Novel heterocyclic compound and salt thereof, and luminescent substrate composition

Abstract

The present invention addresses the problem of providing a novel compound that can be used as a luminescent substrate in a firefly bioluminescence system. The solution for the problem is a heterocyclic compound characterized by being represented by general formula (1) [wherein Cy has a specific cyclic structure; R1 represents -NR5R6, -OR7 or a hydrogen atom; R5, R6 and R7 each independently represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, in which R5 and R6 may be bound to each other to form a ring and one of R5 and R6 may be bound to Y1 to form a ring; R2 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms; X1 represents S, O, NR8 or CH2; Y1 and Y2 each independently represent N or CR8, in which R8 each independently represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an alkenyl group having 2 to 4 carbon atoms, or an acyl group having 2 to 4 carbon atoms; and n and m each independently represent an integer of 0 to 3].

Description

Novel heterocyclic compound and its salt, and luminescent substrate composition

[0001] The present invention relates to novel heterocyclic compounds and salts thereof, and luminescent substrate compositions.

[0002] Visualization of the inside of a living organism has become a major challenge in the field of life science, and research is being conducted into the use of bioluminescence systems for visualization of the inside of a living organism. Among such bioluminescence systems, the firefly luminescence system is known to have excellent luminescence efficiency. In the firefly luminescence system, firefly luciferin (LH), a luminescence substrate, is used to generate the luminescence. ２ ) is a light-emitting enzyme, firefly luciferase (Luc), adenosine triphosphate (ATP), and magnesium ions (Mg ２＋ When oxyluciferin is deactivated to the ground state, yellow-green light having a wavelength of about 560 nm is emitted.

[0003] Recently, compounds that achieve a variety of emission wavelengths have been synthesized as analogs of the luminescent substrates of the firefly luminescence system. For example, Patent Documents 1 to 4 listed below disclose luminescent substrates having a molecular structure similar to firefly luciferin. Among these firefly luciferin analogs, luminescent substrates that emit long-wavelength light are promising as labeling materials for visualizing lesions deep within the body because long-wavelength light has high transmittance in living organisms. For example, Patent Documents 1 to 3 listed below disclose compounds that exhibit an emission spectrum with a maximum wavelength of approximately 670 nm, and Patent Document 4 listed below discloses a compound that exhibits an emission spectrum with a maximum wavelength of approximately 760 nm. These materials enable the visualization of microscopic cells deep within the body that were previously not possible to optically image.

[0004] JP 2009-184932 A JP 2014-218456 A JP 2015-193584 A International Publication No. 2021 / 193069

[0005] As mentioned above, various compounds with novel structures have been proposed as luminescent substrates in the firefly bioluminescence system, but only a small number of compounds have been put into practical use as materials capable of near-infrared luminescence labeling.

[0006] Therefore, an object of the present invention is to provide a novel compound that can be used as a luminescent substrate in the firefly bioluminescence system.A further object of the present invention is to provide a novel compound that can emit light of a long wavelength and can be used as a luminescent substrate in the firefly bioluminescence system.

[0007] As a result of intensive research to solve the above problems, the present inventors have found that a compound having a specific structure or a salt thereof functions as a luminescence substrate in the firefly bioluminescence system, and have thus completed the present invention. That is, the gist and configuration of the present invention that solves the above problems is as follows.

[0008] [1] The following general formula (1): [In the formula, Cy represents the following general formula (2-1) or (2-2): It is expressed as R １ is -NR ５ R ６ , -OR ７ or hydrogen, R ５ , R ６ and R ７ are each independently hydrogen or an alkyl group having 1 to 4 carbon atoms, provided that R ５ and R ６ may be bonded to each other to form a ring, and R ５ and R ６ One of them is Y １ may be bonded to form a ring, R ２ is hydrogen or an alkyl group having 1 to 4 carbon atoms, and R ３ and R ４ are each independently CH or N, １ and X ２ are each independently S, O, or NR ８ or CH ２ and Y １ , Y ２ , Y ３ and Y ４ are each independently N or CR ８ where R ８are each independently a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an alkenyl group having 2 to 4 carbon atoms, or an acyl group having 2 to 4 carbon atoms, and n and m are each independently an integer of 0 to 3. The heterocyclic compound of the present invention described in [1] above functions as a luminescent substrate in the firefly bioluminescence system.

[0009] [2] The X １ is S, O or NR ８ The heterocyclic compound according to [1], wherein: The heterocyclic compound according to [2] above has improved luminous efficiency.

[0010] [3] The Y １ and Y ２ However, each is independently CR ８ The heterocyclic compound according to [1] or [2], wherein: The heterocyclic compound according to [3] above has improved luminous efficiency.

[0011] [4] The heterocyclic compound according to any one of [1] to [3], wherein Cy is represented by the general formula (2-1). The heterocyclic compound according to [4] is easy to synthesize.

[0012] [5] The following structural formulas (1-1) to (1-10): The heterocyclic compound according to any one of [1] to [4], which is represented by any one of the following: The heterocyclic compound according to [5] above is capable of emitting light of a long wavelength, and is therefore particularly useful for visualization of deep areas in a living body.

[0013] [6] A salt of the heterocyclic compound according to any one of [1] to [5]. The salt of the heterocyclic compound according to [6] has excellent solubility in water and a buffer solution having a pH close to neutral, and can therefore be dissolved at a high concentration.

[0014] [7] A luminescent substrate composition comprising the heterocyclic compound according to any one of [1] to [5] or the salt according to [6]. The luminescent substrate composition of the present invention according to [7] above can constitute a firefly bioluminescence system together with a luminescent enzyme.

[0015] According to the present invention, it is possible to provide a heterocyclic compound and a salt thereof that can be used as a luminescent substrate in a firefly bioluminescence system. Also, according to one embodiment of the present invention, it is possible to provide a heterocyclic compound and a salt thereof that can emit light of a long wavelength and can be used as a luminescent substrate in a firefly bioluminescence system.

[0016] This is an emission spectrum normalized so that the maximum value of emission intensity is 1 in a luminescence system using a compound represented by structural formula (1-1), a compound represented by structural formula (1-2), a compound represented by structural formula (1-3), or a compound represented by structural formula (1-4) as a luminescence substrate. This is an emission spectrum normalized so that the maximum value of emission intensity is 1 in a luminescence system using a compound represented by structural formula (1-5) or a compound represented by structural formula (a) as a luminescence substrate. This is an emission spectrum normalized so that the maximum value of emission intensity is 1 in a luminescence system using a compound represented by structural formula (1-5), a compound represented by structural formula (1-6), a compound represented by structural formula (1-7), a compound represented by structural formula (1-8), a compound represented by structural formula (1-9), or a compound represented by structural formula (1-10) as a luminescence substrate.

[0017] The heterocyclic compound and its salt, and the luminescent substrate composition of the present invention will be illustrated in detail below based on embodiments.

[0018] <Heterocyclic Compound and Salt Thereof> The heterocyclic compound of the present invention is represented by the following general formula (1): The heterocyclic compound of the present invention is characterized by being represented by the formula (1). In addition to the dihydrothiazole ring, the heterocyclic compound of the present invention has another five-membered ring and further has a five- or six-membered ring structure (Cy). Since the molecular structure is similar to that of firefly luciferin, the heterocyclic compound functions as a luminescent substrate in the firefly bioluminescence system. Furthermore, a heterocyclic compound in which at least one of n and m in the general formula (1) is not 0 not only functions as a luminescent substrate in the firefly bioluminescence system but is also capable of emitting light of a long wavelength. The heterocyclic compound represented by the general formula (1) can also be converted into a salt, and the salt also functions as a luminescent substrate in the firefly bioluminescence system.

