Detection reagent for SARS-related coronavirus

Abstract

The present invention relates to a compound represented by a general formula (1) or a salt or a solvate thereof. [In the formula, R1, R2, and R3 are as defined in the description.]

Description

Technical Field

[0001] The present invention relates to a novel compound or a salt or solvate thereof, a method for detecting a SARS-related coronavirus, and a reagent for detecting a SARS-related coronavirus.

Background Art

[0002] The SARS-CoV-2 virus, which is a SARS-related coronavirus, has caused a global pandemic of coronavirus disease 2019 (COVID-19). In the diagnosis of the infectious disease, it is necessary to detect the virus. Current virus detection principles can be broadly classified into polymerase chain reaction (PCR) or antigen-antibody reactions. In PCR, the gene possessed by the virus is amplified, and in antigen-antibody reactions, the specific affinity between viral proteins and immunoglobulins is utilized. Due to the spread of the novel coronavirus, existing devices that have been exclusively used in molecular biology have been intensively miniaturized and made high-throughput suitable for use in medical fields. However, in the extension of existing technologies, simplicity and versatility are reaching their limits, and it is difficult to respond to large-scale pandemics.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] Patent Document 1 discloses that a specific analog of coelenterazine obtained by modifying coelenterazine, which is a substrate in the luciferin-luciferase reaction, can be catalytically oxidized by human serum albumin to emit light. However, there is no disclosure of a luminescent substrate that can specifically detect a specific virus.

[0005] An object of the present invention is to provide a novel compound or a salt or solvate thereof that can be used particularly as a detecting agent for SARS-related coronavirus, a method for detecting SARS-related coronavirus, and a detecting agent for SARS-related coronavirus. **Means for Solving the Problems**

[0006] As a result of intensive studies in view of the above problems, the present inventors have found that a specific compound emits light by reacting with SARS-related coronavirus, and have completed the present invention. The present invention includes the following aspects. [1] A compound represented by the following general formula (1) or a salt or solvate thereof.

[0007] **[Chemical Formula]**

[0008] [In the formula, R 1 is either a methyl group or any one of the groups represented by the following formula (i-1) or (i-2);

[0009] **[Chemical Formula]**

[0010] (In the formula, R 4 is a hydrogen atom, a hydroxyl group, a fluorine atom, a methoxy group, or a trifluoromethyl group.) R 2 is either a hydrogen atom or any one of the groups represented by the following formula (ii);

[0011] **[Chemical Formula]**

[0012] (In the formula, n is an integer of 1 to 5, and R 5 is any one of the groups represented by the following formula (v-1), (v-2), or (v-3).)

[0013] [Chemical formula]

[0014] {wherein X 1 is a nitrogen atom or a CH group.} R 3 is any one of the groups represented by the following formula (iii-1) or (iii-2);

[0015] [Chemical formula]

[0016] (wherein X 2 is a nitrogen atom or a CH group, and R 6 is a hydroxyl group, a methoxy group, a methyl group, a trifluoromethyl group, or any one of the groups represented by the following formula (vi-1), (vi-2), (vi-3), (vi-4), or (vi-5).)

[0017] [Chemical formula]

[0018] and satisfies the following condition (A-i), (A-ii), or (A-iii). (A-i) The said R 1 is a group represented by the following formula (i-2),

[0019] [Chemical formula]

[0020] (wherein R 4 is the same as above.) and the said R 2 is a group represented by the following formula (ii);

[0021] [Chemical formula]

[0022] (wherein n and R 5 are the same as defined above.) (A-ii) The R 1 is a group represented by the following formula (i-1) (however, excluding natural-type marine firefly luciferin);

[0023] [Chemical formula]

[0024] (A-iii) The R 1 is a methyl group, and the R 2 is a group represented by the following formula (ii).

[0025] [Chemical formula]

[0026] (wherein n and R 5 are the same as defined above.)] [2] The compound represented by the general formula (1) is a compound represented by any of the following formulas, the compound according to [1] above or a salt or solvate thereof.

[0027] [Chemical formula]

[0028] [3] A method for detecting SARS-related coronavirus, comprising the step of contacting a compound represented by the following general formula (2) or a salt or solvate thereof with a biological sample collected from a subject.

[0029] [Chemical formula]

[0030] [wherein, R 1is any one of a methyl group or a group represented by the following formula (i-1) or (i-2);

[0031]

Chemical formula

[0032] (In the formula, R 4 is a hydrogen atom, a hydroxyl group, a fluorine atom, a methoxy group, or a trifluoromethyl group.) R 2 is either a hydrogen atom or any one of the groups represented by the following formula (ii);

[0033]

Chemical formula

[0034] (In the formula, n is an integer from 1 to 5, and R 5 is any one of the groups represented by the following formula (v-1), (v-2), or (v-3).)

[0035]

Chemical formula

[0036]

Chemical formula

[0037] (In the formula, X 2 is a nitrogen atom or a CH group, and R 6 is a hydroxyl group, a methoxy group, a methyl group, a trifluoromethyl group, or any one of the groups represented by the following formula (vi-1), (vi-2), (vi-3), (vi-4), or (vi-5).)]]

[0038] [Chemical formula]

[0039] [4] The method for detecting a SARS-related coronavirus according to [3] above, wherein the compound represented by the general formula (2) is a compound represented by any of the following formulas.

[0040] [Chemical formula]

[0041] [Chemical formula]

[0042] [5] The method for detecting a SARS-related coronavirus according to [3] above, wherein the compound represented by the general formula (2) satisfies at least two of the following conditions (B-i), (B-ii), and (B-iii). (B-i) The R 1 is a group represented by the following formula (i-1);

[0043] [Chemical formula]

[0044] (B-ii) The R 2 is a group represented by the following formula (ii);

[0045] [Chemical formula]

[0046] (In the formula, n and R 5 are the same as defined above.) (B-iii) The R 3 is a group represented by the following formula (iii-2-2).

[0047] [Chemical formula]

[0048] [6] The method for detecting a SARS-related coronavirus according to any one of [3] to [5] above, further comprising a step of measuring luminescence after the step of bringing them into contact. [7] A detection agent for a SARS-related coronavirus, comprising a compound represented by the following general formula (2), or a salt or solvate thereof.

[0049] [Chemical formula]

[0050] [In the formula, R 1 is a methyl group or the following formula:

[0051] [Chemical formula]

[0052] (In the formula, R 4 is a hydrogen atom, a hydroxyl group, a fluorine atom, a methoxy group, or a trifluoromethyl group.) and is any one of the groups represented by; R 2 is a hydrogen atom or the following formula:

[0053] [Chemical formula]

[0054] (In the formula, n is an integer from 1 to 5, and R 5 is the following formula:

[0055] [Chemical formula]

[0056] {In the formula, X 1 is a nitrogen atom or a CH group.} and is any one of the groups represented by.) is any one of the groups represented by; R 3 is the following formula:

[0057] [Chemical formula]

[0058] (wherein X 2 is a nitrogen atom or a CH group, and R 6 is a hydroxyl group, a methoxy group, a methyl group, a trifluoromethyl group, or any one of the groups represented by the following formula (vi-1), (vi-2), (vi-3), (vi-4) or (vi-5).)]]

[0059] [Chemical formula] [Advantages of the Invention]

[0060] According to the present invention, a novel compound or a salt or solvate thereof, a method for detecting a SARS-related coronavirus, and a detection agent for a SARS-related coronavirus are provided. [Brief Description of the Drawings]

[0061]

Figure 1

Figure 2

Figure 3

[0062] 1. A compound, or a salt or solvate thereof The compound of the present invention is represented by the following general formula (1). The compound of the present invention has an imidazopyrazinone ring as a main skeleton and includes derivatives of natural Cypridina luciferin. In the present specification, the wavy line in the chemical formula indicates the bonding position.

[0063] [Chemical formula]

[0064] [In the formula, R 1 is any one of a methyl group or a group represented by the following formula (i-1) or (i-2);

[0065] [Chemical formula]

[0066] (In the formula, R 4 is a hydrogen atom, a hydroxyl group, a fluorine atom, a methoxy group, or a trifluoromethyl group.) R 2 is any one of a hydrogen atom or a group represented by the following formula (ii);

[0067] [Chemical formula]

[0068] (In the formula, n is an integer from 1 to 5, and R 5 is any one of a group represented by the following formula (v-1), (v-2), or (v-3).)

