate having a thiazoline ring.
In addition, as a carboxyl compound, a commercially available product name “Aka Lumine” (available from Wako Pure Chemical Industries, Ltd., also available from Kurokin Kasei Co., Ltd., a compound represented by the above chemical formula (III)) is used. A hydrogen halide salt of the compound represented by the above chemical formula (III) can also be obtained by reacting hydrogen halide.

<Hydrogen halide salt of compound represented by general formula (II), hydrohalide salt represented by general formula (V)>
In the above general formula (V), X ² is a halogen element, and examples of the halogen element include fluorine, chlorine, bromine, iodine, etc. Among these, chlorine and bromine are preferable from the viewpoint of water solubility. That is, as the hydrogen halide salt of the compound represented by the general formula (II) and the hydrogen halide salt represented by the general formula (V), hydrochloride and hydrobromide are preferable.

In the general formulas (II) and (V), R ⁴ and R ⁵ are hydrogen or an alkyl group having 1 to 3 carbon atoms, and R ⁴ and R ⁵ may be the same or different. Here, examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, and an i-propyl group. In addition, as R < ⁴ > and R < ⁵ >, a C1-C3 alkyl group is preferable from a viewpoint of luminous efficiency, and a methyl group is especially preferable.

In the general formula (II) and (V), R ⁶ and R ⁷ is an alkenyl group of alkyl or 2 to 8 carbon atoms having 1 to 8 carbon atoms, or different from R ⁶ and R ⁷ are each identical Good. Here, examples of the alkyl group include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, and an octyl group. Examples of the alkenyl group include a vinyl group, an allyl group, a butenyl group, a pentenyl group, and an octenyl group. Is mentioned.
Moreover, in said general formula (II) and (V), R < ⁶ > and R < ⁷ > is a substituent of each benzene ring, p and q which show the number of this substituent are integers of 0-4, p and Each q may be the same or different. In the present invention, from the viewpoint of ease of synthesis, p and q are preferably 0, that is, it is preferable that substituents R ⁶ and R ⁷ do not exist in each benzene ring.

In the above general formula (V), b represents the average addition number of hydrogen halide (HX ² ), which is more than 0 and 2 or less, preferably 1. In the hydrogen halide salt of the compound represented by the general formula (II) and the hydrogen halide salt represented by the general formula (V), hydrogen halide can be added to two nitrogen atoms in the molecule.

  The hydrogen halide salt of the compound represented by the general formula (II) (particularly, the hydrogen halide salt represented by the general formula (V)) can be synthesized as follows. For example, 4- (N, N-dimethylamino) phenylboronic acid is used as a starting material and reacted with ethyl 4-iodobenzoate to obtain a biphenylethyl ester, which is then converted to sodium hydroxide. Convert to carboxyl in aqueous solution. Meanwhile, D-cysteine methyl ester hydrochloride is reacted with triphenylmethanol to prepare a methyl ester form of a D-cysteine-S-trityl compound, and then the methyl ester form is reacted with the carboxyl form, An amide is formed. Next, the amide form is cyclized with triphenylphosphine oxide and trifluoromethanesulfonic anhydride to form a heterocyclic ring to obtain a thiazoline form. Next, the methyl ester portion of the thiazoline body is hydrolyzed to obtain a carboxyl body having a thiazoline ring (that is, a compound represented by the general formula (II)). Subsequently, a carboxylate having a thiazoline ring is reacted with a hydrogen halide (hydrogen chloride, hydrogen bromide, etc.) to give a hydrogen halide salt of the compound represented by the general formula (II) (in particular, the general formula (V ) Can be obtained. In addition, the desired hydrogen halide salt can be obtained by appropriately changing the starting material, introducing various substituents, or utilizing other synthetic routes. In the reaction of a carboxylate having a thiazoline ring and a hydrogen halide, the reaction temperature is not particularly limited, and the amount of hydrogen halide used can be changed according to the purpose. It is preferable to use an excess amount relative to the carboxylate having a thiazoline ring.

  Among the hydrogen halide salts of the compounds represented by the above general formula (I) or general formula (II), from the viewpoints of water solubility, luminous efficiency, and ease of synthesis, represented by the above general formula (I) The hydrogen halide salt of the compound is preferred.