[0019] In the above general formula (1), R１ is -NR ５ R ６ , -OR ７ or hydrogen, R ５ , R ６ and R ７ are each independently hydrogen or an alkyl group having 1 to 4 carbon atoms, provided that R ５ and R ６ may be bonded to each other to form a ring, and R ５ and R ６ One of them is Y １ Here, examples of the alkyl group having 1 to 4 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, and a tert-butyl group. From the viewpoint of luminous efficiency, R ５ and R ６ As R, a methyl group is preferred. ５ and R ６ The ring formed by combining with N is preferably a three- to seven-membered ring. ５ and R ６ Examples of the group formed by bonding with N include groups of the following formula: Preferred are 1-azacyclopropyl groups (three-membered rings), 1-azacyclobutyl groups (four-membered rings), 1-azacyclopentyl groups (five-membered rings), 1-azacyclohexyl groups (six-membered rings), and 1-azacycloheptyl groups (seven-membered rings) represented by the following formula: ５ and R ６ One of them is Y １ The ring formed by bonding with Y is preferably a five-membered ring or a six-membered ring. １ is CR ８ and R ５ and R ６ One of the two and R ８ and bond to form a ring structure.

[0020] In the above general formula (1), R ２is hydrogen or an alkyl group having 1 to 4 carbon atoms. Examples of the alkyl group having 1 to 4 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, and a tert-butyl group. From the viewpoint of luminous efficiency, R ２ As the aryl group, hydrogen is preferred.

[0021] In the above general formula (1), X １ is S, O, NR ８ or CH ２ and Y １ and Y ２ are each independently N or CR ８ From the viewpoint of luminous efficiency, X １ is S, O or NR ８ Also, from the viewpoint of luminous efficiency, it is preferable that Y １ and Y ２ are each independently CR ８ It is preferable that:

[0022] In the above general formula (1), X １ , Y １ and Y ２ Regarding R ８ are each independently hydrogen, an alkyl group having 1 to 4 carbon atoms, an alkenyl group having 2 to 4 carbon atoms, or an acyl group having 2 to 4 carbon atoms. １ and Y ２ One or both of the following are CR ８ If R ８ is preferably hydrogen. Examples of the alkyl group having 1 to 4 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, and a tert-butyl group. Examples of the alkenyl group having 2 to 4 carbon atoms include a vinyl group (CH ２ ═CH—), allyl group (CH ２ =CHCH ２ -), 1-propenyl group (CH ３ CH═CH—), isopropenyl group (CH ２ =C(CH ３ )-), 1-butenyl group (CH ３ CH ２CH═CH—), 2-butenyl group (CH ３ CH=CHCH ２ -), 3-butenyl group (CH ２ =CHCH ２ CH ２ Examples of the acyl group having 2 to 4 carbon atoms include an acetyl group (CH ３ —CO—), propionyl group (CH ３ CH ２ -CO-), butyryl group (CH ３ CH ２ CH ２ -CO-), isobutyryl group ((CH ３ ) ２ CH—CO—), acryloyl group (CH ２ ═CH—CO—), methacryloyl group (CH ２ =C(CH ３ )-CO-) and the like.

[0023] In the general formula (1), n ​​and m are each independently an integer of 0 to 3. n and m represent the number of repeating vinylene units (-CH=CH-), and the larger the number of n and / or m, the longer the emission wavelength. From the viewpoint of increasing the emission wavelength and visualizing deep within a living body, it is preferable that at least one of n and m is not 0. The optimal wavelength for biological imaging (i.e., the wavelength suitable for biological permeability) is 600 to 900 nm, which is less susceptible to the scattering and absorption of hemoglobin, oxyhemoglobin, and water. Therefore, from the viewpoint of visualizing deep within a living body, n is preferably 1, 2, or 3. Meanwhile, from the viewpoint of ease of synthesis, n is preferably 0, 1, or 2. Similarly, from the viewpoint of visualizing deep within a living body, m is preferably 1, 2, or 3. Furthermore, from the viewpoint of ease of synthesis, m is preferably 0, 1, or 2. Note that the respective vinylene units may be linked by trans bonds, may be linked by cis bonds, or may have a mixture of trans bonds and cis bonds. From the viewpoint of luminous efficiency, it is preferable that the vinylene units are linked together via a trans bond.

[0024] In the above general formula (1), Cy is represented by the following general formula (2-1) or (2-2): From the viewpoint of ease of synthesis, Cy is preferably represented by the above general formula (2-1).

[0025] In the above general formula (2-1), R ３ and R ４ are each independently CH or N. ３ and R ４ When at least one of the groups is N, the water solubility of the heterocyclic compound is improved, and the luminescence intensity is also improved.

[0026] In the general formula (2-2), X ２ is S, O, NR ８ or CH ２ and Y ３ and Y ４ are each independently N or CR ８ In the general formula (2-2), X ２ , Y ３ and Y ４ Regarding R ８ are each independently hydrogen, an alkyl group having 1 to 4 carbon atoms, an alkenyl group having 2 to 4 carbon atoms, or an acyl group having 2 to 4 carbon atoms. ３ and Y ４ One or both of the following are CR ８ If R ８ is preferably hydrogen. Examples of the alkyl group having 1 to 4 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, and a tert-butyl group. Examples of the alkenyl group having 2 to 4 carbon atoms include a vinyl group (CH ２ ═CH—), allyl group (CH ２ =CHCH ２ -), 1-propenyl group (CH ３ CH═CH—), isopropenyl group (CH ２ =C(CH ３ )-), 1-butenyl group (CH ３ CH ２ CH═CH—), 2-butenyl group (CH ３ CH=CHCH ２ -), 3-butenyl group (CH ２ =CHCH ２ CH ２Examples of the acyl group having 2 to 4 carbon atoms include an acetyl group (CH ３ —CO—), propionyl group (CH ３ CH ２ -CO-), butyryl group (CH ３ CH ２ CH ２ -CO-), isobutyryl group ((CH ３ ) ２ CH—CO—), acryloyl group (CH ２ ═CH—CO—), methacryloyl group (CH ２ =C(CH ３ )-CO-) and the like.

[0027] The heterocyclic compounds represented by the general formula (1) include those represented by the following structural formulas (1-1) to (1-10): Heterocyclic compounds and salts thereof represented by any of the structural formulae (1-1) to (1-10) above function as luminescent substrates in the firefly bioluminescence system and are capable of emitting light of long wavelengths, and are therefore useful for visualizing deep parts of living organisms. Heterocyclic compounds and salts thereof represented by the structural formulae (1-5) or (1-6) above are particularly useful for visualizing deep parts of living organisms, and are capable of emitting light of particularly long wavelengths. Heterocyclic compounds and salts thereof that emit long wavelength light can be used in combination with luminescent substrates that emit blue light, or firefly luciferin (LH) that emits yellow-green light. ２ ) and other technologies, it becomes possible to perform multicolor multiplexed imaging that simultaneously visualizes multiple biological phenomena.

[0028] The heterocyclic compound represented by the general formula (1) can be synthesized as follows, although not particularly limited thereto. For example, a five-membered ring compound having an aldehyde group, such as pyrrole-2-carboxyaldehyde, is used as a starting material, and if desired, is protected with Boc using di-tert-butyl dicarbonate. Next, a Wittig reagent (CNPhCH ２ PPh ３A Wittig reaction is carried out using a starting compound such as 1-methyl-2-pyrrolecarboxaldehyde or 2-thiophenecarboxaldehyde, followed by the addition of an aqueous sodium hydroxide solution to remove the Boc protection, to obtain a cyano compound (nitrile compound). Alternatively, a five-membered ring compound having an aldehyde group, such as 1-methyl-2-pyrrolecarboxaldehyde or 2-thiophenecarboxaldehyde, is used as a starting material, and a Wittig reaction is carried out with the starting compound to obtain a cyano compound (nitrile compound). Alternatively, a five-membered ring compound having an aldehyde group, such as 1-methyl-2-pyrrolecarboxaldehyde or furfural, is used as a starting material, and a Horner, Wadsworth, and Emmons (HWE) reaction is carried out with the starting compound using an HWE reagent to obtain a cyano compound (nitrile compound). Alternatively, a five-membered ring compound having an aldehyde group, such as 5-bromo-2-thiophenecarboxaldehyde or 5-bromo-2-furaldehyde, can be used as a starting material, and the starting compound can be reacted with dimethylamine or the like to obtain a dimethylamino compound. The dimethylamino compound can be subjected to an HWE reaction using an HWE reagent to obtain a cyano compound (nitrile compound). Five-membered ring compounds having an aldehyde group can also be synthesized using various methods. For example, a five-membered ring compound having an aldehyde group can be synthesized by reacting the five-membered ring compound with a lithium compound such as lithium diisopropylamide (LDA) or lithium bis(trimethylsilyl)amide (LiHMDS), followed by a formylating agent such as N,N-dimethylformamide (DMF). The cyano compound (nitrile compound) obtained as described above can be thiazoline-cyclized with D-cysteine ​​hydrochloride hydrate to obtain a heterocyclic compound represented by general formula (1). Furthermore, the desired heterocyclic compound can be obtained by appropriately changing the starting material, introducing various substituents, or by utilizing other synthetic routes.