[0069] [Chemical formula]

[0070] {In the formula, X 1 is a nitrogen atom or a CH group.} R 3 is any one of the groups represented by the following formula (iii-1) or (iii-2);

[0071]

Chemical formula

[0072] (In the formula, X 2 is a nitrogen atom or a CH group, and R 6 is a hydroxyl group, a methoxy group, a methyl group, a trifluoromethyl group, or any one of the groups represented by the following formula (vi-1), (vi-2), (vi-3), (vi-4), or (vi-5).)

[0073]

Chemical formula

[0074] And satisfies the following condition (A-i), (A-ii), or (A-iii). (A-i) The said R 1 is a group represented by the following formula (i-2),

[0075]

Chemical formula

[0076] (In the formula, R 4 is the same as above.) And the said R 2 is a group represented by the following formula (ii);

[0077]

Chemical formula

[0078] (In the formula, n and R 5 are the same as above.)

[0079] (A-ii) The said R 1is a group represented by the following formula (i-1) (however, excluding native firefly luciferin); [Chemical formula]

[0080] (A-iii) The R 1 is a methyl group, and the R 2 is a group represented by the following formula (ii).

[0081] [Chemical formula]

[0082] (In the formula, n and R 5 are the same as above.)

[0083] The above formula (i-1) has an asymmetric carbon C *1 .

[0084] [Chemical formula]

[0085] C *1 The configuration of may be either the R configuration shown in the following formula (i-1-R) or the S configuration of the following formula (i-1-S). Also, it may be either a single configuration or a mixture thereof. Examples of the mixture of the R configuration and the S configuration include a racemate containing equal amounts of each configuration.

[0086] [Chemical formula]

[0087] In the above formula (i-2), when R 4 is a hydroxyl group, a fluorine atom, a methoxy group, or a trifluoromethyl group, the bonding position to the benzene ring skeleton may be any of the ortho, meta, and para positions with respect to the other methylene group, and preferably the para position. R4 is preferably a hydrogen atom or a hydroxyl group.

[0088] Preferred embodiments of the group represented by the above formula (i-2) include groups represented by the following formulas (i-2-1) and (i-2-2).

[0089]

Chemical formula

[0090] The above n is an integer of 1 to 5, preferably 2 or 3.

[0091] The group represented by the above formula (v-2) is a group represented by the following formula (v-2-1) or (v-2-2).

[0092]

Chemical formula

[0093] The above R 2 Preferred embodiments thereof include a hydrogen atom, or a group represented by the following formula (ii-1) or (ii-2).

[0094]

Chemical formula

[0095] The group represented by the above formula (iii-1) is a group represented by the following formula (iii-1-1) or (iii-1-2).

[0096]

Chemical formula

[0097] In the above formula (iii-2), the bonding position of R 6 to the benzene ring skeleton may be any of the ortho, meta, and para positions with respect to the other imidazopyrazinone ring, and is preferably the para position.

[0098] R 3 As a preferred embodiment, examples of the group represented by the above formula (iii-1-1) or (iii-1-2), or the following formula (iii-2-1), (iii-2-2), (iii-2-3) or (iii-2-4) may be mentioned.

[0099]

Chemical formula

[0100] Natural-type sea pansy luciferin is a compound represented by the following formula and is not included in the compounds satisfying condition (A-ii).

[0101]

Chemical formula

[0102] As a preferred embodiment of the compound represented by the above general formula (1), compounds represented by any of the following formulas may be mentioned.

[0103]

Chemical formula

[0104] The present invention includes salts of the compound represented by the above general formula (1). Such salts are not particularly limited. Specifically, for example, hydrohalic acid salts (e.g., hydrochloride, hydrobromide, and hydroiodide, etc.), inorganic acid salts (e.g., sulfate, nitrate, perchlorate, phosphate, carbonate, and bicarbonate, etc.), organic carboxylates (e.g., acetate, trifluoroacetate, maleate, tartrate, fumarate, and citrate, etc.), organic sulfonates (e.g., methanesulfonate, trifluoromethanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, and camphorsulfonate, etc.) of acid addition salts; quaternary amine salts, alkali metal salts (e.g., sodium salt and potassium salt, etc.), alkaline earth metal salts (e.g., magnesium salt and calcium salt, etc.) and other base addition salts may be mentioned. The present invention includes hydrates and solvates of the compound represented by the above general formula (1). Examples of the solvate include a solvate with ethanol and the like.

[0105] (Production method) The compound of the present invention can be produced, for example, by condensing a selenotelluramine derivative (x1) with a ketoacetal compound (x2-1) or a diacetyl compound (x2-2) as shown in Scheme 1 or 2.

[0106] [Chemical formula]

[0107] [Chemical formula]

[0108] [wherein, R 1 , R 2 and R 3 are the same as defined above.]

[0109] The reaction preferably proceeds by using about 1 to 4 moles of a ketoacetal compound (x2-1) or methylglyoxal (x2-2) per mole of the selenotelluramine derivative (x1) in the presence of water and reacting at a temperature of about 65 to 100 °C for 1 to 5 hours in the presence of an acid. The reaction preferably proceeds by being carried out in a solvent. Examples of the solvent include ethanol and the like.

[0110] The compound of the present invention, or a salt or solvate thereof, is useful, for example, as a detection agent for SARS-related coronavirus, and can be suitably used as a luminescent substrate in the method for detecting SARS-related coronavirus described below. Although the compound of the present invention is a derivative of Cypridina luciferin, it may not function as a luminescent substrate in the luciferin-luciferase reaction.

[0111] 2. Method for detecting SARS-related coronavirus The method for detecting SARS-related coronavirus of the present invention includes a step of bringing a compound represented by the following general formula (2), or a salt or solvate thereof, into contact with a biological sample collected from a subject.

[0112] [Chemical formula] [In the formula, R 1 is any one of a methyl group or a group represented by the following formula (i-1) or (i-2); [Chemical formula]

[0113] (In the formula, R 4 is a hydrogen atom, a hydroxyl group, a fluorine atom, a methoxy group, or a trifluoromethyl group.) R 2 is any one of a hydrogen atom or a group represented by the following formula (ii);

[0114] [Chemical formula]

[0115] (In the formula, n is an integer from 1 to 5, and R 5 is any one of the groups represented by the following formula (v-1), (v-2), or (v-3).) [Chemical formula]

[0116] {In the formula, X 1 is a nitrogen atom or a CH group.} R 3 is any one of the groups represented by the following formula (iii-1) or (iii-2).

[0117] [Chemical formula]

[0118] (In the formula, X 2 is a nitrogen atom or a CH group, and R 6 is a hydroxyl group, a methoxy group, a methyl group, a trifluoromethyl group, or any one of the groups represented by the following formula (vi-1), (vi-2), (vi-3), (vi-4), or (vi-5).)

[0119] [Chemical formula]

[0120] (Compound, etc.) The method for detecting SARS-related coronavirus of the present invention uses the compound represented by the above general formula (2) or a salt or solvate thereof as a luminescent substrate. In the compound represented by the above general formula (2), R 1 , R 2 and R 3 are the same as those in the compound represented by the above general formula (1), and the preferred embodiments are also the same. The compound represented by the general formula (2) above can be produced in the same manner as the compound represented by the general formula (1).

[0121] As a preferred embodiment of the compound represented by the general formula (2) above, compounds represented by any of the following formulas can be mentioned.

[0122]

Chemical formula

[0123]

Chemical formula

[0124] In a more preferred embodiment of the method for detecting SARS-related coronavirus of the present invention, the compound represented by the general formula (2) satisfies at least two of the following conditions (B-i), (B-ii), and (B-iii). (B-i) The R 1 is a group represented by the following formula (i-1);

[0125]

Chemical formula

[0126] (B-ii) The R 2 is a group represented by the following formula (ii);

[0127]

Chemical formula

[0128] (In the formula, n and R 5 are the same as defined above.) (B-iii) The R 3 is a group represented by the following formula (iii-2-2).

