Novel heterocyclic compounds and salts thereof, and luminescent matrix compositions

Abstract

To provide a novel compound that can emit short-wavelength light and can be used as a luminescent substrate in firefly bioluminescence system.SOLUTION: Provided is a compound represented by the following general formula (I). In the formula, R1 is hydrogen or an alkyl group having 1 to 4 carbon atoms, R2 is a divalent substituted hydrocarbon group that forms a 5- or 6-membered ring together with the bonded benzene ring and contains 1 or 2 O and / or S as a constituent element of the ring, X is N or CR3, where R3 is hydrogen or an alkyl group having 1 to 4 carbon atoms, Y is S or O, and n is an integer of 0 to 2.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a novel heterocyclic compound and a salt thereof, and a luminescent substrate composition.

Background Art

[0002] Visualization of deep inside the living body is a major issue in the field of life science, and research using a bioluminescence system for visualizing deep inside the living body has been conducted. Among such bioluminescence systems, the luminescence system of fireflies is known as a system with excellent luminescence efficiency. In the luminescence system of the firefly, the luminescent substrate, firefly luciferin (LH2), is converted into excited-state oxyluciferin in the presence of the luminescent enzyme, firefly luciferase (Luc), adenosine triphosphate (ATP), and magnesium ions (Mg 2+ 2+), and yellow-green light with a wavelength of about 560 nm is emitted when the oxyluciferin deactivates to the ground state.

[0003] In recent years, compounds that achieve various emission wavelengths have been synthesized as analogs of the luminescent substrate of such a firefly luminescence system. For example, Patent Document 1 below discloses a compound represented by the following structural formula (a), Patent Document 2 below discloses a compound represented by the following structural formula (b), and Patent Document 3 below discloses a compound represented by the following structural formula (c). All of them are disclosed to exhibit an emission spectrum with a maximum wavelength of about 670 nm, and these materials enable visualization of microcells deep inside the living body that could not be imaged by light until now.

Chemical Formula

[0004] In addition, Non-Patent Document 1 below discloses a luminescent enzyme (AkaLuc) created by genetic recombination from firefly luciferase (Luc), which is a natural luminescent enzyme of fireflies, as a luminescent enzyme specialized for the compound represented by the above structural formula (a) or (b). By using this specialized enzyme, high brightness can be achieved.

[0005] On the other hand, Non-Patent Document 2 discloses that the compound represented by the following structural formula (d) exhibits an emission spectrum with a maximum wavelength of about 560 nm. Furthermore, it is disclosed that the compound represented by the following structural formula (e) exhibits an emission spectrum with a maximum wavelength of about 640 nm, and the compound represented by the following structural formula (f) exhibits an emission spectrum with a maximum wavelength of about 530 nm.

Chemical Formula

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0008] Recently, there has been an increasing demand for dual, triple, and quadruple measurement technologies that can visualize multiple in-vivo phenomena, and there has been a growing call for luminescent substrates that emit short-wavelength light, particularly blue luminescent substrates. In vivo imaging technology is being increasingly applied to virus labeling, as if dragged by the research on the novel coronavirus. This has led to the need for simultaneous measurement of multiple events, which in turn has led to an expansion of the emission wavelength range. To expand the wavelength range from near-infrared emitting substrates, luminescent substrates that emit short-wavelength light, particularly blue-emitting substrates, are required.

[0009] On the other hand, as disclosed in Non-Patent Document 2, by substituting the dimethylamino group of the compound represented by the above structural formula (a) or (d) with a hydroxyl group (that is, by making it the compound represented by the above structural formula (e) or (f)), the emission spectrum can be shifted to a shorter wavelength, but the degree is not sufficient.

[0010] Therefore, an object of the present invention is to solve the above problems of the prior art and provide a novel compound that can emit short-wavelength light and can be used as a luminescent substrate in the firefly bioluminescence system.

Means for Solving the Problems

[0011] As a result of intensive studies to solve the above problems, the present inventors have found that a compound having a specific structure or a salt thereof functions as a luminescent substrate in the firefly bioluminescence system and emits short-wavelength light, thus completing the present invention.

[0012] That is, according to the present invention, the following general formula (I):

Chemical formula

[0013] In a preferred example of the heterocyclic compound of the present invention, R in the general formula (I) 2 is represented by any one of -CH2-CH2-O-, -O-CH2-O-, -CH2-CH2-CH2-O-, -O-CH2-CH2-O-. In this case, the luminescence efficiency (luminance) is improved.

[0014] In another preferred example of the heterocyclic compound of the present invention, X in the general formula (I) is N. In this case, the luminescence efficiency (luminance) is improved.

[0015] In another preferred example of the heterocyclic compound of the present invention, Y in the general formula (I) is S. In this case, the luminescence efficiency (luminance) is improved.

[0016] In another preferred example of the heterocyclic compound of the present invention, n in the general formula (I) is 1 or 2. In this case, the luminescence efficiency (luminance) is improved.

[0017] Among the heterocyclic compounds of the present invention, the following structural formulas (I-1) to (I-5):

Chemical formula

[0018] Further, according to the present invention, there is provided a salt of the heterocyclic compound, and the salt also functions as a luminescent substrate in the firefly bioluminescence system and can emit short-wavelength light.

[0019] Furthermore, according to the present invention, there is provided a luminescent substrate composition containing the heterocyclic compound or a salt thereof, which can form a firefly bioluminescence system together with a luminescent enzyme and can emit short-wavelength light.

Advantages of the Invention

[0020] According to the present invention, it is possible to provide a heterocyclic compound and a salt thereof that can emit short-wavelength light and can be used as a luminescent substrate in a firefly bioluminescence system.

Brief Description of the Drawings

[0021]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Embodiments for Carrying Out the Invention

[0022] Hereinafter, the heterocyclic compound and a salt thereof, and the luminescent substrate composition of the present invention will be exemplified and described in detail based on their embodiments.

[0023] <Heterocyclic Compound and a Salt Thereof> The heterocyclic compound of the present invention has the following general formula (1):

Chemical Formula

[0024] In the above general formula (1), R 1 is hydrogen or an alkyl group having 1 to 4 carbon atoms. 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, a tert-butyl group, etc. From the viewpoint of luminous efficiency (luminance), R 1 is preferably hydrogen.

