Reagent for detecting 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

SARS-related coronavirus detection reagent

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

[0002] The SARS-CoV-2 virus, a SARS-related coronavirus, has caused the global COVID-19 pandemic. Virus detection is necessary for the diagnosis of infectious diseases. Current virus detection methods can be broadly divided into polymerase chain reaction (PCR) and antigen-antibody reaction. PCR amplifies viral genes, while antigen-antibody reaction utilizes the specific affinity between viral proteins and immunoglobulins. Due to the spread of COVID-19, efforts are being made to miniaturize and increase the throughput of existing equipment that has been used primarily in molecular biology, making it suitable for use in medical settings. However, existing technologies are reaching their limits in terms of simplicity and versatility, making it difficult to respond to large-scale epidemics.

[0003] International Publication No. 2021 / 187531

[0004] Patent Document 1 discloses that a specific coelenterazine analog, which is a modified version of luciferin, a substrate in the luciferin-luciferase reaction, can be oxidized and catalyzed by human serum albumin to emit light, but does not disclose any 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 as a detection agent for SARS-associated coronavirus, a method for detecting SARS-associated coronavirus, and a detection agent for SARS-associated coronavirus.

[0006] The present inventors have conducted extensive research in light of the above-mentioned problems and have found that a specific compound emits luminescence upon reaction with SARS-associated coronavirus, thereby completing the present invention. The present invention encompasses the following aspects: [1] A compound represented by the following general formula (1), or a salt or solvate thereof:

[0007]

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

[0009]

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

[0011]

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

[0013]

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

[0015]

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

[0017]

[0018] And, the following condition (A-i), (A-ii), or (A-iii) is satisfied: (A-i) The R １ is a group represented by the following formula (i-2):

[0019]

[0020] (In the formula, R ４ is the same as above.) And the R２ is a group represented by the following formula (ii):

[0021]

[0022] (Wherein, n and R ５ (A-ii) The above R １ is a group represented by the following formula (i-1) (excluding natural Cypridina luciferin);

[0023]

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

[0025]

[0026] (Wherein, n and R ５ The same as above.) [2] The compound according to the above [1], wherein the compound represented by the general formula (1) is a compound represented by any one of the following formulae:

[0027]

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

[0029]

[0030] [In the formula, R １ is a methyl group or one of groups represented by the following formula (i-1) or (i-2):

[0031]

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

[0033]

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

[0035] {In the formula, X １ is a nitrogen atom or a CH group. ３ is any one of groups represented by the following formula (iii-1) or (iii-2):

[0036]

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

[0038]

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

[0040]

[0041]

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

[0043]

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

[0045]

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

[0047]

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

[0049]

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

[0051]

[0052] (In the formula, R ４ is a hydrogen atom, a hydroxyl group, a fluorine atom, a methoxy group, or a trifluoromethyl group; ２ is a hydrogen atom or the following formula:

[0053]

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

[0055]

[0056] {In the formula, X １ is a nitrogen atom or a CH group.}; ３ is the following formula:

[0057]

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

[0059]

[0060] The present invention provides a novel compound or a salt or solvate thereof, a method for detecting SARS-associated coronavirus, and an agent for detecting SARS-associated coronavirus.

[0061] Figure 1 shows the results of measuring the luminescence activity of natural Cypridina luciferin with (i) each domain of the spike protein of the SARS-CoV-2 virus and (ii) spike proteins of other viruses. Figure 2(i) shows the cross-reactivity of natural Cypridina luciferin with the immunoglobulin protein IgA. Figure 2(ii) shows the correlation between the concentration of the spike protein of the SARS-CoV-2 virus and the luminescence intensity of natural Cypridina luciferin. Figure 3 shows the cross-reactivity of natural Cypridina luciferin with various proteins.

[0062] 1. Compound or 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 the main skeleton and includes derivatives of natural Cypridina luciferin. In this specification, the wavy lines in the chemical formula indicate the bond positions.

[0063]

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

[0065]

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

[0067]

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

[0069]

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

[0071]

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

[0073]

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

[0075]

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

[0077]

[0078] (Wherein, n and R ５ is the same as above.)

[0079] (A-ii) The R １ is a group represented by the following formula (i-1) (excluding natural Cypridina luciferin);

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

[0081]

[0082] (Wherein, n and R ５ is the same as above.)

[0083] The above formula (i-1) is a compound having an asymmetric carbon C ＊１ It has.