［式中、ａは０を超え且つ２以下である］で表される塩酸塩、及び、
下記一般式（VII）：

［式中、ａは０を超え且つ２以下である］で表される臭化水素酸塩が特に好ましい。なお、一般式（VI）及び一般式（VII）中、ａは塩化水素（ＨＣｌ）又は臭化水素（ＨＢｒ）の平均付加数を示し、０を超え且つ２以下であり、好ましくは１である。 Further, among the hydrogen halide salts of the compound represented by the general formula (I), the compound represented by the above chemical formula (III) from the viewpoint of water solubility, light emission efficiency, visualization in the living body, and availability. Hydrochloride and hydrobromide are preferred, and the following general formula (VI):

[Wherein a is greater than 0 and less than or equal to 2], and
The following general formula (VII):

The hydrobromide represented by the formula [wherein a is greater than 0 and not greater than 2] is particularly preferred. In general formula (VI) and general formula (VII), a represents the average addition number of hydrogen chloride (HCl) or hydrogen bromide (HBr), which exceeds 0 and is 2 or less, preferably 1. .

The hydrogen halide salt of the present invention emits light by being oxidized by a luminescent beetle luciferase when added to a system in which luminescent beetle luciferase, adenosine triphosphate (ATP) and magnesium ion (Mg ²⁺ ) are present. The hydrogen halide salt of the present invention can also be provided as a luminescence detection kit together with ATP and Mg ²⁺ , and the luminescence detection kit includes other luminescent substrates and solutions adjusted to an appropriate pH. May be.

  When the hydrogen halide salt of the present invention is applied to a light emitting system, it is preferable to use the hydrogen halide salt of the present invention at a concentration of 1 μM or more in order to obtain a suitable light emission intensity. More preferably. The pH of the luminescent system is preferably 4 to 10, more preferably 6 to 8. If necessary, a buffering agent such as potassium phosphate, Tris-HCl, glycine, HEPES is used to stabilize the pH. May be used.

  Moreover, the hydrogen halide salt of the present invention can be made to emit light by various oxidases in the firefly luminescence beetle luciferase luminescence system. Luciferase has been isolated from North American firefly (Photinus pyralis), railroad worm (Railroad worm), etc., and any of them can be used. Examples of the oxidase that can be used include Hikari Komekimushiluciferase, Iriomote luciferase, and flavin-containing monooxygenase.

Bioluminescence using the hydrogen halide salt of the present invention as a luminescent substrate is enhanced when coenzyme A (CoA), pyrophosphate, or magnesium ion (Mg ²⁺ ) is present in the luminescence system. The luminescence enhancement effect of these compounds is remarkable when the concentration of CoA, pyrophosphate or Mg ²⁺ in the luminescence system is 5 μM or more, respectively, and the luminescence is enhanced as the concentration increases.

  In order to use the firefly bioluminescence system for measurement / detection, it is preferable to stabilize the luminescence so as to prevent the inactivation of the enzyme and show a plateau luminescence behavior, for example, the presence of magnesium ions in the luminescence system It is preferable to coexist magnesium ions and pyrophosphate. In the case of magnesium ion alone, from the viewpoint of light emission stabilization, the magnesium ion concentration of the light emitting system is preferably 0.5 mM or more, and the stability of light emission improves as the concentration increases. When using magnesium pyrophosphate, the concentration of the magnesium pyrophosphate in the luminescent system is preferably 10 μM or more, and more preferably 100 μM or more, from the viewpoint of stabilization of light emission. In addition, the ratio of pyrophosphate and magnesium ion may not be an equivalent ratio. Suitable magnesium salts include inorganic acid salts such as magnesium sulfate and magnesium chloride, and organic acid salts such as magnesium acetate. Suitable pyrophosphates include alkali metal pyrophosphates such as sodium and potassium, alkaline earth metal pyrophosphates such as magnesium and calcium, and iron pyrophosphate.

  The hydrogen halide salt of the present invention can be used as a luminescent label in biological measurement / detection, and can be used, for example, for labeling amino acids, polypeptides, proteins, nucleic acids and the like. The method for bonding the hydrogen halide salt of the present invention to these substances is well known to those skilled in the art. For example, using methods well known to those skilled in the art, the carboxyl group and amino group of the target substance can be bonded. Thus, the hydrogen halide salt of the present invention can be bound.