[0025] In the above general formula (1), R 2 forms a 5-membered or 6-membered ring together with the benzene ring to which it is bonded, and is a divalent substituted hydrocarbon group containing one or two O and / or S as the ring-constituting elements (ring-skeleton constituting elements). Here, the elements forming the 5-membered or 6-membered ring skeleton are O, S, and C, preferably O and C. The C forming the ring skeleton may have a substituent bonded thereto in addition to hydrogen. Examples of the substituent include an alkyl group having 1 to 4 carbon atoms. R 2Examples include -CH2-CH2-O-, -O-CH2-O-, -CH2-CH2-CH2-O-, -O-CH2-CH2-O-, -CH2-CH2-S-, -S-CH2-S-, -CH2-CH2-CH2-S-, -S-CH2-CH2-S-, -O-CH2-S-, -O-CH2-CH2-S-, etc., and groups in which hydrogen in these groups is substituted with an alkyl group or the like. These divalent groups may have the left end bonded to the meta position of the benzene ring (meta position with respect to the vinylene unit that can be bonded to the benzene ring) and the right end bonded to the para position of the benzene ring (para position with respect to the vinylene unit that can be bonded to the benzene ring), or the left end may be bonded to the para position of the benzene ring and the right end bonded to the meta position of the benzene ring. From the viewpoints of synthesis and luminous efficiency (luminance), R et al is preferably a divalent group represented by any of -CH2-CH2-O-, -O-CH2-O-, -CH2-CH2-CH2-O-, -O-CH2-CH2-O-. 2 In the general formula (1) above, X is N or CR

[0026] wherein R 3 is 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, a tert-butyl group, etc. From the viewpoint of luminous efficiency, X is preferably N. When X is N, the molecular structure is more similar to that of firefly luciferin. 3 In the general formula (1) above, Y is S or O, and S is preferred. When Y is S, the molecular structure is more similar to that of firefly luciferin, so the luminous efficiency (luminance) is improved.

[0027] In the general formula (1) above, n represents the repeating number of vinylene units (-CH=CH-), and is an integer from 0 to 2. Here, the smaller the number of n, the shorter the emission wavelength. From the viewpoint of luminous efficiency (luminance), n is preferably 1 or 2.

[0028]

[0029] ​As the heterocyclic compound represented by the general formula (1) above, the following structural formulas (1-1) to (1-5): [Chemical formula] Compounds represented by any of them are particularly preferred. Compounds represented by any of the above structural formulas (1-1) to (1-5) and their salts function as luminescent substrates in the firefly bioluminescence system, can emit light with a short wavelength, and are also excellent in luminescence efficiency (luminance).

[0030] The heterocyclic compound represented by the general formula (1) is not particularly limited, but can be synthesized as follows. For example, using an aldehyde having a heterocycle such as 2,3-dihydrobenzofuran-5-carboxaldehyde, 3,4-ethylenedioxybenzaldehyde, piperonal, etc. as a starting material, reacting the aldehyde having the heterocycle with (carbethoxymethylene)triphenylphosphorane, 4-phosphonocrotonic acid, etc., and obtaining an ester form while increasing the number of olefins as desired. Next, the ester form is hydrolyzed to obtain a carboxyl form. Next, the carboxyl form is amidated with S-trityl-D-cysteine methyl ester (D-cys(Trt)-OMe), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N,N-dimethylaminopyridine (DMAP) to obtain an amide form. Next, the amide form is cyclized to thiazoline with triphenylphosphine oxide (Ph3PO) and trifluoromethanesulfonic anhydride (Tf2O) to obtain a thiazoline methyl ester form. Then, if desired, the methyl ester part of the thiazoline methyl ester form is hydrolyzed to obtain a carboxyl form having a thiazoline ring. Also, by appropriately changing the starting material, introducing various substituents, etc., or using other synthetic routes, a desired heterocyclic compound can be obtained.

[0031] The heterocyclic compound represented by the above general formula (1) can also be in the form of a salt. That is, the salt of the heterocyclic compound of the present invention is a salt of the heterocyclic compound represented by the above general formula (1). Such a salt of the heterocyclic compound of the present invention can also function as a luminescent substrate in the firefly bioluminescence system and can emit short-wavelength light. 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, as the acid in the addition salt of the heterocyclic compound of the present invention and an acid, there are 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, trifluoroacetic acid, etc. Examples of the acid addition 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, as the base in the addition salt of the heterocyclic compound of the present invention and a base, there are sodium hydroxide, potassium hydroxide, calcium hydroxide, etc. Examples of the base addition salt include sodium salt, potassium salt, calcium salt, etc.

[0032] The salt of the heterocyclic compound represented by the above general formula (1) has excellent solubility in water and a buffer solution with a pH near neutral. Therefore, the salt of the heterocyclic compound represented by the above general formula (1) can be dissolved at a high concentration in water and a buffer solution with a pH near neutral, and the luminescence brightness can be improved.

[0033] <Luminescent Substrate Composition> The luminescent substrate composition of the present invention contains the heterocyclic compound represented by the general formula (1) described above or a salt thereof, and may consist only of the heterocyclic compound represented by the general formula (1) described above or a salt thereof. The luminescent substrate composition of the present invention can form a firefly bioluminescence system together with a luminescent enzyme such as natural firefly luciferase (Luc) or its mutant enzyme, and can emit short-wavelength light.

[0034] The above-described heterocyclic compound of the present invention and a salt thereof are oxidized and emit light by firefly luciferase when added to a system in which firefly luciferase, adenosine triphosphate (ATP), and magnesium ions (Mg 2+ ) are present. Incidentally, the heterocyclic compound of the present invention and a salt thereof can also be provided as a luminescence detection kit (luminescent substrate composition) together with ATP and Mg 2+ , and the luminescence detection kit may include other luminescent substrates and a solution adjusted to an appropriate pH.

[0035] When applying the heterocyclic compound of the present invention and a salt thereof to a luminescence system, in order to obtain a suitable luminescence intensity, it is preferable to use the heterocyclic compound of the present invention and a salt thereof at a concentration of 1 μM or more, and more preferably at a concentration of 5 μM or more. That is, the luminescent substrate composition of the present invention preferably contains the heterocyclic compound represented by the general formula (1) described above or a salt thereof at a concentration of 1 μM or more, and more preferably at a concentration of 5 μM or more. Further, 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, and, if necessary, may contain a buffer such as potassium phosphate, Tris-HCl, glycine, HEPES, etc. to stabilize the pH. Further, when the luminescent substrate composition (luminescence detection kit) contains ATP, the concentration of the ATP is preferably 4 μM or more, and more preferably 20 μM or more.