[0084]

[0085] C ＊１ The configuration of may be either the R configuration shown in the following formula (i-1-R) or the following formula (i-1-S). In addition, it may be either one configuration alone or a mixture thereof. A mixture of the R and S configurations includes a racemate containing equal amounts of each configuration.

[0086]

[0087] In the above formula (i-2), R ４ When R 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 relative to the other methylene group, and is preferably the para position. ４ is preferably a hydrogen atom or a hydroxyl group.

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

[0089]

[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]

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

[0094]

[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]

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

[0098] R ３ Preferred embodiments of the formula (iii-1-1) or (iii-1-2) above, or groups represented by the following formula (iii-2-1), (iii-2-2), (iii-2-3) or (iii-2-4) can be mentioned.

[0099]

[0100] Natural Cypridina luciferin is a compound represented by the following formula, and is not included in the compounds that satisfy condition (A-ii).

[0101]

[0102] Preferred embodiments of the compound represented by the general formula (1) include compounds represented by any of the following formulae:

[0103]

[0104] The present invention encompasses salts of the compound represented by the general formula (1). Such salts are not particularly limited. Specific examples include acid addition salts of hydrohalides (e.g., hydrochlorides, hydrobromides, and hydroiodides), inorganic acid salts (e.g., sulfates, nitrates, perchlorates, phosphates, carbonates, and bicarbonates), organic carboxylates (e.g., acetates, trifluoroacetates, maleates, tartrates, fumarates, and citrates), and organic sulfonates (e.g., methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, benzenesulfonates, toluenesulfonates, and camphorsulfonates); and base addition salts such as quaternary amine salts, alkali metal salts (e.g., sodium salts and potassium salts), and alkaline earth metal salts (e.g., magnesium salts and calcium salts). The present invention encompasses hydrates and solvates of the compound represented by the general formula (1). Examples of such solvates include solvates with ethanol.

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

[0106]

[0107]

[0108] [In the formula, R １ , R ２ and R ３ is the same as above.]

[0109] The reaction proceeds advantageously in the presence of water, using about 1 to 4 moles of ketoacetal compound (x2-1) or methylglyoxal (x2-2) per mole of coelenteramine derivative (x1), and reacting in the presence of acid at a temperature of about 65 to 100°C for 1 to 5 hours. The reaction proceeds advantageously in a solvent, such as ethanol.

[0110] The compound of the present invention, or a salt or solvate thereof, is useful, for example, as a detection agent for SARS-associated coronavirus, and can be suitably used as a luminescent substrate in the method for detecting SARS-associated coronavirus described below. Note that 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-associated coronavirus The method for detecting SARS-associated coronavirus of the present invention comprises the step of contacting a biological sample collected from a subject with a compound represented by the following general formula (2) or a salt or solvate thereof:

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

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

[0114]

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

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

[0117]

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

[0119]

[0120] (Compounds, etc.) The method for detecting SARS-associated coronavirus of the present invention uses a 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 The compound represented by the general formula (2) can be produced in the same manner as the compound represented by the general formula (1).

[0121] Preferred embodiments of the compound represented by the general formula (2) include compounds represented by any of the following formulae:

[0122]

[0123]

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

[0125]

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

[0127]

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

[0129]

[0130] Examples of compounds that satisfy at least two of the above conditions (Bi), (B-ii), and (B-iii) include compounds represented by any of the following formulae:

[0131]

[0132] (SARS-associated coronavirus) SARS-associated coronaviruses belong to the Betacoronavirus genus and include severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS coronavirus (SARS-CoV). SARS-CoV-2 is believed to be the cause of acute respiratory disease (COVID-19) and has caused the COVID-19 pandemic. SARS-associated coronaviruses are distinct from Middle East respiratory syndrome (MERS), the cause of MERS coronavirus.

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

[0134] (Biological Sample) As the sample, a biological sample derived from a subject can be used as long as it is a sample in which SARS-associated coronavirus may be present. Specific examples include cells and body fluid samples derived from a subject. Examples of cells and body tissues include oral mucosal cells, nasal mucosal cells, and epidermal tissue, which are easy to collect. Examples of body fluid samples include blood-derived samples, lymph, urine, sweat, saliva, nasal mucus, tears, etc.

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

[0136] The concentration of the compound represented by the general formula (2) or its salt or solvate used is not particularly limited, but is preferably 5 to 100 μM, more preferably 20 to 50 μM. A concentration equal to or higher than the lower limit is preferred because it is believed that a sufficient luminescence reaction rate can be achieved. A concentration equal to or lower than the upper limit is preferred from the viewpoint of the solubility of the compound, etc.