[0129]

Chemical formula

[0130] Examples of the compound that satisfies at least two of the above conditions (B-i), (B-ii), and (B-iii) include compounds represented by any of the following formulas.

[0131] [Chemical formula]

[0132] (SARS-related coronavirus) SARS-related coronavirus belongs to the genus Betacoronavirus and includes SARS coronavirus-2 (Severe acute respiratory syndrome coronavirus 2; SARS-CoV-2) and SARS coronavirus (SARS-CoV). SARS-CoV-2 is thought to be the cause of the acute respiratory disease (COVID-19) and has caused a worldwide pandemic of COVID-19. SARS-related coronavirus is distinguished from MERS coronavirus, which causes Middle East respiratory syndrome (MERS).

[0133] (Subject) The subject is not particularly limited as long as it is a subject that can be infected with SARS-related coronavirus. Specifically, it includes humans; non-human primates other than humans, including chimpanzees, other apes, and monkey species; livestock such as cows, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; and small animals or laboratory animals including rodents such as mice, rats, and guinea pigs, and preferably humans. In addition, the subject includes adults, infants, and newborns. One preferred embodiment of the subject is a subject suspected of being infected with SARS-related coronavirus.

[0134] (Biological sample) As a sample, a biological sample derived from a subject can be used as long as it is a sample in which a SARS-related coronavirus may be present. Specifically, examples include cells and body fluid samples derived from a subject. Examples of cells and biological tissues include cells of oral mucosa, cells of nasal mucosa, and epidermis, which are easy to collect. Examples of body fluid samples include samples derived from blood, lymph, urine, sweat, saliva, nasal discharge, and tears.

[0135] (Contact step) The method for detecting a SARS-related coronavirus of the present invention includes a step of contacting a compound represented by the above general formula (2) or a salt or solvate thereof with a biological sample collected from a subject. The contact conditions can be set according to the luciferin-luciferase reaction, such as the pH and salt concentration of the reaction solution, and reaction conditions such as reaction temperature.

[0136] The concentration of the compound represented by the above general formula (2) or a salt or solvate thereof used is not particularly limited, and is preferably 5 to 100 μM, more preferably 20 to 50 μM. If it is above the above lower limit, it is considered preferable because a sufficient luminescence reaction rate can be achieved. If it is below the above upper limit, it is preferable from the viewpoint of the solubility of the compound and the like.

[0137] The reaction time is not particularly limited. Since the luminescence reaction proceeds rapidly, for example, it can be 10 seconds to 10 minutes, more preferably 30 seconds to 5 minutes, and even more preferably 1 to 3 minutes.

[0138] It is considered that the above compound reacts with the spike protein of the SARS-related coronavirus, and when the oxidized form of the above compound transitions to an excited state and then transitions from the excited state to the ground state, luminescence is emitted. Therefore, when a SARS-related coronavirus is present in the biological sample, luminescence is observed after contact. That is, when luminescence is measured, it can be determined that the subject is infected with the SARS-related coronavirus.

[0139] The method for detecting SARS-related coronavirus of the present invention preferably further includes a step of measuring luminescence after the step of bringing them into contact. The step of measuring luminescence can be carried out using a general luminescence measuring device. Examples of the luminescence measuring device include a luminometer, a microscope equipped with a luminescence detecting means, a luminescence imaging device, and the like.

[0140] Thus, the SARS-related coronavirus is detected. For the subject determined to be infected with the SARS-related coronavirus, necessary measures such as infection prevention measures and treatment for other subjects may be taken.

[0141] 3. Detection agent for SARS-related coronavirus The present invention also provides a detection agent containing the compound represented by the above general formula (2) or a salt or solvate thereof. The detection agent is provided, for example, in the form of a composition. The composition may contain other components as necessary. Examples of other components include a base, a carrier, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant, a thickener, a humectant, a coloring agent, a fragrance, a chelating agent, and the like. In addition, the detection agent is also provided, for example, in the form of a kit for detecting SARS-related coronavirus. The kit may contain various reagents (for example, reaction solutions, etc.), instruments (for example, instruments for collecting and storing biological samples), and the like. Further, as the method of using the kit, an instruction manual describing the method for detecting SARS-related coronavirus of the present invention may be included.

Examples

[0142] Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.

[0143] 1. Synthesis example Each reagent was purchased from Fujifilm Wako Pure Chemical Corporation, Kanto Chemical Co., Inc., Tokyo Chemical Industry Co., Ltd., BLD Pharmatech Co., Ltd., or Sigma-Aldrich Co., and used as it was without purification. 1H-NMR and 13 13 C-NMR was measured using a Bruker Avance III-500 spectrometer. Tetramethylsilane (TMS, 0 ppm) was used as an internal standard. Coupling constants (J) are reported in Hz. The abbreviations s, d, t, q, m, and br denote singlet, doublet, triplet, quartet, multiplet, and broad singlet, respectively.

[0144] General Discussion The compounds of the present invention were synthesized by condensing selenotelluramine derivatives with ketoacetal compounds or diacetyl as shown in Scheme 1 or 2.

[0145] [Chemical formula]

[0146] [Chemical formula]

[0147] [wherein, R 1 , R 2 and R 3 are the same as defined above.]

[0148] The selenotelluramine derivatives were synthesized according to the following Reaction Schemes 3-5.

[0149] [Chemical formula]

[0150] [Chemical formula]

[0151] [Chemical formula]

[0152] [wherein, R 1 , R 2 and R 3Same as above.

[0153] Synthesis Example 1: Synthesis of 2-benzyl-6-(1H-indol-3-yl)imidazo[1,2-a]pyrazin-3(7H)-one (CLA1)

[0154] [Chemical formula]

[0155] Under an argon atmosphere, 5-(1H-indol-3-yl)pyrazine-2-amine (40.0 mg, 0.19 mmol, 1 eq.) and 1,1-diethoxy-3-phenylpropan-2-one (50.7 mg, 0.22 mmol, 1.2 eq.) were dissolved in ethanol (3 ml) and ultrapure water (Milli-Q) (0.3 ml), and cooled to 0 °C. The reaction solution was degassed under vacuum, concentrated hydrochloric acid (0.1 ml) was added, and the mixture was stirred at 80 °C overnight. After allowing to cool to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: methylene chloride / methanol = 20 / 1) to obtain 2-benzyl-6-(1H-indol-3-yl)imidazo[1,2-a]pyrazin-3(7H)-one (CLA1). (49.8 mg, 77%) 1 1H-NMR (500 MHz, CD3OD): δ (ppm) = 7.78 (t, J = 11.8 Hz, 2H), 7.66 (s, 1H), 7.56 (s, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.23 (d, J = 7.2 Hz, 2H), 7.18 - 7.05 (m, 5H), 4.04 (s, 2H). 13 13C-NMR (125 MHz, CD3OD): δ (ppm) = 35.43, 108.70, 113.93, 120.80, 122.59, 124.64, 126.42, 128.32, 130.39, 130.83, 139.33, 140.45.

[0156] Synthesis Example 2: Synthesis of 2-(sec-butyl)-6-(1H-indol-3-yl)imidazo[1,2-a]pyrazin-3(7H)-one (CLA19)

[0157] [Chemical formula]

[0158] Under an argon atmosphere, 5-(1H-indol-3-yl)pyrazin-2-amine (40.0 mg, 0.19 mmol, 1 eq.) and 1,1-diethoxy-3-methylpentan-2-one (107 mg, 0.57 mmol, 2 eq.) were dissolved in ethanol (2 ml) and ultrapure water (Milli-Q) (0.2 ml), and cooled to 0 °C. The reaction solution was degassed under vacuum, concentrated hydrochloric acid (0.1 ml) was added, and the mixture was stirred at 80 °C for 4 hours. After allowing to cool to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: chloroform / methanol = 10 / 1) to obtain 2-(sec-butyl)-6-(1H-indol-3-yl)imidazo[1,2-a]pyrazin-3(7H)-one (CLA19). (19.3 mg, 33%) 1 1H-NMR (500 MHz, CD3OD): δ (ppm) = 7.80 (t, J = 5.9 Hz, 1H), 7.74 (s, 1H), 7.63 (s, 1H), 7.58 (s, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.15 - 7.07 (m, 2H), 3.20 (quintet, J = 3.2 Hz, 3H), 1.24 (d, J = 6.9 Hz, 2H), 0.81 (t, J = 7.4 Hz, 3H). 13 13C-NMR (125 MHz, CD3OD): δ (ppm) = 12.40, 19.02, 29.58, 35.36, 107.18, 113.08, 119.95, 121.72, 123.78, 125.50, 125.62, 138.49.