[0036] In addition, the heterocyclic compound and its salt of the present invention can be caused to emit light by various luminescent enzymes (oxidoreductases) in the firefly luciferase luminescence system. Luciferase has been isolated from fireflies (Photinus pyralis) native to North America, railroad worms, etc., and any of them can be used. Examples of usable oxidoreductases include Photuris lucif erase, Iriomotebotaru luciferase, flavin-containing monooxygenase, etc. In addition, a mutant enzyme of natural firefly luciferase can also be used as a luminescent enzyme.

[0037] In bioluminescence using the heterocyclic compound and its salt of the present invention as a luminescent substrate, when coenzyme A (CoA), pyrophosphate or magnesium ion (Mg 2+ ) is present in the luminescence system, the luminescence is enhanced. 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.

[0038] In order to use the firefly bioluminescence system for measurement / detection, it is preferable to stabilize the luminescence so as to prevent inactivation of the enzyme and exhibit a plateau-like luminescence behavior. For example, it is preferable to have magnesium ions present in the luminescence system, and it is more preferable to have magnesium ions and pyrophosphate coexist. When only magnesium ions are present, 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 as the concentration increases. 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, and more preferably 100 μM or more. Note that the ratio of pyrophosphate to magnesium ions does not have to be an equivalent ratio. Examples of suitable magnesium salts include inorganic acid salts such as magnesium sulfate and magnesium chloride, and organic acid salts such as magnesium acetate. Examples of suitable pyrophosphates include pyrophosphates of alkali metals such as sodium and potassium, pyrophosphates of alkaline earth metals such as magnesium and calcium, and pyrophosphate of iron.

[0039] The heterocyclic compound of the present invention and its salts can be used as a luminescent label in biological measurements / detections, and can be used, for example, to label amino acids, polypeptides, proteins, nucleic acids, etc. In addition, the method for binding the heterocyclic compound or its salt of the present invention to these substances is well known to those skilled in the art. For example, using a method well known to those skilled in the art, the heterocyclic compound or its salt of the present invention can be bound to the carboxyl group or amino group of the target substance.

[0040] Furthermore, the heterocyclic compound of the present invention and its salts can be used in measurements / detections that utilize the detection of firefly luciferase activity by the luminescence of a luminescent substrate. For example, by administering the heterocyclic compound or its salt of the present invention to cells or animals into which the luciferase gene has been introduced, the expression of target genes or proteins in vivo can be measured / detected.

Examples

[0041] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples in any way.

[0042] <Synthesis of the compound represented by structural formula (I-1)> The synthetic route of the compound of structural formula (I-1) is as follows.

Chemical formula

[0043] A toluene solution (10 mL) of 2,3-dihydrobenzofuran-5-carboxaldehyde (1) (0.500 mL, 4.05 mmol) was added with a Wittig reagent ((carbethoxymethylene)triphenylphosphorane: Ph3P=CHCO2Et) (2.82 g, 8.10 mmol). After stirring at 120 °C for 1.5 hours, it was returned to room temperature and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel: 16.8 g, hexane:ethyl acetate = 4:1) to obtain the ester form (2) (429 mg, 1.97 mmol, 49%) as a white solid. The identification results of the ester form (2) are as follows.

[0044] 1 1H-NMR (500 MHz, CDCl3) δ = 7.59 (d, J = 16.0 Hz, 1H), 7.33 (s, 1H), 7.24 (d, J = 8.0, Hz, 1H), 6.73 (d, J = 8.0 Hz, 1H), 6.24 (d, J = 16.0 Hz, 1H), 4.54 (t, J = 8.9 Hz, 2H), 4.22 (q, J = 7.1 Hz, 2H), 3.14 (t, J = 8.9 Hz, 2H), 1.31 (t, J = 7.2 Hz, 3H) ESI-MS [M + H] + : m / z 219.10

[0045] The ester form (2) (429 mg, 1.97 mmol) was dissolved in isopropanol (25 mL) and 5M aqueous sodium hydroxide solution (12 mL). After stirring at room temperature for 15 hours, 100 mL of chloroform was added. This solution was extracted with distilled water (100 mL×3), and the aqueous layer was acidified with 6M hydrochloric acid and then extracted with ethyl acetate (50 mL×3). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to obtain the carboxyl form (3) (205 mg, 1.08 mmol, 55%) as a white solid. The identification results of the carboxyl form (3) are as follows.

[0046] 11H-NMR (500 MHz, CDCl3) δ = 7.73 (d, J = 16.0 Hz, 1H), 7.44 (s, 1H), 7.33 (d, J = 8.0 Hz, 1H), 6.80 (d, J = 7.7 Hz, 1H), 6.29 (d, J = 16.0 Hz, 1H), 4.64 (t, J = 8.6 Hz, 2H), 3.25 (t, J = 8.6 Hz, 2H) HR-ESI-MS: m / z: [M + H] + C 11 H 11 Calculated value of C11H11O3: 191.07082; Measured value: 191.07253

[0047] The carboxyl compound (3) (180 mg, 0.947 mmol) was dissolved in N,N-dimethylformamide solution (6 mL). To this solution, S-trityl-D-cysteine methyl ester (D-cys(Trt)-OMe) (450 mg, 1.09 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (259 mg, 1.35 mmol), and N,N-dimethylaminopyridine (DMAP) (220 mg, 1.80 mmol) were added under an argon atmosphere. After stirring at room temperature for 24 hours, it was quenched with saturated aqueous sodium bicarbonate (10 mL) and extracted with ethyl acetate (50 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: 62.8 g, hexane:ethyl acetate = 2:1) to obtain the amide compound (4) (390 mg, 0.709 mmol, 75%) as a white solid. The identification results of the amide compound (4) are as follows.

[0048] 11H-NMR (500 MHz, CDCl3) δ = 7.54 (d, J = 15.5 Hz, 1H), 7.30 - 7.26 (comp, 17H), 6.79 (d, J = 8.6 Hz, 1H), 6.20 (d, J = 15.5 Hz, 1H), 4.77 (m, 1H), 4.63 (t, J = 8.9 Hz, 2H), 4.12 (m, 1H), 3.24 (t, J = 8.9 Hz, 2H), 2.75 - 2.69 (m, 1H) ESI-MS [M + Na] + : m / z 572.21

[0049] To a solution of amide (4) (108 mg, 0.197 mmol) and triphenylphosphine oxide (Ph3PO) (60.8 mg, 0.219 mmol) in dichloromethane (2 mL) was added a solution of trifluoromethanesulfonic anhydride (Tf2O) (0.105 mL, 0.637 mmol) dissolved in dichloromethane (2 mL) at 0 °C under an argon atmosphere, and then the mixture was stirred at room temperature for 45 minutes. Saturated aqueous sodium bicarbonate (20 mL) was added to the reaction mixture to quench it, and the mixture was extracted with chloroform (20 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: 20.0 g, hexane:ethyl acetate = 2:1) to obtain thiazoline methyl ester (5) (43.3 mg, 0.150 mmol, 76%) as a white solid. The identification results of thiazoline methyl ester (5) are as follows.