  Further, the hydrogen halide salt of the present invention can be used for measurement / detection utilizing detection of luminescent beetle luciferase activity by luminescence of a luminescent substrate. For example, by administering the hydrogen halide salt of the present invention to a cell or animal into which a luciferase gene has been introduced, the expression of the target gene or protein in vivo can be measured / detected. Note that light having a long wavelength has high light permeability and high tissue permeability. Therefore, among the hydrogen halide salts of the present invention, a hydrogen halide salt having long-wavelength light emission is useful as a labeling material for visualizing a deep part in a living body.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples.

(1) Instrument analysis and measuring device pH measurement: Measured using pH test paper UNIV manufactured by Toyo Roshi Kaisha, Ltd. Moreover, it measured using HORIBA pH / ION METER F-23 as a pH meter.

¹ H nuclear magnetic resonance spectrum ( ¹ H NMR): Measured using a Lambda-270 type apparatus (270 MHz) manufactured by JEOL. It was described as “ ¹ H NMR (measurement frequency, measurement solvent): δ chemical shift value (number of hydrogen, multiplicity, spin coupling constant)”. The chemical shift value (δ) is expressed in pp using tetramethylsilane (δ = 0) as an internal standard. The multiplicity is displayed as s (single line), d (double line), t (triple line), q (quadruple line), m (multiple line or complicated overlapping signal), and a wide range of signals is represented by br It was written. The spin coupling constant (J) is described in Hz.

(2) Chromatography Analytical thin layer chromatography (TLC): A Merck TLC plate, silica gel 60F ₂₅₄ (Art. 5715) thickness 0.25 mm was used. Detection of the compound on TLC was performed by irradiating with UV irradiation (254 nm or 365 nm) and a color former, followed by heating to cause color development. As the color former, p-anisaldehyde (9.3 ml) and acetic acid (3.8 ml) dissolved in ethanol (340 ml) and concentrated sulfuric acid (12.5 ml) were added.

Preparative thin layer chromatography (PTLC): A Merck TLC plate, silica gel 60F ₂₅₄ (Art. 5744) 0.5 mm thick is used, or E.I. A silica gel 60GF ₂₅₄ (Art. 7730) for thin layer chromatography manufactured by Merck was used on a 20 cm × 20 cm glass plate adjusted to a thickness of 1.75 mm.

Silica gel column chromatography: This was performed using Merck silica gel 60F ₂₅₄ (Art. 7734).

(3) Basic operation The reaction solution was cooled by immersing the reaction vessel in a dewar filled with a refrigerant. From room temperature to 4 ° C., ice water, and from 4 ° C. to −90 ° C., liquid nitrogen-acetone was used as a refrigerant. The extracted solution after the reaction was dried by washing with saturated brine and then adding anhydrous sodium sulfate or anhydrous magnesium sulfate. About what performed neutralization after reaction with resin, the cation exchange resin Amberlite IR120B NA or the anion exchange resin Amberlite IRA400 OH AG by Organo Corporation was used. The solution was concentrated under reduced pressure using a rotary evaporator under reduced pressure of an aspirator (20 to 30 mmHg). The trace amount of the solvent was removed using a vacuum pump (about 1 mmHg) equipped with a trap cooled in a liquid nitrogen bath. All solvent mixing ratios were expressed in volume ratios.

<Synthesis of biphenyl-type luminescent substrate>
(Synthesis of biphenyl derivative (2))
Ethyl 4-iodobenzoate (250 μl, 1.5 mmol), tetrakis (triphenylphosphine) palladium (0) (100 mg, 5%), potassium carbonate (539.0 mg, 3.8 mmol) were added to N, N-dimethylformamide (6 ml) and placed under an argon atmosphere. 4- (N, N-dimethylamino) phenylboronic acid (1) (315.0 mg, 1.9 mmol) was added, and the mixture was stirred with heating at 90 ° C. for 14 hr. Furthermore, tetrakis (triphenylphosphine) palladium (0) (27 mg, 2%), potassium carbonate (125.0 mg, 0.90 mmol), 4- (N, N-dimethylamino) phenylboronic acid (1) (97.0 mg, 0.58 mmol) was added and stirred for 24 hours. The reaction mixture was subjected to silica gel column chromatography [ethyl acetate] to remove insoluble matters, and then concentrated under reduced pressure. The obtained crude product was purified by silica gel column chromatography [hexane-ethyl acetate (4: 1) → (0: 1)], and the biphenyl derivative (2) (229.0 mg, 0.85 mmol, 56%) was white. Obtained as a solid.