[0029] The heterocyclic compound represented by the general formula (1) above can also be converted into a salt. That is, the salt of the heterocyclic compound of the present invention is a salt of the heterocyclic compound represented by the general formula (1). Such a salt of the heterocyclic compound of the present invention also functions as a luminescent substrate in the firefly bioluminescence system. Here, the salt of the heterocyclic compound of the present invention may be an addition salt with an acid or an addition salt with a base. For example, examples of the acid in the addition salt of the heterocyclic compound of the present invention with an acid include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, phosphorous acid, nitrous acid, citric acid, formic acid, acetic acid, oxalic acid, maleic acid, lactic acid, tartaric acid, fumaric acid, benzoic acid, mandelic acid, cinnamic acid, pamoic acid, stearic acid, glutamic acid, aspartic acid, methanesulfonic acid, ethanedisulfonic acid, p-toluenesulfonic acid, salicylic acid, succinic acid, and trifluoroacetic acid. Examples of the salt include hydrochloride, hydrobromide, hydroiodide, sulfate, sulfamate, phosphate, nitrate, phosphite, nitrite, citrate, formate, acetate, oxalate, maleate, lactate, tartrate, fumarate, benzoate, mandelate, cinnamate, pamoate, stearate, glutamate, aspartate, methanesulfonate, ethanedisulfonate, p-toluenesulfonate, salicylate, succinate, trifluoroacetate, etc. On the other hand, examples of the base in the addition salt of the heterocyclic compound of the present invention with a base include sodium hydroxide, potassium hydroxide, calcium hydroxide, etc., and examples of the base addition salt include sodium salt, potassium salt, calcium salt, etc.

[0030] The salt of the heterocyclic compound represented by the general formula (1) has excellent solubility in water or a buffer solution having a pH close to neutral, and therefore can be dissolved at a high concentration in water or a buffer solution having a pH close to neutral, thereby improving the luminance of the emitted light.

[0031] <Luminescent substrate composition> The luminescent substrate composition of the present invention contains the heterocyclic compound represented by the above-mentioned general formula (1) or a salt thereof, and may consist solely of the heterocyclic compound represented by the above-mentioned general formula (1) or a salt thereof. The luminescent substrate composition of the present invention can constitute a firefly bioluminescence system together with a luminescent enzyme such as natural firefly luciferase (Luc) or a mutant enzyme thereof.

[0032] The heterocyclic compound and salt thereof of the present invention as described above react with luminescent beetle luciferase, adenosine triphosphate (ATP), and magnesium ions (Mg ２＋ When added to a system containing ATP and Mg, the compound is oxidized by luminescent beetle luciferase to emit light. ２＋ The luminescence detection kit may also contain other luminescence substrates and a solution adjusted to an appropriate pH.

[0033] When the heterocyclic compound and its salt of the present invention are applied to a luminescence system, in order to obtain a suitable luminescence intensity, the heterocyclic compound and its salt of the present invention are preferably used at a concentration of 1 μM or higher, more preferably 5 μM or higher. That is, the luminescent substrate composition of the present invention preferably contains the heterocyclic compound represented by the above-described general formula (1) or its salt at a concentration of 1 μM or higher, more preferably 5 μM or higher. Furthermore, the pH of the luminescent substrate composition of the present invention and the pH of the luminescence system are preferably 4 to 10, more preferably 6 to 8. If necessary, a buffer such as potassium phosphate, Tris-HCl, glycine, or HEPES may be added to stabilize the pH. Furthermore, when the luminescent substrate composition (luminescence detection kit) contains ATP, the concentration of the ATP is preferably 4 μM or higher, more preferably 20 μM or higher.

[0034] Furthermore, the heterocyclic compounds and salts thereof of the present invention can be used in a firefly luciferase luminescence system with various luminescent enzymes (oxidases). Luciferases have been isolated from North American fireflies (Photinus pyralis), railroad worms, and the like, and any of these can be used. Usable oxidases include click beetle luciferase, Iriomote firefly luciferase, and flavin-containing monooxygenase. Mutant enzymes of natural firefly luciferase can also be used as luminescent enzymes.

[0035] Bioluminescence using the heterocyclic compound and its salt of the present invention as a luminescent substrate involves the addition of coenzyme A (CoA), pyrophosphate, or magnesium ions (Mg ２＋ The luminescence enhancement effect of these compounds is enhanced by the presence of CoA, pyrophosphate, or Mg in the luminescence system. ２＋ The luminescence was remarkable when the concentration of each was 5 μM or more, and the luminescence increased with increasing concentration.

[0036] In order to use the firefly bioluminescence system for measurement / detection, it is preferable to stabilize the luminescence so as to prevent enzyme inactivation and exhibit plateau luminescence behavior. For example, it is preferable to have magnesium ions present in the luminescence system, and it is even more preferable to have magnesium ions coexist with pyrophosphate. When magnesium ions are used alone, from the viewpoint of luminescence stabilization, the magnesium ion concentration in the luminescence system is preferably 0.5 mM or more, and the luminescence stability improves with increasing concentration. When magnesium pyrophosphate is used, from the viewpoint of luminescence stabilization, the magnesium pyrophosphate concentration in the luminescence system is preferably 10 μM or more, more preferably 100 μM or more. The ratio of pyrophosphate to magnesium ions does not have to 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 pyrophosphates of alkali metals such as sodium and potassium, pyrophosphates of alkaline earth metals such as magnesium and calcium, and iron pyrophosphate.

[0037] The heterocyclic compound of the present invention and its salts can be used as luminescent labels in biological measurements / detections, and can be used to label, for example, amino acids, polypeptides, proteins, nucleic acids, etc. Methods for binding the heterocyclic compound of the present invention or its salts to these substances are well known to those skilled in the art. For example, the heterocyclic compound of the present invention or its salts can be bound to a carboxyl group or an amino group of a target substance using methods well known to those skilled in the art.

[0038] Furthermore, the heterocyclic compounds and salts thereof of the present invention can be used for measurement / detection by detecting luminescent beetle luciferase activity through the luminescence of a luminescent substrate. For example, by administering the heterocyclic compounds or salts thereof of the present invention to cells or animals into which a luciferase gene has been introduced, the expression of target genes or proteins in vivo can be measured / detected. Note that light with long wavelengths has high optical transparency and high tissue penetration. Therefore, among the heterocyclic compounds and salts thereof of the present invention, heterocyclic compounds and salts thereof having long-wavelength luminescence are useful as labeling materials for visualizing deep areas within the body.

[0039] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples in any way.

[0040] <Instrumental analysis, measurement equipment> (1H nuclear magnetic resonance spectrum (1H-NMR)) Measurements were performed using a JEOL ECA 500 (500 MHz), and the measurement results were reported as "1H-NMR (measurement frequency, measurement solvent) chemical shift value (number of hydrogen atoms, multiplicity, spin coupling constant)." Chemical shift values ​​(δ) were expressed in ppm using tetramethylsilane (δ = 0) as the internal standard. Multiplicity was indicated as s (singlet), d (doublet), t (triplet), q (quartet), or m (multiplet or complex overlapping signals), with broad signals indicated as br. Spin coupling constants (J) were reported in Hz.

[0041] (Mass spectrum (MS): electron spray ionization (ESI)) Measurements were performed using a JEOL JMS-T100LC TOF mass spectrometer AccuTOF by electron spray ionization (ESI). The instrument settings were: desolvation gas temperature 250°C, needle voltage 2000 V, ring lens voltage 10 V, orifice 1 voltage 80 V, and orifice 2 voltage 5 V. Sample delivery was performed by the infusion method, with a flow rate of 20 μl / min. Measurement results were reported as "ESI-MS: m / z [M + adduct ion] mass number."