[0050] 11H-NMR (500 MHz, CDCl3) δ = 7.36 (m, 1H), 7.27 - 7.24 (comp, 1H), 7.08 (d, J = 16.0 Hz, 1H), 6.94 (d, J = 16.0 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 5.19 (t, J = 8.9 Hz, 1H), 4.62 (t, J = 8.6 Hz, 2H), 3.59 (m, 2H), 3.22 (t, J = 8.6 Hz, 2H) 13 13C-NMR (126 MHz, CDCl3) δ = 171.5, 170.4, 161.9, 142.6, 129.0, 128.2, 128.0, 124.0, 119.4, 109.8, 77.9, 71.9, 52.9, 34.6, 29.4 HR-ESI-MS: m / z: [M + H] + C 15 H 16 Calculated value for C15H14NO3S: 291.08448; Measured value: 291.07584

[0051] To a solution of thiazoline methyl ester (5) (21.8 mg, 0.0753 mmol) in tetrahydrofuran (0.5 mL) was added 6M hydrochloric acid (0.5 mL) under an argon atmosphere. After stirring at room temperature for 18 hours, it was quenched with sodium hydrogen carbonate. The reaction mixture was purified by automated preparative medium-pressure column chromatography (smart flash EPCLC Al-580S ULTRAPACK COLUMNS C 18 H2O / methanol = 95 / 5) to obtain the compound represented by structural formula (I-1) (6.60 mg, 0.0240 mmol, 32%) as a white solid. The identification results of the compound represented by structural formula (I-1) are as follows.

[0052] 1H-NMR (500 MHz, CD3OD) δ = 7.48 (s, 1H), 7.30 (d, J = 8.3 Hz, 1H), 7.09 (d, J = 16.0 Hz, 1H), 6.96 (d, J = 16.0 Hz, 1H), 6.74 (d, J = 8.5 Hz, 1H), 4.97 (t, J = 9.5 Hz, 1H), 4.59 (t, J = 8.9 Hz, 2H), 3.61 - 3.47 (m, 2H), 3.24 - 3.20 (m, 2H) ESI-MS [M + H] - : m / z 274.07

[0053] <Synthesis of the compound represented by Structural Formula (I-2)> The synthetic route of the compound of Structural Formula (I-2) is as follows.

Chemical formula

[0054] Sodium hydride was added to a solution of 4-phosphonocrotonic acid (2.99 mL, 13.50 mmol) in tetrahydrofuran, and the mixture was stirred at 0 °C for 1 hour. Then, 2,3-dihydrobenzofuran-5-carboxaldehyde (1) (0.83 ml, 6.75 mmol) was added, and the mixture was stirred at room temperature for 30 minutes and then quenched with ethanol. After extraction with chloroform (100 ml × 3), the organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 4:1) to obtain the ester form (6) (1.86 mg, 7.61 mmol, 56%) as a yellow solid. The identification results of the ester form (6) are as follows.

[0055] 11H-NMR (500 MHz, CDCl3) δ = 7.44 (dd, J = 10.9, 15.45 Hz, 1H), 7.35 (s, 1H), 7.22 (d, J = 13.3Hz, 1H), 6.77 - 6.70 (m, 2H), 5.92 (d, J = 14.9 Hz, 1H), 4.62 (t, J = 17.2, 8.6 Hz, 2H), 4.22 (q, J = 6.85, 14.3 Hz, 2H), 3.23 (t, J =8.3, 17.15 Hz, 2H),1.31 (t, J = 14.3, 7.45 Hz, 3H)

[0056] The ester form (6) (1 g, 4.09 mmol) was dissolved in isopropanol (30 mL) and 5M aqueous sodium hydroxide solution (15 mL). After stirring at 110 °C for a certain time, 100 mL of chloroform was added. This solution was extracted with distilled water (100 mL), the aqueous layer was acidified with 6M hydrochloric acid, and then extracted with chloroform (100 mL × 3). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to obtain the carboxyl form (7) (813 mg, 3.76 mmol, 92%) as a white solid. The identification results of the carboxyl form (7) are as follows.

[0057] 1 1H-NMR (500 MHz, CD3OD) δ = 7.44 (s, 1H), 7.41 (dd, J = 10.3, 14.9 Hz, 1H), 7.26 (d, J = 8.0 Hz, 1H), 6.92 - 6.82 (m, 2H), 6.71 (d, J = 8.6 Hz, 1H), 5.91 (d, J = 8.6 Hz, 1H), 4.56 (t, J = 8.6, 17.75 Hz, 2H), 3.21 (t, J = 8.6, 17.2 Hz, 2H)

[0058] The carboxyl compound (7) (350 mg, 1.62 mmol) was dissolved in an N,N-dimethylformaldehyde solution (5 mL). To this solution, S-trityl-D-cysteine methyl ester (D-cys(Trt)-OMe) (801 mg, 1.93 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (460 mg, 2.39 mmol), and N,N-dimethylaminopyridine (DMAP) (390 mg, 3.19 mmol) were added. After stirring at room temperature for 3 hours under an argon atmosphere, it was quenched with distilled water (5 mL) and extracted with chloroform (50 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: 60 g, hexane:ethyl acetate = 2:1) to obtain the amide compound (8) (521 mg, 0.907 mmol, 56%) as a yellow solid. The identification results of the amide compound (8) are as follows.

[0059] 1 H-NMR (500 MHz, CDCl3) δ = 7.39-7.20 (m, 17H), 6.81-6.20 (m, 3H), 5.96 (d, J = 8.05 Hz, 1H), 5.86 (d, J = 14.85 Hz, 1H), 4.74-4.72 (m, 1H), 4.61 (t, J = 8.6, 17.2 Hz, 2H), 3.22 (t, J = 8.6, 17.2 Hz, 2H), 2.74-2.67 (m, 2H)

[0060] A dehydrated methylene chloride solution (2 mL) of amide compound (8) (150 mg, 0.261 mmol) and triphenylphosphine oxide (Ph3PO) (87.1 mg, 0.312 mmol) was added to a solution prepared by dissolving trifluoromethanesulfonic anhydride (Tf2O) (0.05 mL, 0.313 mmol) in a dehydrated methylene chloride solution (2 mL) at 0 °C under an argon atmosphere, and then stirred at room temperature for 15 minutes. Saturated aqueous sodium bicarbonate (20 mL) was added to the reaction mixture to quench it, and the mixture was extracted with chloroform (20 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: 40.0 g, hexane:ethyl acetate = 2:1) to obtain thiazoline methyl ester compound (9) (46 mg, 0.145 mmol, 55%) as a yellow solid. The identification results of thiazoline methyl ester compound (9) are as follows.