[0159] Synthesis Example 3: Synthesis of 2-benzyl-6-(1H-pyrrolo[2,3-b]pyridin-3-yl)imidazo[1,2-a]pyrazin-3(7H)-one (CLA9)

[0160] [Chemical formula]

[0161] (1) Under an argon atmosphere, 5-bromopyrazine-2-amine (100.0 mg, 0.57 mmol, 1 eq.) and 1-((4-methoxyphenyl)sulfonyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (261.0 mg, 0.62 mmol, 1.1 eq.) were dissolved in ethanol (2 ml) and toluene (16 ml). To this, 1 M aqueous sodium carbonate solution (6 ml) was added, and the mixture was stirred at room temperature. The reaction solution was degassed under vacuum, a catalytic amount of tetrakis(triphenylphosphine)palladium(0) was added, and it was degassed again under vacuum and stirred at 100 °C overnight. After cooling to room temperature, the palladium catalyst was removed by filtration through celite. The obtained residue was extracted with ethyl acetate, then washed with distilled water and saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (eluent: hexane / ethyl acetate = 4 / 1) and concentrated under reduced pressure. The obtained residue was dissolved in 1,4-dioxane (4 ml) and methanol (4 ml), 5 N aqueous sodium hydroxide solution (3 ml) was added thereto, and the mixture was stirred at room temperature overnight. The reaction solution was extracted with ethyl acetate, then washed with distilled water and saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by column chromatography (eluent: ethyl acetate / methanol = 9 / 1) to obtain 5-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrazine-2-amine. (88.0 mg, 72%) 1 1H-NMR (500 MHz, CD3OD): δ (ppm) = 8.40 (d, J = 7.7 Hz, 1H), 8.24 (s, 1H), 8.17 (d, J = 4.1 Hz, 1H), 7.98 (s, 1H), 7.66 (s, 1H), 7.10 (q, J = 7.1 Hz, 1H). 13 13C-NMR (125 MHz, CD3OD): δ (ppm) = 112.54, 116.24, 118.34, 122.99, 129.77, 132.03, 137.90, 139.16, 142.52, 148.09, 152.62.

[0162] (2) Under an argon atmosphere, 5-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrazine-2-amine (30.0 mg, 0.14 mmol, 1 eq.) obtained in the above (1) and 1,1-diethoxy-3-phenylpropan-2-one (63 mg, 0.28 mmol, 2 eq.) were dissolved in ethanol (2 ml) and milliQ (0.2 ml), and cooled to 0 °C. The reaction solution was degassed under vacuum, concentrated hydrochloric acid (0.1 ml) was added, and the mixture was stirred at 80 °C for 4 hours. After allowing to cool to room temperature, it was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: chloroform / methanol = 4 / 1) to obtain 2-benzyl-6-(1H-pyrrolo[2,3-b]pyridin-3-yl)imidazo[1,2-a]pyrazin-3(7H)-one (CLA9). (31.7 mg, 65%) 1 H-NMR (500 MHz, CD3OD): δ (ppm) = 9.20 (q, J = 3.0 Hz, 1H), 9.01 (br, 1H), 8.75 (br, 1H), 8.43 (q, J = 2.2 Hz, 1H), 8.36 (s, 1H) 7.58 (q, J = 4.6 Hz, 1H), 7.24 - 7.14 (m, 5H), 4.20 (s, 2H). 13 C-NMR (125 MHz, CD3OD): δ (ppm) = 18.35, 30.84, 58.30, 112.36, 113.56, 118.00, 124.59, 128.29, 128.69, 129.64, 129.74, 130.00, 136.35, 136.57, 137.54, 137.78, 139.70, 140.02, 141.52.

[0163] Synthesis Example 4: Synthesis of 2-(sec-butyl)-6-(4-(diethylamino)phenyl)imidazo[1,2-a]pyrazin-3(7H)-one (CLA21)

[0164]

Chemical Structure

[0165] (1) Under an argon atmosphere, 5-bromopyrazine-2-amine (200.0 mg, 1.14 mmol, 1 eq.) and (4-(diethylamino)phenyl)boronic acid (355.0 mg, 1.83 mmol, 1.6 eq.) were dissolved in ethanol (3.5 ml) and toluene (24 ml), and 1 M aqueous sodium carbonate solution (9 ml) was added thereto, followed by stirring at room temperature. The reaction solution was degassed under vacuum, a catalytic amount of tetrakis(triphenylphosphine)palladium(0) was added, degassed again under vacuum, and stirred at 100 °C overnight. After cooling to room temperature, the palladium catalyst was removed by filtration through celite. The obtained residue was extracted with ethyl acetate, then washed with distilled water and saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by column chromatography (eluent: hexane / ethyl acetate = 1 / 1) to obtain 5-(4-(diethylamino)phenyl)pyrazine-2-amine. (261.9 mg, 94%) 1 1H-NMR (500 MHz, CDCl3): δ (ppm) = 8.36 (d, J = 1.4 Hz, 1H), 8.00 (d, J = 1.5 Hz, 1H), 7.74 (q, J = 3.0 Hz, 2H), 6.74 (d, J = 8.7 Hz, 2H), 3.39 (q, J = 7.0 Hz, 4H), 1.18 (t, J = 7.0 Hz, 6H). 13 13C-NMR (125 MHz, CDCl3): δ (ppm) = 12.68, 44.56, 111.98, 124.16, 126.83, 131.38, 137.86, 143.90, 147.80, 152.14.

[0166] (2) Under an argon atmosphere, 5-(4-(diethylamino)phenyl)pyrazine-2-amine (40.0 mg, 0.16 mmol, 1 eq.) obtained in the above (1) and 1,1-diethoxy-3-methylpentan-2-one (93 mg, 0.49 mmol, 3 eq.) were dissolved in ethanol (2 ml) and ultrapure water (Milli-Q) (0.2 ml), and cooled to 0 °C. The reaction solution was degassed under vacuum, concentrated hydrochloric acid (0.1 ml) was added, and stirred at 80 °C for 4 hours. After allowing to cool to room temperature, it was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: chloroform / methanol = 20 / 1) to obtain 2-(sec-butyl)-6-(4-(diethylamino)phenyl)imidazo[1,2-a]pyrazin-3(7H)-one (CLA21). (38.9 mg, 70%) 1 1H-NMR (500 MHz, CD3OD): δ (ppm) = 8.98 (s, 1H), 8.75 (s, 1H), 8.23 (d, J = 8.6 Hz, 2H), 7.75 (d, J = 8.4 Hz, 2H), 3.64 (q, J = 7.2 Hz, 4H), 1.77 - 1.69 (m, 2H), 1.33 (d, J = 6.9 Hz, 3H), 1.11 (t, J = 7.2 Hz, 6H), 0.85 (t, J = 7.3 Hz, 3H). 13 13C-NMR (125 MHz, CD3OD): δ (ppm) = 10.78, 12.25, 18.36, 19.46, 29.97, 33.09, 54.66, 58.29, 113.28, 124.32, 129.21, 129.97, 135.74, 137.55, 139.69.