・ Identification result of biphenyl derivative (2)
¹ H NMR (500 MHz, CDCl ₃ ): δ 1.40 (3H, t, J = 6.9 Hz), 3.00 (6H, s), 4.38 (2H, q, J = 6.9 Hz), 6.79 (2H, d, J = 9.2 Hz), 7.55 (2H, d, J = 9.2 Hz), 7.61 (2H, d, J = 8.0 Hz), 8.05 (2H, d, J = 8.6 Hz)

(Synthesis of carboxylic acid (3))
The biphenyl derivative (2) (148.0 mg, 0.54 mmol) was dissolved in ethanol (6 ml), 1M aqueous sodium hydroxide solution (6 ml) was added, and the mixture was refluxed for 4 hours. A saturated aqueous ammonium chloride solution (9 ml) was added to the reaction mixture for neutralization, followed by filtration and washing with water to obtain carboxylic acid (3) (122.0 mg, 0.50 mmol, 92%) as a yellow solid.

・ Identification result of carboxylic acid (3)
¹ H NMR (500 MHz, DMSO-D6): δ 2.91 (6H, s), 6.77 (2H, d, J = 6.9 Hz), 7.54 (2H, d, J = 7.5 Hz), 7.49 (2H, d , J = 7.5 Hz), 7.89 (2H, d, J = 7.5 Hz)

(Synthesis of cysteine protector (5))
Trifluoroacetic acid (TFA) (1.5 ml) was added to D-cysteine methyl ester hydrochloride (4) (308 mg, 1.8 mmol) and triphenylmethanol (515 mg, 1.98 mmol) and dissolved. The mixture was stirred at room temperature for 2 hours, concentrated under reduced pressure, and neutralized with an anion exchange resin IRA400 OH AG. The resin was filtered and the resulting solution was concentrated under reduced pressure. The obtained residue was purified by silica gel chromatography [silica gel 30 g; hexane-ethyl acetate (1: 1)] to give a protected cysteine (5) (658 mg, 1.7 mmol, 97%) as a pale yellow oil. It was.

・ Identification result of cysteine protector (5)
¹ H NMR (270 MHz, CDCl ₃ ): δ 2.47 (1H, dd, J = 8.1, 13 Hz), 2.60 (1H, dd, J = 5.1, 13 Hz), 3.21 (1H, dd, J = 5.1 , 8.1 Hz), 3.66 (3H, s), 7.18-7.32 (9H, complex), 7.40-7.54 (6H, complex)

(Synthesis of amide (6))
Carboxylic acid (3) (15.0 mg, 0.062 mmol) and cysteine protector (5) (31.2 mg, 0.082 mmol) were dissolved in N, N-dimethylformamide (2 ml). To this solution was added 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride (DMT-MM) (21.6 mg, 0.073 mmol) and the mixture was added. Stir at room temperature for 3 hours. Thereafter, the mixture was extracted with dichloromethane (20 ml × 3), the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography [hexane-ethyl acetate (2: 1)] to obtain amide compound (6) (32.9 mg, 0.054 mmol, 87%) as a transparent oil.

・ Identification result of amide (6)
¹ H NMR (500 MHz, CDCl ₃ ): δ 2.75 (2H, complex), 3.01 (6H, s), 3.75 (3H, s), 4.85 (1H, dd, J = 12.6, 4.6 Hz), 6.81 ( 2H, d, J = 8.6 Hz), 7.15-7.34 (9H, complex), 7.35-7.42 (6H, complex), 7.55 (2H, d, J = 8.6 Hz), 7.63 (2H, d, J = 8.0 Hz) ), 7.78 (2H, d, J = 8.0 Hz)

(Synthesis of cyclized product (7))
The amide compound (6) (14.0 mg, 0.023 mmol) and triphenylphosphine oxide (19.0 mg, 0.068 mmol) are dissolved in dichloromethane (1.5 ml) under an argon atmosphere, and trifluoromethanesulfonic anhydride (50 μl, 0.29 mmol) And stirred for 2.5 hours. Thereafter, the mixture was extracted with dichloromethane (20 ml × 2), and the organic layer was washed with saturated brine (30 ml). Furthermore, the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by preparative thin layer chromatography [20 cm × 20 cm × 1.75 mm × 1 sheet; hexane-ethyl acetate (1: 1)] to give a cyclized product (7) (7.0 mg, 0.020 mmol, 86%) as a yellow solid.