[0042] (Mass spectrum (MS): matrix-assisted laser desorption / ionization (MALDI)) Measurements were performed using a JEOL matrix-assisted laser desorption / ionization time-of-flight mass spectrometer JMS-S3000 SpiralTOF™-plus 2.0. The matrix used was α-cyano-4-hydroxycinnamic acid. Measurement results were recorded as "HR-MALDI-MS: m / z [M + adduct ion] + calculated value of molecular formula; measured value."

[0043] <Method for Producing a Compound Represented by Structural Formula (1-1)> (Synthesis of Boc-Protected Compound (4)) Pyrrole-2-carboxaldehyde (3) (4.5 g, 47.3 mmol), di-tert-butyl dicarbonate (12.4 g, 56.8 mmol), and triethylamine (23.0 mL, 165.6 mmol) were dissolved in methylene chloride (50 mL) and stirred at room temperature for 4 hours under an argon atmosphere. 1-Methylpiperazine (1 mL, 9.5 mmol) was then added and the mixture was stirred at room temperature for an additional hour. The mixture was then quenched by adding distilled water. After extraction with chloroform (30 mL x 3), the organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel: 36.0 g, hexane:ethyl acetate=5:1) to obtain the Boc-protected compound (4) (6.7 g, 34.5 mmol, 73%) as a yellow oil.

[0044] ・Identification results of Boc protector (4) 1H-NMR (500 MHz, CDCl3) δ=10.31 (s, 1H), 7.43 (dd, J = 1.7, 2.9 Hz, 1H), 7.18 (dd, J = 1.7, 3.5 Hz, 1H), 6.28 (t, J = 3.5 Hz, 1H), 1.64 (s, 9H) ESI-MS : m / z [M+Na]+ ; 218.06

[0045] (Synthesis of phosphorus ylide (6)) 4-Cyanobenzyl bromide (5) (1.00 g, 5.10 mmol) and triphenylphosphine (1.60 g, 6.12 mmol) were dissolved in o-xylene (25 ml) and heated under reflux for 4 hours. The reaction mixture was cooled on ice and washed with toluene (5 ml) to obtain crude phosphorus ylide (6) (2.10 g).

[0046] (Synthesis of Cyano Compound (7a)) Phosphorus ylide compound (6) (469.7 mg, 1.02 mmol) and NaH (80.0 mg, 2.04 mmol) were dissolved in THF, cooled to 0°C, and stirred for 1 hour. After that, Boc-protected compound (4) (100.0 mg, 0.51 mmol) was dissolved and stirred for 3 hours. The mixture was then quenched with 2 mL of MeOH, and 2 mL of 6 M NaOH was added dropwise. After extraction with chloroform (50 mL x 3), the organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel: 50.0 g, hexane:ethyl acetate = 3:1) to obtain cyano compound (7a) (14.7 mg, 0.076 mmol, 15%) as a yellow solid.

[0047] ・Identification results of cyano body (7a) 1H-NMR (500 MHz, CDCl3) δ=8.46(s, 1H), 7.59(d, J=8.5 Hz, 2H), 7.48(d, J=8.5 Hz, 2H), 7.08(d, J=16 Hz, 1H), 6.88(m, 1H), 6.63(d, J=16 Hz, 1H), 6.45(m, 1H), 6.28(m, 1H) HR-MALDI-MS: m / z [M + H]+ C 13 H 11 Calculated N2 value 195.09141; measured value 195.09167

[0048] (Synthesis of the compound represented by structural formula (1-1)) The cyano compound (7a) (20.3 mg, 0.11 mmol), D-cysteine ​​hydrochloride hydrate (20.3 mg, 0.12 mmol), and potassium carbonate (21.7 mg, 0.16 mmol) were dissolved in EtOH (1 ml) and distilled water (1 ml), and the solution was stirred at 70°C for 17 hours. The residue was then purified by automated preparative medium-pressure column chromatography (Smart Flash EPCLC Al-580S ULTRAPACK COLUMN C 18 The residue was purified with a solvent mixture of HCl, HCl / methanol (H2O / methanol = 95 / 5) to obtain the compound represented by structural formula (1-1) (9.6 mg, 0.032 mmol, 31%) as a yellow solid.

[0049] ・Identification results of the compound represented by structural formula (1-1) 1H-NMR (500 MHz, CD3OD) δ=7.81(d, J=9 Hz, 2H), 7.48(d, J=8.5 Hz, 2H), 7.11(d, J=16.5 Hz, 1H), 6.80(m, 1H), 6.78(d, J=16 Hz, 1H), 6.30(m, 1H), 6.123-6.118(m, 1H), 5.08(t, J=9 Hz, 1H), 3.67(t, J=10 Hz, 1H), 3.60(t, J=9 Hz, 1H) HR-MALDI-MS: m / z [M + H] + C 16 H 15 Calculated N2O2S value: 299.08953; Measured value: 299.08487

[0050] <Method for producing the compound represented by structural formula (1-2)> (Synthesis of monobromo compound (9)) 5-Methyl-2-pyridinecarbonitrile (8) (2.0 g, 16.9 mmol), NBS (3.0 g, 16.9 mmol), and AIBN (138.7 mg, 0.85 mmol) were dissolved in carbon tetrachloride (40 ml). The mixture was heated under reflux at 85°C for 2 hours under an argon atmosphere and then quenched with aqueous sodium thiosulfate. Extraction was performed with ethyl acetate (15 ml x 3), and the organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel: 92 g, hexane:ethyl acetate = 3:1) to obtain monobromo compound (9) (1.3 g, crude).

[0051] Identification of monobromo compound (9): 1H-NMR (500 MHz, CDCl3) δ 8.73 (d, J = 2.3 Hz, 1H), 7.88 (dd, J = 8.0, 2.3 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 4.49 (s, 2H)

[0052] (Synthesis of phosphorus ylide (10)) Monobromo compound (9) (1.87 g, 9.5 mmol) and triphenylphosphine (3.73 g, 14.3 mmol) were dissolved in o-xylene (12 ml) and heated under reflux for 6 hours. The reaction mixture was cooled on ice and washed with hexane (10 ml) to obtain crude phosphorus ylide (10).

[0053] (Synthesis of Cyano Compound (7b)) The Boc-protected compound (4) was synthesized using the method described in the section on the preparation method for the compound represented by structural formula (1-1). The phosphorus ylide compound (10) (375.8 mg, 1.02 mmol) and NaH (105.3 mg, 2.14 mmol) were dissolved in THF, cooled to 0°C, and stirred for 1 hour. Then, the Boc-protected compound (4) (100 mg, 0.51 mmol) was dissolved and stirred for 1 hour. The mixture was then quenched with distilled water. After extraction with ethyl acetate (50 ml x 3), the organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel: 30.8 g, hexane:ethyl acetate = 3:1) to obtain the cyano compound (7b) (15.2 mg, 0.078 mmol, 15%) as a brown solid.

[0054] Identification of the cyano compound (7b): 1H-NMR (500 MHz, CDCl3) δ 8.71 (d, J = 2.3 Hz, 1H), 8.45 (s, 1H), 7.80 (dd, J = 8.3, 2.0 Hz, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.15 (d, J = 16.6 Hz, 1H), 6.93-6.91 (m, 1H), 6.60 (d, J = 16.6 Hz, 1H), 6.51 (s, 1H), 6.30 (q, J = 2.9 Hz, 1H).

[0055] (Synthesis of the compound represented by structural formula (1-2)) The cyano compound (7b) (40.0 mg, 0.19 mmol), D-cysteine ​​hydrochloride hydrate (36.9 mg, 0.21 mmol), and potassium carbonate (39.4 mg, 0.29 mmol) were dissolved in EtOH (2 mL), distilled water (1 mL), and THF, and the mixture was stirred at 70°C for 18 hours. The residue was then purified by automated preparative medium-pressure column chromatography (smart flash EPCLC Al-580S ULTRAPACK COLUMN C18, HO / methanol = 95 / 5), yielding the compound represented by structural formula (1-2) (14.7 mg, 0.049 mmol, 63%) as a yellow solid.