[0167] Synthesis Example 5: Synthesis of 1-(3-(2-benzyl-6-(4-(diethylamino)phenyl)-3-oxo-3,7-dihydroimidazo[1,2-a]pyrazin-8-yl)propyl)guanidine (CLA12)

[0168]

Chemical Structure

[0169] (1) Under an argon atmosphere, tert-butyl (3-(3-amino-6-bromopyrazin-2-yl)propyl)carbamate (150.0 mg, 0.45 mmol, 1 eq.) and (4-(diethylamino)phenyl)boronic acid (122.3 mg, 0.63 mmol, 1.4 eq.) were dissolved in ethanol (3 ml) and toluene (24 ml), and 1 M aqueous sodium carbonate solution (9 ml) was added thereto, followed by stirring at room temperature. The reaction solution was degassed under vacuum, a catalytic amount of tetrakis(triphenylphosphine)palladium(0) was added, degassed again under vacuum, and stirred at 100 °C overnight. After cooling to room temperature, the palladium catalyst was removed by filtration through celite. The obtained residue was extracted with ethyl acetate, then washed with distilled water and saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by column chromatography (eluent: hexane / ethyl acetate = 2 / 3) to obtain tert-butyl (3-(3-amino-6-(4-(diethylamino)phenyl)pyrazin-2-yl)propyl)carbamate. (136.9 mg, 75%) 1 1H-NMR (500 MHz, CDCl3): δ (ppm) = 8.15 (s, 1H), 7.68 (d, J = 8.9 Hz, 2H), 6.65 (d, J = 8.8 Hz, 2H), 4.90 (s, 1H), 4.43 (s, 2H), 3.31 (q, J = 3.3 Hz, 4H), 3.18 (q, J = 6.2 Hz, 2H), 2.64 (t, J = 7.2 Hz, 2H), 1.99 (quintet, J = 6.9 Hz, 2H), 1.36 (s, 9H), 1.10 (t, J = 7.0 Hz, 6H). 13 13C-NMR (125 MHz, CDCl3): δ (ppm) = 12.62, 26.38, 28.43, 30.23, 40.24, 44.47, 79.09, 111.89, 124.48, 126.75, 135.34, 140.61, 143.27, 147.66, 150.15, 156.22.

[0170] (2) Under an argon atmosphere, tert-butyl (3-(3-amino-6-(4-(diethylamino)phenyl)pyrazin-2-yl)propyl)carbamate (135.6 mg, 0.33 mmol) obtained in the above (1) was dissolved in TFA (1 ml) and methylene chloride (6 ml), and stirred at room temperature for 4 hours. The reaction solution was extracted with ethyl acetate, then washed with distilled water and saturated brine, dried over sodium sulfate, and concentrated under reduced pressure to obtain 3-(3-aminopropyl)-5-(4-(diethylamino)phenyl)pyrazin-2-amine. It was used in the next reaction without further purification.

[0171] (3) Under an argon atmosphere, 3-(3-aminopropyl)-5-(4-(diethylamino)phenyl)pyrazin-2-amine (375.0 mg, 1.25 mmol, 1 eq.) obtained in the above (2) and 1H-pyrazole-1-carboximidamide hydrochloride (369.0 mg, 2.51 mmol, 2 eq.) were dissolved in DMF (18 ml), and DIEA (621 mg) was added thereto, and stirred at room temperature overnight. Ether was added to obtain a precipitate, which was then washed with an acetone-ethanol mixed solution (acetone / ethanol = 4 / 1) (10 ml) to obtain 1-(3-(3-amino-6-(4-(diethylamino)phenyl)pyrazin-2-yl)propyl)guanidine. (461.6 mg, quant.) It was used in the next reaction without further purification.

[0172] (4) Under an argon atmosphere, 1-(3-(3-amino-6-(4-(diethylamino)phenyl)pyrazin-2-yl)propyl)guanidine (100.0 mg, 0.29 mmol, 1 eq.) obtained in the above (3) and 1,1-diethoxy-3-phenylpropan-2-one (104.0 mg, 0.46 mmol, 2 eq.) were dissolved in ethanol (4 ml) and ultrapure water (Milli-Q) (0.4 ml), and cooled to 0 °C. The reaction solution was degassed under vacuum, concentrated hydrochloric acid (0.2 ml) was added, and the mixture was stirred at 80 °C for 4 hours. After allowing it to cool to room temperature, it was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: ethanol / water = 2 / 1) to obtain 1-(3-(2-benzyl-6-(4-(diethylamino)phenyl)-3-oxo-3,7-dihydroimidazo[1,2-a]pyrazin-8-yl)propyl)guanidine (CLA12). (30.4 mg, 22%) 1 1H-NMR (500 MHz, CD3OD): δ (ppm) = 8.97 (s, 1H), 8.44 (br, 2H), 7.90 (br, 2H), 7.38 - 7.23 (m, 5H), 4.38 (s, 2H), 3.77 (s, 4H), 3.46 (br, 2H), 2.35 (br, 2H), 1.29 - 1.17 (m, 9H). 13 13C-NMR (125 MHz, CD3OD): δ (ppm) = 10.88, 18.33, 26.74, 30.26, 30.87, 41.95, 55.10, 58.28, 112.81, 124.69, 128.18, 128.30, 129.66, 129.94, 130.26, 137.85, 138.51, 138.93, 139.31, 141.12, 149.89, 158.62.

[0173] Synthesis Example 6: Synthesis of 1-(3-(6-(4-(diethylamino)phenyl)-2-methyl-3-oxo-3,7-dihydroimidazo[1,2-a]pyrazin-8-yl)propyl)guanidine (CLA13)

[0174]

Chemical Structure

[0175] 1-(3-(3-Amino-6-(4-(diethylamino)phenyl)pyrazin-2-yl)propyl)guanidine was obtained in the same manner as in (1) to (3) of Synthesis Example 5. Next, under an argon atmosphere, 1-(3-(3-amino-6-(4-(diethylamino)phenyl)pyrazin-2-yl)propyl)guanidine (100.0 mg, 0.29 mmol, 1 eq.) and methylglyoxal (42.0 mg, 0.58 mmol, 2 eq.) were dissolved in ethanol (4 ml) and ultrapure water (Milli-Q) (0.4 ml), and the solution was cooled to 0 °C. The reaction solution was degassed under vacuum, concentrated hydrochloric acid (0.2 ml) was added, and the mixture was stirred at 80 °C for 5 hours. After allowing to cool to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: ethanol / water = 1 / 4) to obtain 1-(3-(6-(4-(diethylamino)phenyl)-2-methyl-3-oxo-3,7-dihydroimidazo[1,2-a]pyrazin-8-yl)propyl)guanidine (CLA13). (22.1 mg, 19%) 1 1H-NMR (500 MHz, CD3OD): δ (ppm) = 8.79 (s, 1H), 8.31 (d, J = 8.5 Hz, 2H), 7.75 (d, J = 8.3 Hz, 2H), 3.65 (q, J = 6.6 Hz, 5H), 3.50 (q, J = 7.0 Hz, 5H), 3.20 (quintet, J = 1.6 Hz, 2H), 2.48 (s, 3H), 2.36 - 2.28 (m, 2H), 1.37 (br, 2H).

[0176] Synthesis Example 7: Synthesis of 1-(3-(6-(4-(diethylamino)phenyl)-2-(4-hydroxybenzyl)-3-oxo-3,7-dihydroimidazo[1,2-a]pyrazin-8-yl)propyl)guanidine (CLA14)

[0177]

Chemical Structure

[0178] 1-(3-(3-Amino-6-(4-(diethylamino)phenyl)pyrazin-2-yl)propyl)guanidine was obtained in the same manner as in (1) to (3) of Synthesis Example 5. Next, under an argon atmosphere, 1-(3-(3-amino-6-(4-(diethylamino)phenyl)pyrazin-2-yl)propyl)guanidine (100.0 mg, 0.29 mmol, 1 eq.) and 3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-diethoxypropan-2-one (162.0 mg, 0.46 mmol, 1.6 eq.) were dissolved in ethanol (4 ml) and ultrapure water (Milli-Q) (0.4 ml), and the solution was cooled to 0 °C. The reaction solution was degassed under vacuum, concentrated hydrochloric acid (0.2 ml) was added, and the mixture was stirred at 80 °C for 4 hours. After allowing to cool to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: ethanol / water = 1 / 4) to obtain 1-(3-(6-(4-(diethylamino)phenyl)-2-(4-hydroxybenzyl)-3-oxo-3,7-dihydroimidazo[1,2-a]pyrazin-8-yl)propyl)guanidine (CLA14). (41.5 mg, 29%) 1 1H-NMR (500 MHz, CD3OD): δ (ppm) = 8.81 (s, 1H), 8.31 (d, J = 6.9 Hz, 2H), 7.77 (d, J = 6.1 Hz, 2H), 7.07 (d, J = 7.5 Hz, 2H), 6.64 (d, J = 7.3 Hz, 2H), 4.13 (s, 2H), 3.64 (br, 5H), 3.50 (q, J = 7.0 Hz, 5H), 3.26 (br, 2H), 3.20 (quintet, J = 1.5 Hz, 2H), 2.21 (br, 2H).