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

[0138] It is believed that the compound reacts with the spike protein of SARS-associated coronavirus, and the oxidized form of the compound transitions to an excited state, and then emits luminescence when it transitions from the excited state to the ground state. Therefore, if SARS-associated coronavirus is present in a biological sample, luminescence is observed after contact. In other words, if luminescence is measured, it can be determined that the subject is infected with SARS-associated coronavirus.

[0139] The method for detecting SARS-associated coronavirus of the present invention preferably further comprises a step of measuring luminescence after the contacting step. The step of measuring luminescence can be performed using a general luminescence measuring device. Examples of the luminescence measuring device include a luminometer, a microscope equipped with a luminescence detection means, and a luminescence imaging device.

[0140] In this way, SARS-associated coronavirus is detected. If a subject is determined to be infected with SARS-associated coronavirus, necessary measures such as measures to prevent infection to other subjects and treatment can be taken.

[0141] 3. SARS-associated coronavirus detection agent The present invention also provides a detection agent comprising a compound represented by 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 needed. Examples of other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, etc. The detection agent may also be provided, for example, in the form of a kit for detecting SARS-associated coronavirus. The kit may also include various reagents (e.g., reaction solutions, etc.), tools (e.g., tools for collecting and preserving biological samples), etc. The kit may also include a manual describing the SARS-associated coronavirus detection method of the present invention as a method for using the kit.

[0142] The present invention will be explained in more detail below by way of examples, but the present invention is not limited to these examples.

[0143] 1. Synthesis Examples Reagents were purchased from Fujifilm Wako Pure Chemical Industries, Ltd., Kanto Chemical Co., Ltd., Tokyo Chemical Industry Co., Ltd., BLD Pharma Tech, Inc., or Sigma-Aldrich, and were used as is without purification. １ H-NMR and １３ C-NMR was performed using a Bruker Avance III-500 as the measuring device. Tetramethylsilane (TMS, 0 ppm) was used as the internal standard. Coupling constants (J) are expressed in Hz. The abbreviations s, d, t, q, m, and br represent singlet, doublet, triplet, quartet, multiplet, and broad line, respectively.

[0144] General Remarks The compounds of the present invention were synthesized by condensing a coelenteramine derivative with a ketoacetal compound or diacetyl as shown in Scheme 1 or 2.

[0145]

[0146]

[0147] [In the formula, R １ , R ２ and R ３ is the same as above.]

[0148] Coelenteramine derivatives were synthesized according to the following reaction schemes 3 to 5.

[0149]

[0150]

[0151]

[0152] [In the formula, R １ , R ２ and R ３ is the same 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]

[0155] 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-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 cooling to room temperature, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography (eluent: methylene chloride / methanol = 20 / 1) to give 2-benzyl-6-(1H-indol-3-yl)imidazo[1,2-a]pyrazin-3(7H)-one (CLA1). (49.8 mg, 77%) １ H-NMR (500MHz, CD ３ OD): δ (ppm) = 7.78 (t, J = 11.8 Hz, 2H), 7.66 (s, 1H), 7.56 (s, 1H), 7.34 (d , J=8.0Hz, 1H), 7.23 (d, J=7.2Hz, 2H), 7.18-7.05 (m, 5H), 4.04 (s, 2H). １３ C-NMR (125MHz, CD ３OD): 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]

[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 cooling to room temperature, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography (eluent: chloroform / methanol = 10 / 1) to give 2-(sec-butyl)-6-(1H-indol-3-yl)imidazo[1,2-a]pyrazin-3(7H)-one (CLA19). (19.3 mg, 33%) １ H-NMR (500MHz, CD ３ OD): δ (ppm) = 7.80 (t, J = 5.9Hz, 1H), 7.74 (s, 1H), 7.63 (s, 1H), 7.58 (s, 1H), 7.36 (d, J = 8.0Hz, 1H) ), 7.15-7.07 (m, 2H), 3.20 (quintet, J=3.2Hz, 3H), 1.24 (d, J=6.9Hz, 2H), 0.81 (t, J=7.4Hz, 3H). １３ C-NMR (125MHz, CD ３ OD): 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]