[0061] 1 1H-NMR (500 MHz, CDCl3) δ = 7.34 (s, 1H), 7.19 (d, J = 8.0 Hz, 1H), 6.92 (dd, J = 9.2, 15.5 Hz, 1H), 6.76 - 6.72 (m, 2H), 6.56 (d, J = 15.5 Hz, 1H), 5.17 (t, J = 9.15, 18.3 Hz, 1H), 4.59 (t, J = 8.6, 17.7 Hz, 2H), 3.62 - 3.51 (m, 2H), 3.21 (t, J = 8.6, 17.2 Hz, 2H)

[0062] 6M hydrochloric acid (0.5 mL) was added to a tetrahydrofuran solution (1 mL) of thiazoline methyl ester compound (9) (40 mg, 0.126 mmol) under an argon atmosphere. After stirring at room temperature for 20 hours, it was quenched with sodium bicarbonate. This reaction mixture was subjected to preparative medium-pressure column chromatography (smart flash EPCLC Al-580S ULTRAPACK COLUMNS C 18Purified with H2O / methanol = 95 / 5), and the compound represented by structural formula (I-2) (24 mg, 0.079 mmol, 62%) was obtained as a yellow solid. The identification results of the compound represented by structural formula (I-2) are as follows.

[0063] 1 H-NMR (500 MHz, CD3OD) δ 7.43 (s, 1H), 7.31 - 7.22 (m, 2H), 6.97 (dd, J = 15.2, 8.9 Hz, 1H), 6.85 (d, J = 9.2 Hz, 1H), 6.76 - 6.69 (m, 2H), 4.99 (t, J = 10.6 Hz, 1H), 4.56 (t, J = 8.3 Hz, 2H), 3.61 - 3.49 (m, 2H), 3.21 (t, J = 8.9 Hz, 2H)

[0064] <Synthesis of the compound represented by structural formula (I-3)> The synthetic route of the compound of structural formula (I-3) is as follows.

Chemical formula

[0065] To a toluene solution (10 mL) of 3,4-ethylenedioxybenzaldehyde (10) (601 mg, 3.66 mmol), the Wittig reagent ((carbethoxymethylene)triphenylphosphorane: Ph3P=CHCO2Et) (2.82 g, 8.10 mmol) was added. After stirring at 120 °C for 1.5 hours, it was returned to room temperature and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel: 16.7 g, hexane:ethyl acetate = 4:1) to obtain the ester form (11) (429 mg, 1.83 mmol, 50%) as a white solid. The identification results of the ester form (11) are as follows.

[0066] 11H-NMR (500 MHz, CDCl3) δ = 7.56 (d, J = 16.0 Hz, 1H), 7.05 (s, 1H), 7.02 (d, J = 8.0 Hz, 1H), 6.85 (d, J = 8.0 Hz, 1H), 6.27 (d, J = 16.0 Hz, 1H), 4.27-4.22 (comp, 6H), 1.32 (t, J = 7.2 Hz, 3H) HR-ESI-MS: m / z: [M + H] + C 13 H 14 Calculated value for C15H16O4: 234.08921; Measured value: 234.08281

[0067] The ester compound (11) (429 mg, 1.83 mmol) was dissolved in isopropanol (40 mL) and 5M aqueous sodium hydroxide solution (16 mL). After stirring at room temperature for 14.5 hours, chloroform (100 mL) was added. The solution was extracted with distilled water (100 mL × 3), the aqueous layer was acidified with 6M hydrochloric acid, and then extracted with ethyl acetate (50 mL × 3). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to obtain the carboxyl compound (12) (330 mg, 1.60 mmol, 87%) as a white solid. The identification results of the carboxyl compound (12) are as follows.

[0068] 1 1H-NMR (500 MHz, CDCl3) δ = 7.67 (d, J = 16.0 Hz, 1H), 7.09 (s, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H), 6.29 (d, J = 16.0 Hz, 1H), 4.29 (q, J = 5.5 Hz, 4H) HR-ESI-MS: m / z: [M + Na] + C 11 H 10 Calculated value for C12H11NaO4: 229.04768; Measured value: 229.05374

[0069] The carboxyl compound (12) (300 mg, 1.58 mmol) was dissolved in an N,N-dimethylformaldehyde solution (8 mL). To this solution, S-trityl-D-cysteine methyl ester (D-cys(Trt)-OMe) (791 mg, 1.91 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (462 mg, 2.41 mmol), and N,N-dimethylaminopyridine (DMAP) (384 mg, 3.16 mmol) were added under an argon atmosphere. After stirring at room temperature for 24 hours, the reaction was quenched with saturated aqueous sodium bicarbonate (10 mL), and the mixture was extracted with ethyl acetate (50 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: 16.2 g, hexane:ethyl acetate = 2:1) to obtain the amide compound (13) (823 mg, 1.46 mmol, 92%) as a white solid. The identification results of the amide compound (13) are as follows.

[0070] 1 H-NMR (500 MHz, CDCl3) δ = 7.48 (d, J = 16.0 Hz, 1H), 7.43-7.15 (comp, 16H), 7.01 (m, 2H), 6.85 (d, J = 8.0 Hz, 1H), 6.19 (d, J = 16.0 Hz, 1H), 4.76 (m, 1H), 4.26 (m, 4H), 2.78-2.57 (m, 2H) ESI-MS [M + Na] + : m / z 588.19

[0071] To a dehydrated methylene chloride solution (3 mL) of amide compound (13) (780 mg, 1.38 mmol) and triphenylphosphine oxide (Ph3PO) (461 mg, 1.66 mmol), a solution prepared by dissolving trifluoromethanesulfonic anhydride (Tf2O) (0.792 mL, 4.83 mmol) in dehydrated methylene chloride solution (3 mL) was added at 0 °C under an argon atmosphere, and then the mixture was stirred at room temperature for 4 hours. Saturated aqueous sodium bicarbonate (20 mL) was added to the reaction mixture to quench it, and the mixture was extracted with chloroform (20 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: 20.1 g, hexane:ethyl acetate = 2:1) to obtain thiazoline methyl ester compound (14) (138 mg, 0.452 mmol, 33%) as a white solid. The identification results of thiazoline methyl ester compound (14) are as follows.