・ Identification result of cyclized product (7)
¹ H NMR (500 MHz, CDCl ₃ ): δ3.00 (6H, s), 3.61-3.73 (2H, complex), 3.83 (3H, s), 5.29 (1H, t, J = 9.2 Hz), 6.79 ( 2H, d, J = 6.3 Hz), 7.54 (2H, d, J = 5.9 Hz), 7.59 (2H, d, J = 4.6 Hz), 7.87 (2H, d, J = 4.6 Hz)

(Synthesis of biphenyl type luminescent substrate (8))
The cyclized product (7) (7.7 mg, 0.023 mmol) was dissolved in tetrahydrofuran (THF): distilled water (2: 1, 2 ml). To this solution was added lithium hydroxide monohydrate (2.0 mg, 0.047 mmol), and the mixture was stirred at room temperature for 30 minutes. Ethyl acetate (30 ml) was added to the reaction mixture, and the product was extracted with water (20 ml × 2). The aqueous layers were combined, acidified with 1M hydrochloric acid, and extracted with ethyl acetate (20 ml × 2). Thereafter, the extract was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain biphenyl type luminescent substrate (8) (4.0 mg, crude) as a red solid.

・ Identification result of biphenyl type luminescent substrate (8)
¹ H NMR (500 MHz, CDCl ₃ ): δ3.01 (6H, s), 3.73 (2H, m), 5.34 (1H, t, J = 9.2 Hz), 6.80 (2H, d, J = 8.0 Hz) , 7.55 (2H, d, J = 8.6 Hz), 7.62 (2H, d, J = 8.5 Hz), 7.86 (2H, d, J = 8.0 Hz)

Example 1
<Formation of salt>
1 mg of Aka Lumine (manufactured by Kurokin Kasei Co., Ltd., Lot No.0022) and 50 μl of the following acid or base aqueous solution were mixed and allowed to stand at room temperature for 1 hour.

·base:
Tertiary butyl sodium (tBuONa) (6.6 × 10 ⁻² M (mol / l))
Tertiary butyl potassium (tBuOK) (6.6 × 10 ⁻² M (mol / l))
Ammonium-methanol (NH ₃ in MeOH) (6.6 × 10 ⁻² M (mol / l))
Lithium hydroxide (LiOH) (6.6 × 10 ⁻² M (mol / l))
Triethylamine (Et ₃ N) (0.1 M (mol / l))

·acid:
Phosphoric acid (H ₃ PO ₄ ) (6.6 × 10 ⁻² M (mol / l))
Hydrochloric acid (HCl) (6.6 × 10 ⁻² M (mol / l))
Hydrobromic acid (HBr) (6.6 × 10 ⁻² M (mol / l))
Trifluoroacetic acid (CF ₃ COOH) (0.1 M (mol / l))
Acetic acid (CH ₃ COOH) (0.1 M (mol / l))
Sulfuric acid (H ₂ SO ₄ ) (6.6 × 10 ⁻² M (mol / l))

  Then, in order to precipitate an insoluble matter, centrifugation was performed at 13,000 rpm for 3 minutes. The supernatant was collected to obtain an alumine chloride solution.

<Solubility measurement>
25 μl of the supernatant (saturated solution) obtained as described above was diluted 100 times with pure water, and quantitative measurement was performed using high performance liquid chromatography (HPLC) (n = 3). The results are shown in Table 1.