[0161] (1) Under an argon atmosphere, 5-bromopyrazin-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), and 1 M aqueous sodium carbonate solution (6 ml) was added thereto and stirred at room temperature. The reaction solution was degassed under vacuum, and a catalytic amount of tetrakis(triphenylphosphine)palladium(0) was added thereto. The solution was degassed under vacuum again and stirred at 100°C overnight. After cooling to room temperature, the palladium catalyst was removed by filtration through Celite. The resulting residue was extracted with ethyl acetate, washed with distilled water and saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by column chromatography (eluent: hexane / ethyl acetate = 4 / 1) and concentrated under reduced pressure. The resulting residue was dissolved in 1,4-dioxane (4 ml) and methanol (4 ml), to which 5N aqueous sodium hydroxide solution (3 ml) was added and stirred at room temperature overnight. The reaction solution was diluted with ethyl acetate, then washed with distilled water and saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by column chromatography (eluent: ethyl acetate / methanol = 9 / 1) to give 5-(1H-pyrrole[2,3-b]pyridin-3-yl)pyrazin-2-amine. (88.0 mg, 72%) １ H-NMR (500MHz, CD ３ OD): δ (ppm) = 8.40 (d, J = 7.7Hz, 1H), 8.24 (s, 1H), 8.17 (d, J = 4.1Hz, 1H), 7.98 (s, 1H), 7.66 (s, 1H), 7.10 (q, J = 7.1Hz, 1H). １３ C-NMR (125MHz, CD ３OD): 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-pyrrole[2,3-b]pyridin-3-yl)pyrazin-2-amine (30.0 mg, 0.14 mmol, 1 eq.) obtained in (1) above 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 cooling to room temperature, the mixture 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). (31.7mg, 65%) １ H-NMR (500MHz, CD ３ OD): δ (ppm) = 9.20 (q, J = 3.0Hz, 1H), 9.01 (br, 1H), 8.75 (br, 1H), 8.43 (q, J = 2 .2Hz, 1H), 8.36 (s, 1H) 7.58 (q, J=4.6Hz, 1H), 7.24-7.14 (m, 5H), 4.20 (s, 2H). １３ C-NMR (125MHz, CD ３ OD): 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]

[0165] (1) Under an argon atmosphere, 5-bromopyrazin-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). 1 M aqueous sodium carbonate solution (9 ml) was added to the solution and stirred at room temperature. The reaction solution was degassed under vacuum, and a catalytic amount of tetrakis(triphenylphosphine)palladium(0) was added. The solution was degassed under vacuum again and stirred at 100°C overnight. After cooling to room temperature, the palladium catalyst was removed by filtration through Celite. The resulting residue was extracted with ethyl acetate, washed with distilled water and saturated brine, dried over sodium sulfate, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography (eluent: hexane / ethyl acetate = 1 / 1) to give 5-(4-(diethylamino)phenyl)pyrazin-2-amine. (261.9 mg, 94%) １ H-NMR (500MHz, CDCl ３ ): 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 = 8.7Hz, 2H), 3.39 (q, J = 7.0Hz, 4H), 1.18 (t, J = 7.0Hz, 6H). １３ C-NMR (125MHz, CDCl ３ ): 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)pyrazin-2-amine (40.0 mg, 0.16 mmol, 1 eq.) obtained in (1) above 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 the solution was cooled to 0°C. The reaction solution was degassed under vacuum, and concentrated hydrochloric acid (0.1 ml) was added, followed by stirring at 80°C for 4 hours. After cooling to room temperature, the solution 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 H-NMR (500MHz, CD ３ OD): δ (ppm) = 8.98 (s, 1H), 8.75 (s, 1H), 8.23 ​​(d, J = 8.6Hz, 2H), 7.75 (d, J = 8.4Hz, 2H), 3.64 (q, J = 7 .2Hz, 4H), 1.77-1.69 (m, 2H), 1.33 (d, J=6.9Hz, 3H), 1.11 (t, J=7.2Hz, 6H), 0.85 (t, J=7.3Hz, 3H). １３ C-NMR (125MHz, CD ３ OD): 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]