[0072] 1 1H-NMR (500 MHz, CDCl3) δ = 7.04-7.00 (m, 2H), 6.98 (d, J = 8.4 Hz, 1H), 6.93 (d, J = 16.0 Hz, 1H), 6.84 (d, J = 8.6 Hz, 1H), 5.18 (t, J = 9.2 Hz, 1H), 4.27-4.23 (m, 4H), 3.65-3.52 (m, 2H) ESI-MS [M + H] + : m / z 306.06

[0073] To a tetrahydrofuran solution (1 mL) of thiazoline methyl ester compound (14) (60.0 mg, 0.196 mmol) was added 6M hydrochloric acid (1 mL) under an argon atmosphere. After stirring at room temperature for 16.5 hours, the reaction was quenched with powdered sodium bicarbonate. This reaction mixture was subjected to automated preparative medium-pressure column chromatography (smart flash EPCLC Al-580S ULTRAPACK COLUMNS C 18Purified with H2O / methanol = 95 / 5), and the compound represented by the structural formula (I-3) (10.6 mg, 0.0360 mmol, 19%) was obtained as a white solid. The identification results of the compound represented by the structural formula (I-3) are as follows.

[0074] 1 1H-NMR (500 MHz, CD3OD) δ = 7.07-7.02 (m, 3H), 6.96 (d, J = 16.5 Hz, 1H), 6.83 (d, J = 8.6 Hz, 1H), 4.98 (t, J = 9.5 Hz, 1H), 4.25 (q, J = 4.6 Hz, 4H), 3.61-3.46 (m, 2H) HR-ESI-MS : m / z : [M + H] + C 14 H 13 Calculated value of C12H13NO4S 292.05592; Measured value 292.05883

[0075] <Synthesis of the compound represented by the structural formula (I-4)> The synthetic route of the compound of the structural formula (I-4) is as follows.

Chemical formula

[0076] Sodium hydride was added to a tetrahydrofuran solution of 4-phosphonocrotonic acid (2.70 mL, 12.18 mmol), and the mixture was stirred at 0 °C for 1 hour. Then, 3,4-ethylenedioxybenzaldehyde (10) (1 g, 6.091 mmol) was added, and the mixture was stirred at room temperature for 30 minutes and then quenched with ethanol. After extraction with chloroform (50 ml × 3), the organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 4:1) to obtain the ester form (15) (1.42 g, 5.45 mmol, 89%) as a yellow solid. The identification results of the ester form (15) are as follows.

[0077] 1H-NMR (500 MHz, CDCl3) δ = 7.41 (dd, J = 10.3, 15.45 Hz, 1H), 6.98 (d, J = 1.75 Hz, 1H), 6.96 (dd, J = 2.3, 8.6 Hz, 1H), 6.84 (d, J = 8.0 Hz, 1H), 6.80 - 6.68 (m, 2H), 5.93 (d, J = 14.9 Hz, 1H), 4.28 - 4.25 (m, 4H), 4.22 (q, J = 6.85, 14.3 Hz, 2H), 1.31 (t, J = 7.4 Hz, 3H)

[0078] The ester compound (15) (1 g, 4.31 mmol) was dissolved in isopropanol (30 mL) and 5M aqueous sodium hydroxide solution (2 mL). After stirring at 80 °C for 3 hours, 100 mL of chloroform was added. This solution was extracted with distilled water (100 mL), the aqueous layer was acidified with 6M hydrochloric acid, and then extracted with chloroform (100 mL × 3). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to obtain the carboxyl compound (16) (832 mg, 3.58 mmol, 83%) as a white solid. The identification results of the carboxyl compound (16) are as follows.

[0079] 1 H-NMR (500 MHz, CDCl3) δ = 7.42 - 7.37 (m 1H), 7.02 (d, J = 2.3 Hz 1H), 7.01 (dd, J = 2.3, 8.6 Hz, 1H), 6.84 - 6.79 (m, 3H), 5.93 (d, J = 14.9 Hz, 1H), 4.26 - 4.22 (m, 4H)

[0080] The carboxyl compound (16) (360 mg, 1.55 mmol) was dissolved in an N,N-dimethylformaldehyde solution (5 mL). To this solution were added S-trityl-D-cysteine methyl ester (D-cys(Trt)-OMe) (770 mg, 1.86 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (450 mg, 2.34 mmol), and N,N-dimethylaminopyridine (DMAP) (380 mg, 3.11 mmol). After stirring at room temperature for 3 hours under an argon atmosphere, it was quenched with distilled water (10 mL) and extracted with chloroform (50 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: 70.0 g, hexane:ethyl acetate = 2:1) to obtain the amide compound (17) (696 mg, 1.17 mmol, 76%) as a yellow solid. The identification results of the amide compound (17) are as follows.

[0081] 1 H-NMR (500 MHz, CDCl3) δ = 7.39-7.20 (m, 15H), 6.99 (d, J = 1.7 Hz, 1H), 6.96 (dd, J = 2.3, 8.6 Hz, 1H), 6.84 (d, J = 15.45 Hz, 1H), 6.78-6.67 (m, 2H), 5.95 (d, J = 7.45 Hz, 2H), 4.75-4.71 (m, 1H), 4.28-4.26 (m, 1H), 3.72 (s, 1H), 2.73-2.66 (m, 2H)

[0082] A dehydrated methylene chloride solution (2 mL) of amide compound (17) (150 mg, 0.253 mmol) and triphenylphosphine oxide (Ph3PO) (84.5 mg, 0.303 mmol) was added to a solution prepared by dissolving trifluoromethanesulfonic anhydride (Tf2O) (0.051 mL, 0.303 mmol) in a dehydrated methylene chloride solution (2 mL) at 0 °C under an argon atmosphere, and then the mixture was stirred at room temperature for 30 minutes. Saturated aqueous sodium bicarbonate (10 mL) was added to the reaction mixture to quench it, and the mixture was extracted with chloroform (20 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: 60.0 g, hexane:ethyl acetate = 2:1) to obtain thiazoline methyl ester compound (18) (63 mg, 0.191 mmol, 75%) as a yellow solid. The identification results of thiazoline methyl ester compound (18) are as follows.