All the acalumine salts prepared from the acidic aqueous solution and the aqueous base solution showed improved solubility over the acalumine (before preparation of the salt), but the salts prepared from the aqueous solution of hydrogen chloride and hydrogen bromide in particular were more than 50 times. Solubility improved.
In addition, for Akalumine hydrochloride and hydrobromide, the color of the aqueous solution is dark red and opaque derived from Acalumine hydrochloride and hydrobromide, and it is difficult to determine whether a precipitate is formed. The solution may not be saturated. Therefore, hydrochloride and hydrobromide may be higher than the measured solubility.

<Substance measurement>
It was confirmed by IR measurement and UV measurement that the produced substance was an acalumine salt.

IR measurement IR measurement was performed only on Aka Lumine (manufactured by Kurokin Kasei Co., Ltd.) (without medium) and on Aka Lumine hydrochloride. As a measuring instrument, Nicolet 6700 manufactured by Thermo Scientific was used, and a sample of 1 mg or less was measured by the ATR method. FIG. 1 shows the IR spectrum of Acalumine, and FIG. 2 shows the IR spectrum of Acalumine hydrochloride.

From comparison between FIG. 1 and FIG. 2, it can be seen that the fingerprint region (1300 to 650 cm ⁻¹ ) of IR differs between Akalumine and Acalumine hydrochloride. Further, in the Akarumine, 1550 cm ^-1, the hydrochloride salt of Akarumine, absorption was observed at 1750 cm ^-1, which are derived from carboxylic acids in the molecular structure (-COOH), in Akarumine, dimethylamino group in the molecule ( This is probably because absorption of —COO— deprotonated by —NMe ₂ group) is observed, whereas absorption of COOH is observed in Acalumine hydrochloride without deprotonation by HCl.

-UV measurement UV measurement was performed on Aka Lumine (manufactured by Kurokin Kasei Co., Ltd.) and Aka Lumine Hydrochloride. As a measuring instrument, a Varian Cary 50 is used, a 1.0 × 10 ⁻⁵ M substrate solution is used, a cell holder temperature is 25 ° C., and a quartz cell for UV measurement (optical path length is 1 cm) is used. did. FIG. 3 shows the UV spectrum of Acalumine, and FIG. 4 shows the UV spectrum of Acalumine hydrochloride.

  A comparison between FIG. 3 and FIG. 4 shows that Acaluminescence has two absorption maxima at 320 nm and 490 nm, whereas Acaluminum hydrochloride has one absorption maximum at 460 nm and the spectra are different.

<Luminescence measurement>
The emission intensity and emission spectrum were measured using Akalumine (500 μM), and Akalumine hydrochloride (500 μM) and Akaluminescence hydrobromide (500 μM) obtained in the examples.

Measurement of luminescence intensity 20 μl of 0.5 M potassium phosphate buffer (KPB solution) at pH 8.0, 20 μl of lysate solution, 0.01 mg / ml of photinus pyralis (an enzyme that degrades luminescence substrates) 20 μl of Promega) and 40 μl of 0.2 mM Mg-ATP were mixed, and luminescence was measured for 180 seconds with a luminescence intensity measuring apparatus (AB-2270, manufactured by ATTO Corporation). The results are shown in FIG.

  From FIG. 5, it can be seen that Akalumine hydrochloride and hydrobromide have substantially the same emission intensity as that of Acalumine. In addition, when the total light emission amount of Acalumine was 100, the total light emission amount of Acalumine hydrochloride was 91, and the total light emission amount of Acalumine hydrobromide was 81.

Emission spectrum measurement 5 μl of 0.5 M potassium phosphate buffer (KPB solution) at pH 8.0, 5 μl of lysate solution, 0.1 mg / ml of photinus pyralis (enzyme that degrades luminescence substrate) 5 μl of Promega) and 10 μl of 0.2 mM Mg-ATP were mixed, and the emission spectrum was measured for 180 seconds with an emission spectrum measuring device (AB-1850, manufactured by ATTO Corporation). The results are shown in FIG.

  From FIG. 6, the emission maximum in the emission spectrum of acaluminescence is 675 nm, the emission maximum in the emission spectrum of acaluminescence hydrochloride is 675 nm, and the emission maximum in the emission spectrum of acaluminescence hydrobromide is 680 nm. It can be seen that the salt and hydrobromide have almost the same emission spectrum as the acaluminescence.