[0056] ・Identification results of the compound represented by structural formula (1-2) 1H-NMR (500 MHz, CD3OD) δ 8.56 (d, J = 1.7 Hz, 1H), 8.13 (d, J = 8.6 Hz, 1H), 7.96 (dd, J = 8.3, 2.0 Hz, 1H), 7.23 (d, J = 16.6 Hz, 1H), 6.83 (m, 1H), 6.78 (d, J = 16.6 Hz, 1H), 6.37 (dd, J = 3.4, 1.7 Hz, 1H), 6.14 (t, J = 3.2 Hz, 1H), 5.17 (t, J = 9.5 Hz, 1H), 3.65-3.52 (m, 2H) HR-MALDI-MS: m / z [M + H] + C 15 H 14 Calculated N3O2S value: 300.08012; Measured value: 300.08012

[0057] <Method for Producing a Compound Represented by Structural Formula (1-3)> (Synthesis of Cyano Compound (12a)) Phosphorus ylide compound (6) was synthesized by the method described in the section on the method for producing a compound represented by structural formula (1-1). Phosphorus ylide compound (6) (1.68 g, 3.66 mmol) and NaH (87.9 mg, 3.66 mmol) were dissolved in THF, cooled to 0°C, and stirred for 1 hour. N-methylpyrrole-2-carboxaldehyde (11) (100.0 mg, 0.916 mmol) was then dissolved in the solution, and the mixture was stirred for 5 hours. The mixture was then quenched with distilled water. After extraction with chloroform (50 ml x 3), the organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel: 50.0 g, hexane:ethyl acetate=5:1) to obtain the cyano compound (12a) (27.5 mg, 0.13 mmol, 14%) as a yellow solid.

[0058] ・Identification results of cyano body (12a) 1H-NMR (500 MHz, CDCl3) δ=7.59(d, J=9 Hz, 2H), 7.50(d, J=8 Hz, 2H), 7.08(d, J=16 Hz, 1H), 6,83(d, J=16 Hz, 1H), 6.69(m, 1H), 6.57(m, 1H), 6.18(m, 1H), 3.72(s, 3H) HR-MALDI-MS: m / z [M + H] + C 14 H 13 Calculated N2 value 209.10708; measured value 209.10732

[0059] (Synthesis of Compound Represented by Structural Formula (1-3)) The cyano compound (12a) (20.0 mg, 0.10 mmol), D-cysteine ​​hydrochloride hydrate (17.5 mg, 0.10 mmol), and sodium bicarbonate (24.2 mg, 0.23 mmol) were dissolved in MeOH (1 ml) and distilled water (1 ml), and 0.1 ml of 1M NaOH was added dropwise. The mixture was stirred at 60°C for 23 hours. The residue was then purified by automated preparative medium-pressure column chromatography (Smart Flash EPCLC Al-580S ULTRAPACK COLUMN C). 18 The residue was purified with a solvent mixture of HCl, HCl / methanol (HO / methanol = 95 / 5) to obtain the compound represented by structural formula (1-3) (5.8 mg, 0.02 mmol, 21%) as a yellow solid.

[0060] ・Identification results of the compound represented by structural formula (1-3) 1H-NMR (500 MHz, CD3OD) δ 7.80 (d, J = 8.0 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 16.6 Hz, 1H), 6.86 (d, J = 16.0 Hz, 1H), 6.68 (t, J = 2.0 Hz, 1H), 6.49-6.48 (m, 1H), 6.05 (dd, J = 3.7, 2.6 Hz, 1H), 5.07 (t, J = 9.2 Hz, 1H), 3.70 (s, 3H), 3.68-3.56 (m, 2H) HR-MALDI-MS: m / z [M + H]+ C 17 H 17 Calculated N2O2S value: 313.10016; Measured value: 313.10052

[0061] <Method for Producing a Compound Represented by Structural Formula (1-4)> (Synthesis of Cyano Compound (6b)) N-methylpyrrole-2-carboxaldehyde (11) (100.0 mg, 0.916 mmol) and diethyl P-[(6-cyano-3-pyridinyl)methyl]phosphonate (349.3 mg, 1.37 mmol) were dissolved in THF (5 ml) and cooled to 0°C. NaH (76.9 mg, 3.21 mmol) was then dissolved and the mixture was heated to reflux for 1 hour. The mixture was then quenched with distilled water. After extraction with ethyl acetate (5 ml x 3), the organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel: 56.5 g, hexane:ethyl acetate = 3:1) to obtain cyano compound (12b) (147.2 mg, 0.703 mmol, 77%) as an orange solid.

[0062] ・Identification results of cyano body (12b) 1H-NMR (500 MHz, CDCl3) δ=8.73 (d, J=2 Hz, 1H), 7.81(d, J=8 Hz, 1H), 7.62(d, J=8 H, 1H), 7.15(d, J=16 Hz, 1H), 6.79(d, J=16 Hz, 1H), 6.73(m, 1H), 6.63-6.62(m, 1H), 6.20(m, 1Hz ), 3.74(s, 3H) HR-MALDI-MS: m / z [M + H] + C 13 H 12 Calculated N3 value: 210.10286; Measured value: 210.10257

[0063] (Synthesis of Compound Represented by Structural Formula (1-4)) The cyano compound (12b) (40.0 mg, 0.19 mmol), D-cysteine ​​hydrochloride hydrate (36.9 mg, 0.21 mmol), and potassium carbonate (39.4 mg, 0.29 mmol) were dissolved in MeOH (2 ml), distilled water (1 ml), and THF (1 ml), and the solution was stirred at 60°C for 24 hours. The residue was then purified by automated preparative medium-pressure column chromatography (Smart Flash EPCLC Al-580S ULTRAPACK COLUMN C 18 The residue was purified with HCl / H2O / methanol (95 / 5) to give the compound represented by structural formula (1-4) (15.9 mg, 0.051 mmol, 27%) as a yellow solid.

[0064] ・Identification results of the compound represented by structural formula (1-4) 1H-NMR (500 MHz, CD3OD) δ 8.60 (d, J = 1.7 Hz, 1H), 8.13 (d, J = 8.0 Hz, 1H), 8.03 (dd, J = 8.3, 2.0 Hz, 1H), 7.34 (d, J = 16.6 Hz, 1H), 6.89 (d, J = 16.6 Hz, 1H), 6.73 (s, 1H), 6.58 (d, J = 2.3 Hz, 1H), 6.09 (t, J = 3.4 Hz, 1H), 5.17 (t, J = 9.5 Hz, 1H), 3.74 (s, 3H), 3.65-3.53 (m, 2H) HR-MALDI-MS: m / z [M + H] + C 16 H 16 Calculated N3O2S value: 314.09586; Measured value: 314.09577

[0065] <Method for producing the compound represented by structural formula (1-5)> (Synthesis of dimethylamino compound (14)) 5-Bromo-2-thiophenecarboxaldehyde (13) (1.00 g, 5.23 mmol) and dimethylamine (1.30 ml, 13.1 mmol) were dissolved in HO (8 ml) and DMSO (2 ml) and stirred at 80°C for 22 hours. The mixture was then quenched with distilled water. After extraction with ethyl acetate (5 ml x 3), the organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel: 25.6 g, hexane:ethyl acetate = 3:1) to obtain the dimethylamino compound (14) (710.0 mg, 4.57 mmol, 87%) as a brown solid.

[0066] Identification of the dimethylamino derivative (14): 1H-NMR (500 MHz, CDCl3) δ = 9.50 (s, 1H), 7.48 (d, J = 4.5 Hz, 1H), 5.93 (d, J = 4 Hz, 1H), 3.10 (s, 6H) HR-MALDI-MS: m / z [M + H] + C 13 Calculated value for H8NS: 210.03568; Measured value: 210.03720

[0067] (Synthesis of cyano compound (15)) Dimethylamino compound (14) (50.0 mg, 0.32 mmol) and diethyl p-[(6-cyano-3-pyridinyl)methyl]phosphonate (122.8 mg, 0.48 mmol) were dissolved in THF (5 ml) and cooled to 0°C. NaH (27.0 mg, 1.13 mmol) was then dissolved and the mixture was heated to reflux for 1 hour. The mixture was then quenched with distilled water. After extraction with ethyl acetate (5 ml × 3), the organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel: 46.5 g, hexane:ethyl acetate = 2:1) to obtain cyano compound (15) (69.6 mg, 0.087 mmol, 27%) as a red solid.