[0083] 1 1H-NMR (500 MHz, CDCl3) δ = 6.97 (d, J = 2.25 Hz, 1H), 6.95 (dd, J = 2.3, 8.6 Hz, 1H), 6.91 (dd, J = 7.45, 17.75 Hz, 1H), 6.83 (d, J = 8 Hz, 1H), 6.76 - 6.68 (m, 2H), 6.57 (d, J = 14.9 Hz, 1H), 5.17 (t, J = 8.6, 17.75 Hz, 2H), 4.27 - 4.34 (m, 4H), 3.62 - 3.51 (m, 2H)

[0084] 6M hydrochloric acid (1 mL) was added to a tetrahydrofuran solution (1 mL) of thiazoline methyl ester compound (18) (46 g, 0.138 mmol) under an argon atmosphere. After stirring at room temperature for 18 hours, it was quenched with sodium bicarbonate. This reaction mixture was subjected to preparative medium-pressure column chromatography (smart flash EPCLC Al-580S ULTRAPACK COLUMNS C 18Purified with H2O / methanol = 95 / 5), and a compound represented by the structural formula (I-4) (28 mg, 0.088 mmol, 63%) was obtained as a yellow solid. The identification results of the compound represented by the structural formula (I-4) are as follows.

[0085] 1 1H-NMR (500 MHz, CD3OD) δ = 6.99 - 6.90 (m, 3H), 6.85 - 6.72 (m, 3H), 6.58 (d, J = 14.85 Hz, 1H), 4.95 (t, J = 9.15, 18.35 Hz, 1H), 4.25 - 4.22 (m, 4H), 3.59 - 3.46 (m, 2H)

[0086] <Synthesis of the compound represented by the structural formula (I-5)> The synthetic route of the compound of the structural formula (I-5) is as follows.

Chemical formula

[0087] Sodium hydride (60%) (1.62 g, 40.6 mmol) was added to a tetrahydrofuran solution of 4-phosphonocrotonic acid (2.85 mL, 12.8 mmol), and the mixture was stirred at 0 °C for 1 hour. Then, piperonal (19) (1.51 g, 10.0 mmol) was added, and the mixture was stirred at room temperature for 1.5 hours. After that, distilled water was added dropwise to quench the reaction. The mixture was extracted with ethyl acetate (75 ml × 3), and the organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 4:1) to obtain a crude product (2.51 g) of the ester compound (20) as a pale yellow solid. The identification results of the crude product of the ester compound (20) are as follows.

[0088] 1H-NMR (500 MHz, CHLOROFORM-D) δ 7.41 (dd, J = 15.5, 10.9 Hz, 1H), 6.99 (d, J = 1.7 Hz, 1H), 6.91 (dd, J = 8.0, 1.7 Hz, 1H), 6.82 - 6.78 (m, 2H), 6.70 (dd, J = 15.5, 10.9 Hz, 1H), 5.98 (s, 2H), 5.94 (d, J = 14.9 Hz, 1H), 4.22 (q, J = 7.3 Hz, 2H), 1.31 (t, J = 7.2 Hz, 3H) 13 C-NMR (126 MHz, CHLOROFORM-D) δ 167.3, 148.6, 148.4, 144.8, 140.2, 130.7, 124.6, 123.0, 120.5, 108.6, 105.9, 101.5, 60.4, 14.4

[0089] The crude product (2.34 g) of the ester form (20) was dissolved in isopropanol (20 mL) and 5M aqueous sodium hydroxide solution (5 mL), and distilled water (15 mL) was added. After stirring at 80 °C for 9 hours, it was acidified with 6M hydrochloric acid and extracted with chloroform (100 mL × 3). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to obtain the carboxyl form (21) (1.60 g, 7.33 mmol, 68% (two steps)) as a pale yellow solid. The identification results of the carboxyl form (21) are as follows.

[0090] 1 H-NMR (500 MHz, ACETONE-D6) δ 7.44 - 7.39 (m, 1H), 7.18 (d, J = 1.7 Hz, 1H), 7.05 (dd, J = 8.0, 1.1 Hz, 1H), 6.99 - 6.98 (m, 2H), 6.87 (d, J = 8.0 Hz, 1H), 6.05 (s, 2H), 5.99 (d, J = 15.5 Hz, 1H) 13C-NMR (126 MHz, ACETONE-D6) δ 167.0, 148.7, 148.5, 145.2, 140.1, 131.0, 124.8, 123.1, 120.5, 108.4, 105.7, 101.6

[0091] The carboxyl compound (21) (270 mg, 12.4 mmol) was dissolved in N,N-dimethylformaldehyde solution (10 mL). To this solution were added S-trityl-D-cysteine methyl ester (D-cys(Trt)-OMe) (562 mg, 1.36 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (607 mg, 3.17 mmol), and N,N-dimethylaminopyridine (DMAP) (609 mg, 4.99 mmol). After stirring at room temperature for 7 hours under an argon atmosphere, it was quenched with distilled water (30 mL) and extracted with chloroform (75 mL × 3). The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 2:1) to obtain the amide compound (22) (588 mg, 1.02 mmol, 82%) as a pale yellow solid. The identification results of the amide compound (22) are as follows.

[0092] 1 H-NMR (500 MHz, CHLOROFORM-D) δ 7.38 (d, J = 8.0 Hz, 6H), 7.34 (dd, J = 14.9, 10.9 Hz, 1H), 7.30 - 7.26 (m, 6H), 7.24 - 7.20 (m, 3H), 6.99 (d, J = 1.1 Hz, 1H), 6.90 (dd, J = 8.0, 1.7 Hz, 1H), 6.80 - 6.77 (m, 2H), 6.68 (dd, J = 15.5, 10.9 Hz, 1H), 6.07 (d, J = 7.4 Hz, 1H), 5.98 (s, 2H), 5.90 (d, J = 14.9 Hz, 1H), 4.75 - 4.71 (m, 1H), 3.72 (s, 3H), 2.74 - 2.66 (m, 2H) 13C-NMR (126 MHz, CHLOROFORM-D) δ 171.1, 165.6, 148.5, 148.3, 144.4, 142.0, 139.6, 130.9, 129.6, 128.1, 127.0, 124.6, 122.9, 122.4, 108.6, 105.9, 101.5, 67.0, 52.8, 51.2, 34.1

[0093] A dehydrated methylene chloride solution (5 mL) of the amide compound (22) (373 mg, 0.646 mmol) was added to a dehydrated methylene chloride solution (5 mL) of triphenylphosphine oxide (Ph3PO) (222 mg, 0.799 mmol) and trifluoromethanesulfonic anhydride (Tf2O) (0.250 mL, 1.52 mmol) at 0 °C under an argon atmosphere, and then stirred at room temperature for 25 minutes. Saturated aqueous sodium bicarbonate (50 mL) was added to the reaction mixture to quench it, and the mixture was extracted with chloroform (50 mL × 3). The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 1:1) to obtain the thiazoline methyl ester compound (23) (123 mg, 0.388 mmol, 60%) as a pale yellow solid. The identification results of the thiazoline methyl ester compound (23) are as follows.