[0179] Synthesis Example 8: Synthesis of 1-(3-(2-(sec-butyl)-6-(4-(diethylamino)phenyl)-3-oxo-3,7-dihydroimidazo[1,2-a]pyrazin-8-yl)propyl)guanidine (CLA20)

[0180]

Chemical formula

[0181] 1-(3-(3-Amino-6-(4-(diethylamino)phenyl)pyrazin-2-yl)propyl)guanidine was obtained in the same manner as in (1) to (3) of Synthesis Example 5. Next, under an argon atmosphere, 1-(3-(3-amino-6-(4-(diethylamino)phenyl)pyrazin-2-yl)propyl)guanidine (100.0 mg, 0.29 mmol, 1 eq.) and 1,1-diethoxy-3-methylpentan-2-one (110.0 mg, 0.58 mmol, 2 eq.) were dissolved in ethanol (4 ml) and ultrapure water (Milli-Q) (0.4 ml), and the solution was cooled to 0 °C. The reaction solution was degassed under vacuum, concentrated hydrochloric acid (0.2 ml) was added, and the mixture was stirred at 80 °C for 4 hours. After allowing to cool to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: ethanol / water = 1 / 4) to obtain 1-(3-(2-(sec-butyl)-6-(4-(diethylamino)phenyl)-3-oxo-3,7-dihydroimidazo[1,2-a]pyrazin-8-yl)propyl)guanidine (CLA20). (61.2 mg, 47%) 1 1H-NMR (500 MHz, CD3OD): δ (ppm) = 8.96 (s, 1H), 8.45 (d, J = 6.3 Hz, 2H), 7.93 (d, J = 5.5 Hz, 2H), 3.77 (br, 4H), 3.50 (br, 4H), 2.37 (s, 2H), 1.98 - 1.87 (m, 2H), 1.51 (d, J = 6.6 Hz, 3H), 1.23 (br, 6H), 0.98 (d, J = 6.8 Hz, 3H). 13 13C-NMR (125 MHz, CD3OD): δ (ppm) = 10.78, 12.50, 19.64, 26.80, 29.86, 30.97, 32.92, 41.87, 49.84, 54.96, 112.43, 124.65, 127.02, 128.29, 130.08, 137.53, 138.54, 139.29, 141.22, 149.84, 158.64.

[0182] Synthesis Example 9: Synthesis of 1-(3-(2-(4-hydroxybenzyl)-6-(1H-indol-3-yl)-3-oxo-3,7-dihydroimidazo[1,2-a]pyrazin-8-yl)propyl)guanidine (CLA15)

[0183]

Chemical formula

[0184] Under an argon atmosphere, 1-(3-(3-amino-6-(1H-indol-3-yl)pyrazin-2-yl)propyl)guanidine (100.0 mg, 0.32 mmol, 1 eq.) and 3-(4-((tert-butyldimethylsilyl)oxy)phenyl)-1,1-diethoxypropan-2-one (182.0 mg, 0.51 mmol, 1.6 eq.) were dissolved in ethanol (4 ml) and ultrapure water (Milli-Q) (0.4 ml), and cooled to 0 °C. The reaction solution was degassed under vacuum, concentrated hydrochloric acid (0.2 ml) was added, and the mixture was stirred at 80 °C for 4 hours. After allowing to cool to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: ethanol / water = 1 / 2) to obtain 1-(3-(2-(4-hydroxybenzyl)-6-(1H-indol-3-yl)-3-oxo-3,7-dihydroimidazo[1,2-a]pyrazin-8-yl)propyl)guanidine (CLA15). (45.3 mg, 30%) 1 1H-NMR (500 MHz, CD3OD): δ (ppm) = 7.73 (s, 1H), 7.63 (s, 1H), 7.52 (d, J = 7.6 Hz, 1H), 7.14 (d, J = 8.3 Hz, 2H), 7.00 (d, J = 7.8 Hz, 1H), 6.72 - 6.63 (m, 4H), 3.50 (q, J = 7.0 Hz, 2H), 3.27 (t, J = 6.5 Hz, 2H), 2.82 (br, 2H), 1.98 (br, 2H).

[0185] Synthesis Example 10: 2-benzyl-6-(4-(diethylamino)phenyl)-8-(2-(pyridin-4-yl)ethyl)imidazo[1,2-a]pyrazin-3(7H)-one (CLA18)

[0186]

Chem.

[0187] (1) Under an argon atmosphere, 3,5-dibromopyrazine-2-amine (1000.0 mg, 3.95 mmol, 1 eq.) and 4-ethynylpyridine (448.5 mg, 4.34 mmol, 1.1 eq.) were dissolved in DMF (30 ml), and TEA (15 ml) was added thereto, followed by stirring at room temperature. The reaction solution was degassed under vacuum, a catalytic amount of tetrakis(triphenylphosphine)palladium(0) and copper chloride were added, and the mixture was degassed again and stirred at 120 °C for 40 minutes. After cooling to room temperature, the catalyst was removed by filtration through Celite. The obtained residue was extracted with ethyl acetate, washed successively with distilled water and saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by column chromatography (eluent: ethyl acetate / methanol = 10 / 1) to obtain 5-bromo-3-(pyridin-4-ylethynyl)pyrazine-2-amine. (748 mg, 68%) 1 1H-NMR (500 MHz, CD3OD, CDCl3): δ (ppm) = 8.50 (q, J = 2.0 Hz, 2H), 7.99 (s, 1H), 7.48 (q, J = 2.0 Hz, 2H), 4.41 (s, 2H). 13 13C-NMR (125 MHz, CD3OD, CDCl3): δ (ppm) = 87.70, 93.00, 122.39, 124.66, 125.99, 130.52, 144.99, 149.17, 155.18.

[0188] (2) Under an argon atmosphere, 5-bromo-3-(pyridin-4-yl ethynyl)pyrazine-2-amine (720 mg, 2.61 mmol) obtained in the above (1) was dissolved in ethanol (25 ml), a catalytic amount of platinum oxide was added, and after the reaction solution was degassed under vacuum, it was stirred at room temperature overnight. The catalyst was removed by filtration through Celite. The obtained residue was purified by column chromatography (eluent: ethyl acetate / methanol = 10 / 1) to obtain 5-bromo-3-(2-(pyridin-4-yl)ethyl)pyrazine-2-amine. (470 mg, 64%) 1 1H-NMR (500 MHz, CDCl3): δ (ppm) = 8.42 (q, J = 1.9 Hz, 2H), 7.91 (s, 1H), 7.09 (q, J = 1.9 Hz, 2H), 4.52 (s, 2H), 3.06 (t, J = 7.8 Hz, 2H), 2.81 (t, J = 7.8 Hz, 2H). 13 13C-NMR (125 MHz, CDCl3): δ (ppm) = 31.47, 33.32, 124.02, 126.77, 141.40, 142.17, 149.96, 150.15, 151.66.