[0068] ・Identification results of cyano body (15) 1H-NMR (500 MHz, CDCl3) δ 8.64 (d, J = 2.3 Hz, 1H), 7.72 (dd, J = 8.0, 2.3 Hz, 1H), 7.56 (d, J = 8.6 Hz, 1H), 7.27 (d, J = 16.0 HR-MALDI-MS: m / z [M + H] + C 14 H 14 Calculated N3S value: 256.09033; measured value: 256.09029

[0069] (Synthesis of Compound Represented by Structural Formula (1-5)) The cyano compound (15) (45.0 mg, 0.18 mmol), D-cysteine ​​hydrochloride hydrate (34.0 mg, 0.19 mmol), and potassium carbonate (36.5 mg, 0.27 mmol) were dissolved in MeOH (2 ml), distilled water (1 ml), and THF (1 ml), and the solution was stirred at 60°C for 18 hours. The residue was then purified by automated preparative medium-pressure column chromatography (Smart Flash EPCLC Al-580S ULTRAPACK COLUMN C 18 The residue was purified with HCl / H2O (HO / methanol = 95 / 5) to obtain the compound represented by structural formula (1-5) (33.7 mg, 0.094 mmol, 53%) as a red solid.

[0070] ・Identification results of the compound represented by structural formula (1-5) 1H-NMR (500 MHz, CD3OD) δ 8.52 (d, J = 1.7 Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.91 (dd, J = 8.3, 2.0 Hz, 1H), 7.38 (d, J = 15.5 Hz, 1H), 6.89 (d, J = 4.0 Hz, 1H), 6.48 (d, J = 16.0 Hz, 1H), 5.83 (d, J = 4.0 Hz, 1H), 5.16 (t, J = 9.5 Hz, 1H), 3.64-3.53 (m, J= 2H), 2.98 (s, 6H) HR-MALDI-MS: m / z [M + H] + C 17 H 18 Calculated value of N3O2S2: 360.08296; Measured value: 360.08349

[0071] <Method for producing the compound represented by structural formula (1-6)> (Synthesis of dimethylamino compound (17)) 5-Bromo-2-furaldehyde (16) (700 mg, 4.00 mmol) and dimethylamine (1.26 ml, 12.0 mmol) were dissolved in HO (8 ml) and DMSO (2 ml) and stirred at 80°C for 22 hours. The mixture was then quenched with distilled water. After extraction with ethyl acetate (25 ml × 3), the organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel: 41.4 g, hexane:ethyl acetate = 1:1) to obtain the dimethylamino compound (17) (387 mg, 2.21 mmol, 55%) as a blue oil.

[0072] Identification of the dimethylamino derivative (17): 1H-NMR (500 MHz, CDCl3) δ 8.97 (s, 1H), 7.20 (s, 1H), 5.23 (d, J = 4.0 Hz, 1H), 3.08 (s, 6H)

[0073] (Synthesis of cyano compound (18)) Dimethylamino compound (17) (50.0 mg, 0.36 mmol) and diethyl p-[(6-cyano-3-pyridinyl)methyl]phosphonate (137.0 mg, 0.54 mmol) were dissolved in THF (5 ml) and cooled to 0°C. NaH (30.2 mg, 1.26 mmol) was then dissolved and the mixture was heated to reflux at 80°C for 1 hour. The mixture was then quenched with distilled water. After extraction with ethyl acetate (25 ml × 3), the organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel: 21.5 g, hexane:ethyl acetate = 4:1) to obtain cyano compound (18) (37.5 mg, 0.16 mmol, 44%) as a red solid.

[0074] Identification of the cyano compound (18): 1H-NMR (500 MHz, CDCl3) δ 8.65 (d, J = 2.3 Hz, 1H), 7.71 (dd, J = 8.0, 2.3 Hz, 1H), 7.55 (d, J = 8.6 Hz, 1H), 6.87 (d, J = 16.0 Hz, 1H), 6.53 (d, J = 15.5 Hz, 1H), 6.46 (d, J = 3.4 Hz, 1H), 5.10 (d, J = 3.4 Hz, 1H), 2.97 (s, 6H).

[0075] (Synthesis of the compound represented by structural formula (1-6)) The cyano compound (18) (25.0 mg, 0.10 mmol), D-cysteine ​​hydrochloride hydrate (27.5 mg, 0.16 mmol), and potassium carbonate (22.1 mg, 0.16 mmol) were dissolved in MeOH (2 ml), distilled water (1 ml), and THF (1 ml), and the solution was stirred at 60°C for 18 hours. The residue was then purified by automated preparative medium-pressure column chromatography (Smart Flash EPCLC Al-580S ULTRAPACK COLUMN C 18 The resulting mixture was purified with a solvent mixture containing 1,2-dimethyl-3,4-dichloro-2 ...

[0076] ・Identification results of the compound represented by structural formula (1-6) 1H-NMR (500 MHz, CD3OD) δ 8.53 (d, J = 9.2 Hz, 1H), 8.08-8.07 (m, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.02 (d, J = 16.0 Hz, 1H), 6.62 (d, J = 16.0 Hz, 1H), 6.45 (d, J = 2.3 Hz, 1H), 5.18 (t, J = 9.2 Hz, 1H), 3.64-3.52 (m, 2H), 2.93 (s, 6H)

[0077] <Method for Producing a Compound Represented by Structural Formula (1-7)> (Synthesis of N,N-Dimethylthiazol-2-amine (20)) Thiazol-2-amine (19) (1.00 g, 10.0 mmol) was dissolved in THF (30 mL), and iodomethane (1.80 mL, 29.7 mmol) was added and stirred. This mixture was cooled to 0°C, and NaH (1.50 g, 39.6 mmol) was added portionwise. The mixture was stirred for 70 minutes. Methanol was added in an ice bath to terminate the reaction. The mixture was extracted with chloroform (100 mL x 3), washed with saturated brine, and then concentrated under reduced pressure. The resulting residue was then purified by silica gel column chromatography (silica gel: 51.2 g, hexane:ethyl acetate=2:1) ​​to obtain N,N-dimethylthiazol-2-amine (20) (842 mg, 6.57 mmol, 66%) as a brown oil.

[0078] Identification of N,N-dimethylthiazol-2-amine (20): 1H-NMR (500 MHz, CDCl3) δ 7.19 (d, J = 3.4 Hz, 1H), 6.50 (d, J = 3.4 Hz, 1H), 3.11 (s, 6H)

[0079] (Synthesis of 2-(dimethylamino)thiazole-5-carbaldehyde (21)) N,N-Dimethylthiazol-2-amine (20) (461 mg, 3.60 mmol) was dissolved in anhydrous THF (30 mL), cooled to -80°C, and stirred under an Ar atmosphere. LDA (2.0 M, 3.60 mL, 7.19 mmol) was added dropwise to this mixed solution, and the mixture was stirred for 60 minutes. Anhydrous DMF (1.25 mL) was then added, and the mixture was warmed to room temperature and stirred for an additional 90 minutes. After confirming the disappearance of the raw materials, water was added in an ice bath to terminate the reaction. The mixture was extracted with chloroform (50 mL x 3), washed with saturated brine, and concentrated under reduced pressure. The resulting residue was then purified by silica gel column chromatography (silica gel: 21.5 g, hexane:ethyl acetate=4:1) to obtain 2-(dimethylamino)thiazole-5-carbaldehyde (21) (256.9 mg, 1.64 mmol, 46%) as a yellow solid.