[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 and stirred at room temperature. The reaction solution was degassed under vacuum, and a catalytic amount of tetrakis(triphenylphosphine)palladium(0) was added thereto. The solution was degassed under vacuum again and stirred at 100°C overnight. After cooling to room temperature, the palladium catalyst was removed by filtration through Celite. The resulting residue was extracted with ethyl acetate, washed with distilled water and saturated brine, dried over sodium sulfate, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography (eluent: hexane / ethyl acetate=2 / 3) to give tert-butyl(3-(3-amino-6-(4-(diethylamino)phenyl)pyrazin-2-yl)propyl)carbamate (136.9 mg, 75%). １ H-NMR (500MHz, CDCl ３ ): δ (ppm) = 8.15 (s, 1H), 7.68 (d, J = 8.9Hz, 2H), 6.65 (d, J = 8.8Hz, 2H), 4.90 (s, 1H), 4.43 (s, 2H), 3.31 (q, J = 3.3Hz, 4 H), 3.18 (q, J=6.2Hz, 2H), 2.64 (t, J=7.2Hz, 2H), 1.99 (quintet, J=6.9Hz, 2H), 1.36 (s, 9H), 1.10 (t, J=7.0Hz, 6H). １３ C-NMR (125MHz, CDCl ３ ): 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 (1) above was dissolved in TFA (1 ml) and methylene chloride (6 ml), and the mixture was 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. This 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 (2) above and 1H-pyrazole-1-carboxamidine hydrochloride (369.0 mg, 2.51 mmol, 2 eq.) were dissolved in DMF (18 ml), DIEA (621 mg) was added to the solution, and the mixture was 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.). This 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 (3) above 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 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 cooling to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: ethanol / water = 2 / 1) to give 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 H-NMR (500MHz, CD ３ OD): 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 C-NMR (125MHz, CD ３ OD): δ (ppm) = 10.88, 18.33, 26.74, 30.26, 30.87, 41.95, 55.10, 58.28, 112.81, 124.69, 128.1 8,128.30,129.66,129.94,130.26,137.85,138.51,138.93,139.31,141.12,149.89,158.62.

[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]

[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 cooling to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: ethanol / water = 1 / 4) to give 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 H-NMR (500MHz, CD ３ OD): δ (ppm) = 8.79 (s, 1H), 8.31 (d, J = 8.5Hz, 2H), 7.75 (d, J = 8.3Hz, 2H), 3.65 (q, J = 6.6Hz, 5H), 3. 50 (q, J=7.0Hz, 5H), 3.20 (quintet, J=1.6Hz, 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]

[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 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 cooling to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: ethanol / water = 1 / 4) to give 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 H-NMR (500MHz, CD ３ OD): δ (ppm) = 8.81 (s, 1H), 8.31 (d, J = 6.9Hz, 2H), 7.77 (d, J = 6.1Hz, 2H), 7.07 (d, J = 7.5Hz, 2H), 6.64 (d, J = 7.3Hz, 2H), 4.13 (s, 2H), 3.64 (br, 5H), 3.50 (q, J = 7.0Hz, 5H), 3.26 (br, 2H), 3.20 (quintet, J = 1.5Hz, 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]

[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 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 cooling to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: ethanol / water = 1 / 4) to give 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 H-NMR (500MHz, CD ３ OD): δ (ppm) = 8.96 (s, 1H), 8.45 (d, J = 6.3Hz, 2H), 7.93 (d, J = 5.5Hz, 2H), 3.77 (br, 4H), 3.50 (br, 4H), 2.37 (s, 2H), 1.98-1.87 (m, 2H), 1.51 (d, J = 6.6Hz, 3H), 1.23 (br, 6H), 0.98 (d, J = 6.8Hz, 3H). １３ C-NMR (125MHz, CD ３ OD): 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]

[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 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 cooling to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluent: ethanol / water = 1 / 2) to give 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 H-NMR (500MHz, CD ３ OD): δ (ppm) = 7.73 (s, 1H), 7.63 (s, 1H), 7.52 (d, J = 7.6Hz, 1H), 7.14 (d, J = 8.3Hz, 2H), 7.00 (d, J = 7.8H) z, 1H), 6.72-6.63 (m, 4H), 3.50 (q, J=7.0Hz, 2H), 3.27 (t, J=6.5Hz, 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]