[0094] 1 H-NMR (500 MHz, CHLOROFORM-D) δ 6.99 (d, J = 1.1 Hz, 1H), 6.93-6.89 (m, 2H), 6.78 (d, J = 8.0 Hz, 1H), 6.73-6.71 (m, 2H), 6.58 (d, J = 15.5 Hz, 1H), 5.98 (s, 2H), 5.17 (t, J = 9.2 Hz, 1H), 3.82 (d, J = 6.3 Hz, 3H), 3.60 (dd, J = 10.9, 9.2 Hz, 1H), 3.54 (dd, J = 10.9, 9.2 Hz, 1H) 13C-NMR (126 MHz, CHLOROFORM-D) δ 171.4, 170.0, 148.5, 148.3, 142.7, 138.5, 130.8, 125.4, 124.7, 122.8, 108.6, 105.9, 101.4, 78.0, 52.9, 34.7

[0095] To a solution of the thiazoline methyl ester derivative (23) (53.1 mg, 0.167 mmol) in tetrahydrofuran (1 mL) was added 4M hydrochloric acid (1 mL) under an argon atmosphere. After stirring at room temperature for 13 hours, the reaction was quenched with sodium hydrogen carbonate. The reaction mixture was purified by medium-pressure preparative liquid chromatography (smart flash EPCLC Al-580S ULTRAPACK COLUMNS C 18 H2O / methanol = 95 / 5) to obtain the compound represented by the structural formula (I-5) (37.7 mg, 0.124 mmol, 74%) as a pale yellow solid. The identification results of the compound represented by the structural formula (I-5) are as follows.

[0096] 1 H-NMR (500 MHz, METHANOL-D4) δ 7.08 (d, J = 1.7 Hz, 1H), 6.96 (dd, J = 8.0, 1.7 Hz, 1H), 6.94-6.91 (m, 1H), 6.86 (dd, J = 15.2, 10.0 Hz, 1H), 6.80-6.77 (m, 2H), 6.59 (d, J = 14.9 Hz, 1H), 5.96 (s, 2H), 4.96 (t, J = 9.2 Hz, 1H), 3.58 (dd, J = 10.9, 9.2 Hz, 1H), 3.49 (dd, J = 10.6, 9.5 Hz, 1H) 13 C-NMR (126 MHz, METHANOL-D4) δ 176.8, 169.3, 148.4, 142.1, 138.1, 131.1, 125.3, 124.3, 122.5, 108.0, 105.4, 101.4, 80.8, 35.5

[0097] <Measurement of Emission Spectrum> Using the compounds (luminescent substrates) represented by Structural Formulas (1-1) to (1-5) synthesized as described above, the emission spectrum was measured.

[0098] <<Measuring Device>> ·Measurement of Emission Spectrum The emission spectrum was measured using the weak emission fluorescence spectrum device AB-1850 manufactured by ATTO Corporation. All the measured spectra are spectra corrected for the characteristics of the detector (data interval 0.25 nm, measurement range 400 - 750 nm).

[0099] ·pH Measurement pH measurement was performed using the F-23 type glass electrode hydrogen ion concentration indicator manufactured by Horiba, Ltd.

[0100] <<Reagents>> ·Ultra-pure Water Water collected from MILLIPORE's Milli-RX12α was used.

[0101] ·Ppy Luciferase (derived from North American firefly Photinus pyralis) Solution The recombinant type (QuantiLum (registered trademark), catalog number E1701) manufactured by Promega was used.

[0102] ·ATP-Mg Solution The product manufactured by Sigma (catalog number 00386-41) was used.

[0103] ·Potassium Phosphate Buffer (KPB Solution) Dipotassium hydrogen phosphate · 12 hydrate (special grade) and potassium dihydrogen phosphate · 2 hydrate (special grade) manufactured by Wako Pure Chemical Industries, Ltd. were dissolved in ultra-pure water and used after adjusting the pH.

[0104] <<Measurement Method>> At room temperature, 5 μL of a KPB solution (pH 8.0, 500 mM), 5 μL of a luminescent substrate solution (100 μM), 5 μL of a 1 mg / mL Ppy luciferase solution, and 10 μL of a 200 μM ATP-Mg solution were mixed, and the luminescence spectrum was measured for 180 seconds using a luminescence spectrum measuring device (AB-1850). The results are shown in FIGS. 1 to 5. FIGS. 1 to 5 are luminescence spectra normalized so that the maximum value of the luminescence intensity is 1.

[0105] <<Results>> From FIGS. 1 to 5, for the benzene ring in the general formula (I), R 2 forms a 5-membered or 6-membered ring, and further, by including one or two O's as ring-constituting elements, as described in Non-Patent Document 2, it can be seen that the emission wavelength becomes shorter than when the dimethylamino group is substituted with a hydroxyl group.

Industrial Applicability

[0106] 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 (I): 【Chemical 1】 [wherein, R １ is hydrogen or an alkyl group having 1 to 4 carbon atoms, R ２ is a group represented by any one of -CH₂-CH₂-O-, -O-CH₂-O-, -CH₂-CH₂-CH₂-O-, -O-CH₂-CH₂-O-, -O-CH₂-S-, -O-CH₂-CH₂-S-, or a group in which hydrogen in these groups is substituted with an alkyl group having 1 to 4 carbon atoms, n is an integer from 0 to 2], a heterocyclic compound characterized by being represented by

2. R in the general formula (I) above ２ is represented by any one of -CH ２ -CH ２ -O-, -O-CH ２ -O-, -CH ２ -CH ２ -CH ２ -O-, -O-CH ２ -CH ２ -O-, and is the heterocyclic compound according to claim 1.

3. The heterocyclic compound according to Claim 1 or 2, wherein n in the general formula (I) is 1 or 2.

4. The following structural formulas (I-1) to (I-5): [Chemical Formula 2] The heterocyclic compound according to any one of Claims 1 to 3, represented by any of them.

5. A salt of the heterocyclic compound according to any one of Claims 1 to 4.

6. A light-emitting matrix composition characterized by containing the heterocyclic compound according to any one of Claims 1 to 4 or the salt according to Claim 5.

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