[0080] Identification of 2-(dimethylamino)thiazole-5-carbaldehyde (21): 1H-NMR (500 MHz, CDCl3) δ 9.68 (s, 1H), 7.87 (s, 1H), 3.23 (s, 6H)

[0081] (Synthesis of cyano compound (22a)) 2-(dimethylamino)thiazole-5-carbaldehyde (21) (50.0 mg, 0.36 mmol) and diethyl p-[(6-cyano-3-pyridinyl)methyl]phosphonate (137.0 mg, 0.54 mmol) were dissolved in THF (5 ml) and cooled to 0°C. NaH (30.2 mg, 1.26 mmol) was then dissolved and the mixture was heated to reflux at 80°C for 1 hour. The mixture was then quenched with distilled water. After extraction with ethyl acetate (25 ml × 3), the organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel: 21.5 g, hexane:ethyl acetate = 4:1) to obtain cyano compound (22a) (37.5 mg, 0.16 mmol, 44%) as a red solid.

[0082] Identification of the cyano derivative (22a): 1H-NMR (500 MHz, CDCl3) δ 8.66 (d, J = 2.3 Hz, 1H), 7.76 (dd, J = 8.3, 2.0 Hz, 1H), 7.60 (d, J = 8.6 Hz, 1H), 7.30 (d, J = 5.2 Hz, 1H), 7.27 (s, 1H), 6.38 (d, J = 16.0 Hz, 1H), 3.18 (s, 6H).

[0083] (Synthesis of Compound Represented by Structural Formula (1-7)) The cyano compound (22a) (22.1 mg, 0.086 mmol), D-cysteine ​​hydrochloride hydrate (22.7 mg, 0.13 mmol), and potassium carbonate (18.0 mg, 0.13 mmol) were dissolved in MeOH (2 ml), distilled water (1 ml), and THF (1 ml), and the solution was stirred at 60°C for 20 hours. The residue was then purified by automated preparative medium-pressure column chromatography (Smart Flash EPCLC Al-580S ULTRAPACK COLUMN C 18 The residue was purified with a solvent mixture containing 1,2-dimethyl-3,4-dimethyl-2,4-dimethyl-1,2 ...

[0084] ・Identification results of the compound represented by structural formula (1-7) 1H-NMR (500 MHz, CD3OD) δ 8.58 (d, J = 1.7 Hz, 1H), 8.12 (d, J = 8.6 Hz, 1H), 7.97 (dd, J = 8.3, 2.0 Hz, 1H), 7.45 (d, J = 16.0 Hz, 1H), 7.27 (s, 1H), 6.55 (d, J = 16.0 Hz, 1H), 5.23 (t, J = 9.5 Hz, 1H), 3.63 (t, J = 10.3 Hz, 1H), 3.56 (t, J = 10.5 Hz, 1H), 3.15 (s, 6H)

[0085] <Method for Producing a Compound Represented by Structural Formula (1-8)> (Synthesis of Cyano Compound (22b)) 2-(Dimethylamino)thiazole-5-carbaldehyde (21) (50.0 mg, 0.32 mmol) and diethyl p-[(6-cyanophenyl)methyl]phosphonate (131.6 mg, 0.48 mmol) were dissolved in THF (5 ml) and cooled to 0°C. NaH (26.9 mg, 1.12 mmol) was then dissolved and the mixture was heated to reflux at 80°C for 3 hours. The mixture was then quenched with distilled water. After extraction with ethyl acetate (30 ml x 3), the organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel: 17.5 g, hexane:ethyl acetate = 4:1) to obtain cyano compound (22b) (69.7 mg, 0.27 mmol, 85%) as a yellow solid.

[0086] Identification of the cyano compound (22b): 1H-NMR (500 MHz, CDCl3) δ 7.57 (d, J = 8.6 Hz, 2H), 7.44 (d, J = 8.6 Hz, 2H), 7.25 (s, 1H), 7.20 (d, J = 15.5 Hz, 1H), 6.42 (d, J = 16.0 Hz, 1H), 3.16 (s, 6H)

[0087] (Synthesis of the compound represented by structural formula (1-8)) The cyano compound (22b) (25.0 mg, 0.098 mmol), D-cysteine ​​hydrochloride hydrate (25.8 mg, 0.15 mmol), and sodium bicarbonate (24.7 mg, 0.29 mmol) were dissolved in MeOH (2 ml), distilled water (1 ml), and THF (1 ml), and two drops of 1M NaOH were added. The mixture was stirred at 60°C for 15 hours. The residue was then purified by automated preparative medium-pressure column chromatography (Smart Flash EPCLC Al-580S ULTRAPACK COLUMN C). 18 The residue was purified with a solvent mixture of HCl, HCl / methanol (H2O / methanol = 95 / 5) to obtain the compound represented by structural formula (1-8) (20.5 mg, 0.057 mmol, 58%) as a yellow solid.

[0088] ・Identification results of the compound represented by structural formula (1-8) 1H-NMR (500 MHz, CD3OD) δ 7.81 (d, J = 8.6 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 15.5 Hz, 1H), 7.21 (s, 1H), 6.54 (d, J = 16.0 Hz, 1H), 5.08 (t, J = 9.5 Hz, 1H), 3.69-3.58 (m, 2H), 3.14 (s, 7H)

[0089] <Method for producing a compound represented by structural formula (1-9)> (Synthesis of N,N-dimethylthiazol-5-amine (24)) 5-Bromothiazole (23) (750 mg, 4.57 mmol) was dissolved in 1,2-dimethoxyethane (DME) (25 mL) and DMF (15 mL), and NHMe2 (50% in water, 1.52 mL, 13.7 mmol) was added and stirred. tBuONa (887.5 mg, 9.14 mmol), Rh(Cod)2BF4 (18.6 mg, 457 μmol), and 1,3-diisopropylimidazolium chloride (34.5 mg, 0.18 mmol) were added to the solution, which was then stirred at 80°C under an Ar atmosphere for 16 hours. After confirming the disappearance of the raw materials, the mixture was cooled to room temperature and suction filtered using silica gel. The filtrate was washed with water and brine, and the organic layer was concentrated. The residue was purified by silica gel column chromatography (silica gel: 25.6 g, hexane:ethyl acetate=3:1) to obtain N,N-dimethylthiazol-5-amine (24) (365 mg, 2.85 mmol, 62%) as a brown oil.

[0090] Identification of N,N-dimethylthiazol-5-amine (24): 1H-NMR (500 MHz, CDCl3) δ 8.08 (s, 1H), 6.85 (s, 1H), 2.92 (s, 6H)

[0091] (Synthesis of 5-(dimethylamino)thiazole-2-carbaldehyde (25)) N,N-Dimethylthiazol-5-amine (24) (450 mg, 3.51 mmol) was dissolved in anhydrous THF (20 mL), cooled to -80 °C, and stirred under an Ar atmosphere. LiHMDS (1.3 M, 3.51 mL, 4.56 mmol) was added dropwise to this mixture, and the mixture was stirred for 60 minutes. Anhydrous DMF (1.5 mL) was then added, and the mixture was warmed to room temperature and stirred for an additional 90 minutes. After confirming the disappearance of the raw materials, water was added in an ice bath to terminate the reaction. The mixture was extracted with ethyl acetate (50 mL × 3), washed with saturated brine, and concentrated under reduced pressure to obtain 5-(dimethylamino)thiazole-2-carbaldehyde (25) (446.5 mg, 2.86 mmol, 81%) as a brown solid.

[0092] Identification of 5-(dimethylamino)thiazole-2-carbaldehyde (25): 1H-NMR (500 MHz, CDCl3) δ 9.68 (s, 1H), 7.06 (s, 1H), 3.13 (s, 6H)

[0093] (Synthesis of cyano compound (26a)) 5-(dimethylamino)thiazole-2-carbaldehyde (25) (75.0 mg, 0.48 mmol) and diethyl p-[(6-cyano-3-pyridinyl)methyl]phosphonate (183 mg, 0.72 mmol) were dissolved in THF (5 ml) and cooled to 0°C. NaH (40.3 mg, 1.68 mmol) was then dissolved and the mixture was heated to reflux at 80°C for 20 minutes. The mixture was then quenched with distilled water. After extraction with ethyl acetate (30 ml × 3), the organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel: 24.5 g, hexane:ethyl acetate = 2:1) to obtain cyano compound (26a) (44.6 mg, 0.17 mmol, 36%) as an orange solid.