[0189] (3) Under an argon atmosphere, 5-bromo-3-(2-(pyridin-4-yl)ethyl)pyrazine-2-amine (200.0 mg, 0.71 mmol, 1 eq.) obtained in the above (2) and (4-(diethylamino)phenyl)boronic acid (193.0 mg, 0.85 mmol, 1.2 eq.) were dissolved in ethanol (3 ml) and toluene (20 ml), 1 M aqueous sodium carbonate solution (8 ml) was added thereto, and the mixture was stirred at room temperature. The reaction solution was degassed under vacuum, a catalytic amount of tetrakis(triphenylphosphine)palladium(0) was added, and after degassing under vacuum again, the mixture was stirred at 100 °C for 5 hours. After cooling to room temperature, the palladium catalyst was removed by filtration through Celite. The obtained residue was extracted with ethyl acetate, then washed with distilled water and saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by column chromatography (eluent: ethyl acetate / methanol = 10 / 1) to obtain 5-(4-(diethylamino)phenyl)-3-(2-(pyridin-4-yl)ethyl)pyrazine-2-amine. (224 mg, 90%)

[0190] (4) Under an argon atmosphere, 5-(4-(diethylamino)phenyl)-3-(2-(pyridin-4-yl)ethyl)pyrazin-2-amine (60.0 mg, 0.17 mmol, 1 eq.) obtained in the above (3) and 1,1-diethoxy-3-phenylpropan-2-one (76.0 mg, 0.34 mmol, 2 eq.) were dissolved in ethanol (4 ml) and milliQ (0.4 ml), and cooled to 0 °C. The reaction solution was degassed under vacuum, concentrated hydrochloric acid (0.2 ml) was added, and the mixture was stirred at 80 °C for 4 hours. After allowing to cool to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by chromatography (eluent: chloroform / methanol = 5 / 1) to obtain 2-benzyl-6-(4-(diethylamino)phenyl)-8-(2-(pyridin-4-yl)ethyl)imidazo[1,2-a]pyrazin-3(7H)-one (CLA18). (71.2 mg, 86%) 1 H-NMR (500 MHz, CD3OD): δ (ppm) = 8.65 (d, J = 5.8 Hz, 2H), 8.62 (s, 1H), 8.14 (d, J = 8.7 Hz, 2H), 8.06 (d, J = 5.6 Hz, 2H), 7.73 (br, 2H), 7.22 - 7.07 (m, 5H), 4.17 (s, 2H), 3.67 - 3.59 (m, 8H), 1.09 (t, J = 7.1 Hz, 6H). 13 C-NMR (125 MHz, CD3OD): δ (ppm) = 10.84, 18.38, 30.89, 31.99, 33.18, 49.84, 54.49, 58.28, 112.05, 124.10, 127.84, 127.96, 128.79, 129.57, 129.76, 129.78, 137.70, 138.53, 139.64, 142.11, 147.37, 164.77.

[0191] Synthesis Example 11: Synthesis of 2-benzyl-6-(4-(diphenylamino)phenyl)-8-(2-(pyridin-4-yl)ethyl)imidazo[1,2-a]pyrazin-3(7H)-one (CLA17)

[0192] [Chem.]

[0193] (1) Under an argon atmosphere, 5-bromo-3-(2-(pyridin-4-yl)ethyl)pyrazine-2-amine (200.0 mg, 0.71 mmol, 1 eq.) and (4-(diphenylamino)phenyl)boronic acid (245.0 mg, 0.85 mmol, 1.2 eq.) were dissolved in ethanol (3 ml) and toluene (20 ml), and 1 M aqueous sodium carbonate solution (8 ml) was added thereto, followed by stirring at room temperature. The reaction solution was degassed under vacuum, a catalytic amount of tetrakis(triphenylphosphine)palladium(0) was added, degassed again under vacuum, and stirred at 100 °C for 4 hours. After cooling to room temperature, the palladium catalyst was removed by filtration through celite. The obtained residue was extracted with ethyl acetate, then washed with distilled water and saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by column chromatography (eluent: ethyl acetate / methanol = 10 / 1) to obtain 5-(4-(diphenylamino)phenyl)-3-(2-(pyridin-4-yl)ethyl)pyrazine-2-amine. (302 mg, 95%)

[0194] (2) Under an argon atmosphere, 5-(4-(diphenylamino)phenyl)-3-(2-(pyridin-4-yl)ethyl)pyrazine-2-amine (60.0 mg, 0.13 mmol, 1 eq.) obtained in the above (1) and 1,1-diethoxy-3-phenylpropan-2-one (60.0 mg, 0.27 mmol, 2 eq.) were dissolved in ethanol (4 ml) and milliQ (0.4 ml), and cooled to 0 °C. The reaction solution was degassed under vacuum, concentrated hydrochloric acid (0.2 ml) was added, and stirred at 80 °C for 4 hours. After allowing to cool to room temperature, concentrated under reduced pressure, and the residue was purified by chromatography (eluent: chloroform / methanol = 10 / 1) to obtain 2-benzyl-6-(4-(diphenylamino)phenyl)-8-(2-(pyridin-4-yl)ethyl)imidazo[1,2-a]pyrazin-3(7H)-one (CLA17). (42.1 mg, 54%) 11H-NMR (500 MHz, CD3OD): δ (ppm) = 8.62 (d, J = 6.4 Hz, 2H), 8.42 (s, 1H), 8.02 (d, J = 6.2 Hz, 2H), 7.70 (d, J = 8.4 Hz, 2H), 7.24 - 6.94 (m, 17H), 4.18 (s, 2H), 3.64 (t, J = 6.8 Hz, 2H), 3.57 (t, J = 6.9 Hz, 2H). 13 13C-NMR (125 MHz, CD3OD): δ (ppm) = 18.36, 30.67, 31.92, 33.10, 49.82, 58.29, 110.07, 123.19, 125.05, 126.30, 127.73, 128.10, 128.41, 128.63, 128.78, 129.60, 129.87, 130.61, 138.17, 142.07, 147.01, 148.46, 150.86, 164.72.

[0195] Synthesis Example 12: Synthesis of 6-(1H-indol-3-yl)-2-methylimidazo[1,2-a]pyrazin-3(7H)-one (CLA2)

[0196]

Chemical Structure

[0197] The target compound was synthesized according to the description in the literature: Photochem. Photobiol. Sci., 2008, 7, 197 - 207.

[0198] Synthesis Example 13: Synthesis of 2-benzyl-6-(4-(diethylamino)phenyl)imidazo[1,2-a]pyrazin-3(7H)-one (CLA3)

[0199]

Chemical Structure

[0200] The target compound was synthesized according to the description in International Publication No. WO 2021 / 187531.

[0201] Synthesis Example 14: Synthesis of 6-(4-(diethylamino)phenyl)-2-methylimidazo[1,2-a]pyrazin-3(7H)-one (CLA4)

[0202]

Chemical Structure

[0203] The target compound was synthesized according to the description in International Publication No. 2021 / 187531.

[0204] Synthesis Example 15: Synthesis of 2-benzyl-6-(4-(dimethylamino)phenyl)imidazo[1,2-a]pyrazin-3(7H)-one (CLA7)

[0205]

Chemical Structure

[0206] The target compound was synthesized according to the description in International Publication No. 2021 / 187531.

[0207] Synthesis Example 16: Synthesis of 6-(4-(dimethylamino)phenyl)-2-methylimidazo[1,2-a]pyrazin-3(7H)-one (CLA8)

[0208]

Chemical Structure

[0209] The target compound was synthesized according to the description in International Publication No. 2021 / 187531.

[0210] Synthesis Example 17: Synthesis of 2-benzyl-6-(4-(pyrrolidin-1-yl)phenyl)imidazo[1,2-a]pyrazin-3(7H)-one (CLA10)

[0211] [Chemical formula]

[0212] The target compound was synthesized according to the description in International Publication No. 2021 / 187531.

[0213] Synthesis Example 18: Synthesis of 1-(3-(6-(4-(diethylamino)phenyl)-2-methyl-3-oxo-3,7-dihydroimidazo[1,2-a]pyrazin-8-yl)propyl)guanidine (CLA16)

[0214] [Chemical formula]

[0215] The target compound was synthesized according to the description in Japanese Patent Application Laid-Open No. 2012-95649.

[0216] 2. Example 1 Native jellyfish luciferin (manufactured by ATTO Corporation, product number 3512055) and the jellyfish luciferin derivative obtained in the synthesis example were reacted with the spike protein derived from the SARS-CoV-2 virus, and luminescence was measured.

[0217] (Measurement conditions) 5 μL of the protein solution (936 μg / mL) and 45 μL of the luciferin-buffer solution (20 μM luciferin shown in Table 1, 20 mM HEPES, pH 7.4) were mixed in a 96-well plate (1 / 2 Area OpticalPlate-96, manufactured by PerkinElmer), and immediately, the luminescence plate reader (GloMax Explorer Multimode Microplate Reader, manufactured by Promega) was used to measure the signal for 1 minute, and the luminescence intensity ([RLU / min]) was determined. (n = 3)

[0218] As the protein solution, a spike protein derived from the SARS-CoV-2 virus (manufactured by MS Techno Systems Co., Ltd., Trimeric SARS-CoV-2 Spike Protein, Full-length, BSV-COV-PR-34; solution of purified protein) was used. The spike protein of the coronavirus is known to normally form a membrane-bound trimer on the virus envelope, and a trimer was used. As the control protein solution, a solution of human serum-derived IgA (manufactured by Fujifilm Wako Pure Chemical Corporation, product number 306-51123) was used to calculate the signal-to-noise ratio (S / N ratio). The results are shown in Table 1.