(Example 2)
<Formation of salt>
1 mg of the biphenyl type luminescent substrate (8) obtained by synthesis as described above and 50 μl of the following acid or base aqueous solution were mixed and allowed to stand at room temperature for 1 hour.

·base:
Tertiary butyl sodium (tBuONa) (6.2 × 10 ⁻² M (mol / l))
Tertiary butyl potassium (tBuOK) (6.2 × 10 ⁻² M (mol / l))
Ammonium-methanol (NH ₃ in MeOH) (6.2 × 10 ⁻² M (mol / l))

·acid:
Phosphoric acid (H ₃ PO ₄ ) (6.2 × 10 ⁻² M (mol / l))
Hydrochloric acid (HCl) (6.2 × 10 ⁻² M (mol / l))
Hydrobromic acid (HBr) (6.2 × 10 ⁻² M (mol / l))

  Then, in order to precipitate an insoluble matter, centrifugation was performed at 13,000 rpm for 3 minutes. The supernatant was collected to obtain a chloride solution of biphenyl type luminescent substrate (8).

<Solubility measurement>
100 μl of the supernatant (saturated solution) obtained as described above was diluted 100 times with pure water, and quantitative measurement was performed using high performance liquid chromatography (HPLC) (n = 3). The results are shown in Table 2.

All the salts of the biphenyl type luminescent substrate (8) prepared from the acidic aqueous solution and the base aqueous solution showed improved solubility over the biphenyl type luminescent substrate (8) (before the salt preparation). Salts prepared from an aqueous solution of hydrogen improved solubility by 20 times or more.
As for the hydrochloride and hydrobromide of the biphenyl type luminescent substrate (8), the color of the aqueous solution is dark and opaque due to the deep yellow color derived from the hydrochloride and hydrobromide of the biphenyl type luminescent substrate (8). Yes, it is difficult to determine whether a precipitate is formed, and the solution may not be saturated. Therefore, hydrochloride and hydrobromide may be higher than the measured solubility.

  As is clear from the results of the above examples, the water solubility of Akalumine is not sufficiently improved with other salts, and the water solubility is significantly improved only with respect to hydrogen halide salts such as hydrochloride and hydrobromide. did. Furthermore, the water solubility level of the hydrochloride and hydrobromide satisfied the level required for practical use (solubility of 10 mg / ml). As for Akalumine, it is the first time that only water halides such as hydrochlorides and hydrobromides are significantly improved in water solubility and have practical value. In addition, when prepared as a hydrogen halide (hydrogen chloride, hydrogen bromide) salt, the solubility of not only a red luminescence but also a biphenyl type luminescent substrate can be improved.

  The hydrogen halide salt of the present invention is excellent in water solubility and can be used as a luminescent substrate in a firefly bioluminescence system.

Claims (6)

The following general formula (I):

[Wherein, R ¹ and R ² are each independently hydrogen or an alkyl group having 1 to 3 carbon atoms, and R ³ is each independently an alkyl group having 1 to 8 carbon atoms or an alkenyl group having 2 to 8 carbon atoms. A group, n is an integer of 0-3, m is an integer of 0-4], or
The following general formula (II):

[Wherein, R ⁴ and R ⁵ are each independently hydrogen or an alkyl group having 1 to 3 carbon atoms, and R ⁶ and R ⁷ are each independently an alkyl group having 1 to 8 carbon atoms or 2 to 2 carbon atoms. 8 is an alkenyl group, and p and q are each independently an integer of 0 to 4.]   2. The hydrohalide salt according to claim 1, which is a hydrochloride or hydrobromide of the compound represented by the general formula (I) or the general formula (II). The R ¹ and R ² in the general formula (I) and the R ⁴ and R ⁵ in the general formula (II) are alkyl groups having 1 to 3 carbon atoms. 2. The hydrogen halide salt according to 2. The hydrogen halide salt according to claim 3, wherein R ¹ and R ² in the general formula (I) and R ⁴ and R ⁵ in the general formula (II) are methyl groups. .   It is a hydrogen halide salt of the compound represented by the said general formula (I), The hydrogen halide salt as described in any one of Claims 1-4 characterized by the above-mentioned. The following chemical formula (III):

The hydrohalide salt according to claim 5, which is a hydrochloride or hydrobromide of a compound represented by the formula:

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