[0094] Identification of the cyano derivative (26a): 1H-NMR (500 MHz, CDCl3) δ 8.73 (d, J = 2.3 Hz, 1H), 7.84 (dd, J = 8.0, 2.3 Hz, 1H), 7.64 (d, J = 8.6 Hz, 1H), 7.31 (d, J = 16.0 Hz, 1H), 6.90 (d, J = 16.0 Hz, 1H), 6.80 (s, 1H), 3.03 (s, 6H).

[0095] (Synthesis of Compound Represented by Structural Formula (1-9)) The cyano compound (26a) (25 mg, 0.098 mmol), D-cysteine ​​hydrochloride hydrate (25.7 mg, 0.15 mmol), and potassium carbonate (20.7 mg, 0.15 mmol) were dissolved in MeOH (2 ml), distilled water (1 ml), and THF (1 ml), and the solution was stirred at 60°C for 20 hours. The residue was then purified by automated preparative medium-pressure column chromatography (Smart Flash EPCLC Al-580S ULTRAPACK COLUMN C 18 The resulting mixture was purified using a solvent mixture containing 1,2-dimethyl-3,4-dimethyl-2,4-dimethyl-1,2 ...

[0096] Identification of the compound represented by structural formula (1-9): 1H-NMR (500 MHz, CD3OD) δ 8.65-8.64 (m, 1H), 8.17 (d, J = 8.0 Hz, 1H), 8.05 (dd, J = 8.6, 2.3 Hz, 1H), 7.35 (d, J = 16.0 Hz, 1H), 7.08 (d, J = 16.6 Hz, 1H), 6.79 (s, 1H), 5.18 (t, J = 9.7 Hz, 1H), 3.63 (t, J = 10.3 Hz, 1H), 3.55 (t, J = 10.0 Hz, 1H), 3.01 (s, 6H).

[0097] <Method for producing the compound represented by structural formula (1-10)> (Synthesis of cyano derivative (26b)) 5-(dimethylamino)thiazole-2-carbaldehyde (25) (50.0 mg, 0.32 mmol) and diethyl P-[(5-cyano-2-pyridinyl)methyl]phosphonate (122 mg, 0.48 mmol) were dissolved in THF (5 ml) and cooled to 0°C. NaH (26.9 mg, 1.12 mmol) was then dissolved in the solution and heated to reflux at 80°C for 30 minutes. The mixture was then quenched with distilled water. After extraction with ethyl acetate (30 ml x 3), the organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel: 19.5 g, hexane:ethyl acetate=1:1) to obtain the cyano compound (26b) (33.3 mg, 0.13 mmol, 41%) as an orange solid.

[0098] Identification of the cyano derivative (26b): 1H-NMR (500 MHz, CDCl3) δ 8.82 (d, J = 1.7 Hz, 1H), 8.06 (dd, J = 8.3, 2.0 Hz, 1H), 7.78 (d, J = 16.0 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.07 (d, J = 15.5 Hz, 1H), 6.86 (s, 1H), 3.04 (s, 6H).

[0099] (Synthesis of the compound represented by structural formula (1-10)) The cyano compound (26b) (20.0 mg, 0.078 mmol), D-cysteine ​​hydrochloride hydrate (20.6 mg, 0.12 mmol), and sodium bicarbonate (19.7 mg, 0.23 mmol) were dissolved in MeOH (2 ml), distilled water (1 ml), and THF (1 ml), and two drops of 1M NaOH were added. The mixture was stirred at 60°C for 20 hours. The residue was then purified by automated preparative medium-pressure column chromatography (Smart Flash EPCLC Al-580S ULTRAPACK COLUMN C). 18 The resulting mixture was purified with a solvent mixture containing 1,2-dimethyl-3,4-dimethyl-2,4-dimethyl-1 ...

[0100] ・Identification results of the compound represented by structural formula (1-10) 1H-NMR (500 MHz, CD3OD) δ 8.95 (d, J = 2.3 Hz, 1H), 8.21 (dd, J = 8.6, 1.7 Hz, 1H), 7.67 (d, J = 15.5 Hz, 1H), 7.58 (d, J = 8.6 Hz, 1H), 7.09 (d, J = 16.0 Hz, 1H), 6.82 (s, 1H), 5.14 (t, J = 8.9 Hz, 1H), 3.46-3.43 (m, 2H), 3.03 (s, 6H)

[0101] <Measurement of Luminescence Spectra> Luminescence spectra were measured using the luminescent substrates represented by structural formulas (1-1) to (1-10) synthesized as described above and a luminescent substrate represented by the following structural formula (a). The luminescent substrate represented by structural formula (a) was prepared according to the "Synthesis of Luminescent Substrate C" in WO 2021 / 193069 (Patent Document 4).

[0102] Specifically, 5 μL of 500 mM potassium phosphate buffer (KPB, pH 8.0), 5 μL of a 100 μM solution of each luminescent substrate, 5 μL of a 1 mg / mL solution of natural firefly luciferase (Fluc), and 10 μL of a 10 mM solution of ATP magnesium salt were mixed, and the luminescence spectrum was measured for 180 seconds using a luminescence measuring device (ATTO Corporation, AB-1850). However, for the luminescent substrate represented by structural formula (a), the substrate concentration was 1 mM, and for the luminescent substrate represented by structural formula (1-6), the substrate concentration was 10 mM. The results are shown in Figures 1 to 3. Note that Figures 1 to 3 show luminescence spectra normalized so that the maximum value of luminescence intensity is 1. Furthermore, the wavelength (λ) of the maximum luminescence intensity was measured. ｍａｘ ) are shown in Table 1.

[0103]

[0104] <Results> As can be seen from Figure 1 and Table 1, the luminescent substrates represented by structural formulas (1-1) to (1-4) can emit light with a long wavelength of about 640 to 660 nm, and are effective for visualizing deep areas in living organisms. Furthermore, from Figures 2, 3, and Table 1, it can be seen that the luminescent substrates represented by structural formulas (1-5) or (1-6) can emit light with a long wavelength of about 790 nm, and are particularly effective for visualizing deep areas in living organisms. Furthermore, since the luminescent substrate represented by structural formula (a), which has previously had the longest emission wavelength, also has an emission wavelength of about 765 nm, it can be seen that the luminescent substrates represented by structural formulas (1-5) or (1-6) have the longest emission wavelength. Furthermore, from Figure 3 and Table 1, it can be seen that the luminescent substrates represented by structural formulas (1-7) to (1-10) can emit light with a long wavelength of about 680 to 760 nm, and are effective for visualizing deep areas in living organisms.

[0105] The heterocyclic compound and its salt of the present invention can be used as a luminescent substrate in the firefly bioluminescence system.

Claims

1. The following general formula (1): 【Chemistry 1】 [In the formula, Cy represents the following general formula (2-1) or (2-2): 【Chemistry 2】 It is expressed as R １ is -NR ５ R ６ , -OR ７ or hydrogen, R ５ , R ６ and R ７ are each independently hydrogen or an alkyl group having 1 to 4 carbon atoms, provided that R ５ and R ６ may be bonded to each other to form a ring, and R ５ and R ６ One of them is Y １ may be bonded to form a ring, R ２ is hydrogen or an alkyl group having 1 to 4 carbon atoms, R ３ and R ４ are each independently CH or N; X １ and X ２ are each independently S, O, or NR ８ or CH ２ And Y, Y ２ , Y ３ and Y ４ each independently represents N or CR ８ where R ８ each independently represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an alkenyl group having 2 to 4 carbon atoms, or an acyl group having 2 to 4 carbon atoms, n and m each independently represent an integer of 0 to 3.

2. The X １ is S, O or NR ８ 2. The heterocyclic compound of claim 1 ,

3. The Y １ and Y ２ However, each is independently CR ８ 2. The heterocyclic compound of claim 1 ,

4. The heterocyclic compound according to claim 1, wherein the Cy is represented by the above general formula (2-1).

5. The heterocyclic compound according to claim 1 , wherein at least one of n and m is not 0.

6. The following structural formulas (1-1) to (1-10): 【Chemistry 3-1】 【Chemistry 3-2】 The heterocyclic compound according to claim 1, wherein the heterocyclic compound is represented by any one of the following formulas:

7. A salt of the heterocyclic compound of claim 1.

8. A luminescent substrate composition comprising the heterocyclic compound according to claim 1 or the salt according to claim 7.

Images