[0219]

Table 1

[0220] 3. Example 2 To identify the luminescence reaction site of native coelenterazine in the spike protein of the SARS-CoV-2 virus, in addition to the full-length SARS-CoV-2 spike protein, the luminescence activities of each of these domains and native coelenterazine were examined to identify the luminescence reaction site of native coelenterazine in the spike protein. The SARS coronavirus spike protein is divided into three domains: S1, S2, and the receptor-binding domain (RBD). The following were used as protein samples. All are manufactured by Sino Biological and are all monomers. They are also provided in a lyophilized state. SARS-CoV2 Spike S1+S2 (product number 40589-V08H04) SARS-CoV2 Spike S1 (product number 40591-V08) SARS-CoV2 Spike S2 (product number 40590-V08H1) SARS-CoV2 Spike RBD (product number 40592-V08H) SARS-CoV2 Spike RBD(Y453F)(Product No. 40592-V08H80) SARS-CoV2 Spike RBD (N501Y)(Product No. 40592-V08H82)

[0221] In addition, the luminescence activities of the spike proteins of the SARS-related coronavirus SARS coronavirus (SARS-CoV), as well as the spike proteins of the Middle East respiratory syndrome coronavirus (MERS-CoV) and human coronavirus (HCoV), which are coronaviruses other than SARS-related coronaviruses, and native coelenterazine were examined. As monomeric protein samples, the following were used. All are manufactured by Sino Biological, and all are monomers. They are also provided in a lyophilized state. SARS-CoV Spike S1(Product No. 40150-V05H1) MERS-CoV Spike S1(Product No. HPLC-40069-V08H) HCoV-HKU1 Spike S1(Product No. 40021-V08H) HCoV-NL63 Spike S1(Product No. 40600-V08H) HCoV-229E Spike S1(Product No. 40601-V08H) HCoV-OC43 Spike S1(Product No. 40607-V08H1)

[0222] The lyophilized protein was dissolved in 10 mM PBS (pH 7.4) to obtain a protein solution. As a control, measurements were also performed on only the solvent (Buffer) instead of the protein sample. The measurement conditions were as follows. (Measurement conditions) 5 μL of protein solution (protein shown in the figure at 720 μM) and 45 μL of luciferin-buffer solution (native firefly luciferin at 20 μM, PBS×1) were mixed in a 96-well plate (1 / 2 Area OpticalPlate-96, manufactured by PerkinElmer). Immediately after mixing, the luminescence signal was measured for 1 minute using a luminescence plate reader (GloMax Explorer Multimode Microplate Reader, manufactured by Promega), and the luminescence intensity ([RLU / min]) was determined. (n = 4) The final concentration of the protein in the reaction solution was 72 μg / mL. The results are shown in Figures 1(i) and (ii).

[0223] Furthermore, as other protein samples, human serum-derived IgA (IgA; manufactured by Fujifilm Wako Pure Chemical Corporation, 306-51123) was used and compared with the SARS coronavirus spike protein (Monomer spike; SARS-CoV2 Spike S1+S2 (product number 40589-V08H04)). The lyophilized protein was dissolved in 10 mM PBS (pH 7.4) to obtain a protein solution. As a control, measurements were also performed for only the solvent (Buffer) instead of the protein sample. The measurement conditions were as follows. (Measurement conditions) 5 μL of protein solution (protein shown in the figure at 100 μg / mL) and 45 μL of luciferin-buffer solution (native firefly luciferin at 20 μM, PBS×1) were mixed in a 96-well plate (1 / 2 Area OpticalPlate-96, manufactured by PerkinElmer). Immediately after mixing, the luminescence signal was measured for 1 minute using a luminescence plate reader (GloMax Explorer Multimode Microplate Reader, manufactured by Promega). (n = 4) The final concentration of the protein in the reaction solution was 10 μg / mL. The results are shown in Figure 2(i).

[0224] As a result, it was found that native jellyfish luciferin causes a luminescence reaction at S1 and S2, while it does not react at all in the RBD domain (Fig. 1(i)). Furthermore, as a result of examining the luminescence activities of different types of coronaviruses, it was found that native jellyfish luciferin reacts with the spike proteins of SARS-CoV-2 and SARS-CoV, but does not react with MERS-CoV (Fig. 1(ii)). From this result, it became clear that native jellyfish luciferin can specifically detect SARS-related coronaviruses among coronaviruses. Furthermore, it was found that native jellyfish luciferin does not cause a luminescence reaction with the immunoglobulin protein IgA, which is abundantly contained in biological samples such as saliva (Fig. 2(i)).

[0225] 4. Example 3 The correlation between the concentration of the viral spike protein and the luminescence intensity of native jellyfish luciferin was verified. As a protein solution, a trimer of the spike protein derived from the SARS-CoV-2 virus (manufactured by MS Technology Systems Co., Ltd., Trimeric SARS-CoV-2 Spike Protein, Full-length, BSV-COV-PR-34) was used, and the measurement was performed in the same manner as in Example 1 except that the concentration was changed. The results are shown in Fig. 2(ii). It became clear that the luminescence intensity of native jellyfish luciferin depends on the concentration of the viral spike protein.

[0226] 5. Example 4 Using various proteins as protein samples, it was verified that native jellyfish luciferin and the spike protein of SARS coronavirus specifically cause a luminescence reaction. The following were used as protein samples. Trimer of Spike Protein Derived from SARS-CoV-2 Virus (3-mer; manufactured by MS Techno Systems Co., Ltd., Trimeric SARS-CoV-2 Spike Protein, Full-length, product number BSV-COV-PR-34) α-Amylase (manufactured by Fujifilm Wako Pure Chemical Corporation, product number 017-26371) Lactoferrin, Human (manufactured by Sigma-Aldrich, product number L4040) Lysozyme, Human, Recombinant (Plant Expression) (manufactured by Fujifilm Wako Pure Chemical Corporation, product number 181-02063) Mucin, Porcine Gastric (manufactured by Fujifilm Wako Pure Chemical Corporation, product number 137-09162) Epidermal Growth Factor (EGF), Human, Recombinant (EGF; manufactured by Fujifilm Wako Pure Chemical Corporation, product number 059-07873) IgA, Human Serum Derived (manufactured by Fujifilm Wako Pure Chemical Corporation, product number 306-51123) (Measurement Conditions) 5 μL of protein solution (100 μg / mL) and 45 μL of luciferin-buffer solution (native firefly luciferin 20 μM, PBS×1) were mixed in a 96-well plate (1 / 2 Area OpticalPlate-96, manufactured by PerkinElmer), and immediately the luminescence signal was measured for 1 minute using a luminescence plate reader (GloMax Explorer Multimode Microplate Reader, manufactured by Promega). (n = 4) The results are shown in Figure 4.

[0227] Native firefly luciferin did not cause a luminescence reaction with the above various proteins.

Claims

1. A compound represented by any of the following formulas, or a salt or solvate thereof. 【Chemical 1】

2. A method for detecting a SARS-related coronavirus, comprising a step of contacting a compound represented by any of the following formulas, or a salt or solvate thereof, with a biological sample collected from a subject. [Chemical Formula 2] [Chemical Formula 3]

3. The method for detecting a SARS-related coronavirus according to Claim 2, wherein the compound is CLA12, CLA13, CLA14, CLA18, CLA20, CLA21, or native coelenterazine.

4. The method for detecting a SARS-related coronavirus according to Claim 2, further comprising a step of measuring luminescence after the step of contacting.

5. A detection agent for a SARS-related coronavirus, comprising a compound represented by any of the following formulas, or a salt or solvate thereof. 【Chemical Formula 4】 [Chemical Formula 5]

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