[0187] (1) Under an argon atmosphere, 3,5-dibromopyrazin-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 and stirred at room temperature. The reaction solution was degassed under vacuum, and catalytic amounts of tetrakis(triphenylphosphine)palladium(0) and copper chloride were added. The mixture was degassed under vacuum again and stirred at 120°C for 40 minutes. After cooling to room temperature, the catalyst was removed by filtration through Celite. The resulting residue was extracted with ethyl acetate, washed with distilled water and saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by column chromatography (eluent: ethyl acetate / methanol = 10 / 1) to give 5-bromo-3-(pyridin-4-ylethynyl)pyrazin-2-amine. (748 mg, 68%) １ H-NMR (500MHz, CD ３ OD, CDCl ３ ): δ (ppm) = 8.50 (q, J = 2.0 Hz, 2H), 7.99 (s, 1H), 7.48 (q, J = 2.0Hz, 2H), 4.41 (s, 2H). １３ C-NMR (125MHz, CD ３ OD, CDCl ３ ): 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-ylethynyl)pyrazin-2-amine (720 mg, 2.61 mmol) obtained in (1) above was dissolved in ethanol (25 ml), a catalytic amount of platinum oxide was added, the reaction solution was degassed under vacuum, and then stirred at room temperature overnight. The catalyst was removed by filtration through Celite. The resulting residue was purified by column chromatography (eluent: ethyl acetate / methanol = 10 / 1) to give 5-bromo-3-(2-(pyridin-4-yl)ethyl)pyrazin-2-amine (470 mg, 64%). １ H-NMR (500MHz, CDCl ３): δ (ppm) = 8.42 (q, J = 1.9Hz, 2H), 7.91 (s, 1H), 7.09 (q, J = 1.9Hz , 2H), 4.52 (s, 2H), 3.06 (t, J=7.8Hz, 2H), 2.81 (t, J=7.8Hz, 2H). １３ C-NMR (125MHz, CDCl ３ ): 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)pyrazin-2-amine (200.0 mg, 0.71 mmol, 1 eq.) obtained in (2) above 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), and 1 M aqueous sodium carbonate solution (8 ml) was added thereto and stirred at room temperature. The reaction solution was degassed under vacuum, and a catalytic amount of tetrakis(triphenylphosphine)palladium(0) was added thereto, followed by degassing under vacuum again and stirring at 100°C for 5 hours. After cooling to room temperature, the palladium catalyst was removed by filtration through Celite. The resulting residue was extracted with ethyl acetate, washed with distilled water and saturated brine, dried over sodium sulfate, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography (eluent: ethyl acetate / methanol=10 / 1) to give 5-(4-(diethylamino)phenyl)-3-(2-(pyridin-4-yl)ethyl)pyrazin-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 (3) above 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 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 cooling to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by chromatography (eluent: chloroform / methanol=5 / 1) to give 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 (500MHz, CD ３ OD): δ (ppm) = 8.65 (d, J = 5.8Hz, 2H), 8.62 (s, 1H), 8.14 (d, J = 8.7Hz, 2H), 8.06 (d, J = 5.6Hz, 2 H), 7.73 (br, 2H), 7.22-7.07 (m, 5H), 4.17 (s, 2H), 3.67-3.59 (m, 8H), 1.09 (t, J=7.1Hz, 6H). １３ C-NMR (125MHz, CD ３ OD): δ (ppm) = 10.84, 18.38, 30.89, 31.99, 33.18, 49.84, 54.49, 58.28, 112.05, 124.10, 127.8 4,127.96,128.79,129.57,129.76,129.78,137.70,138.53,139.64,142.11,147.37,164.77.

[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]

[0193] (1) Under an argon atmosphere, 5-bromo-3-(2-(pyridin-4-yl)ethyl)pyrazin-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 and stirred at room temperature. The reaction solution was degassed under vacuum, and a catalytic amount of tetrakis(triphenylphosphine)palladium(0) was added thereto. The solution was degassed under vacuum again and stirred at 100°C for 4 hours. After cooling to room temperature, the palladium catalyst was removed by filtration through Celite. The resulting residue was extracted with ethyl acetate, washed with distilled water and saturated brine, dried over sodium sulfate, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography (eluent: ethyl acetate / methanol=10 / 1) to give 5-(4-(diphenylamino)phenyl)-3-(2-(pyridin-4-yl)ethyl)pyrazin-2-amine (302 mg, 95%).

[0194] (2) Under an argon atmosphere, 5-(4-(diphenylamino)phenyl)-3-(2-(pyridin-4-yl)ethyl)pyrazin-2-amine (60.0 mg, 0.13 mmol, 1 eq.) obtained in (1) above 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 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 cooling to room temperature, the mixture was concentrated under reduced pressure, and the residue was purified by chromatography (eluent: chloroform / methanol=10 / 1) to give 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%). １ H-NMR (500MHz, CD ３OD): 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=6.8Hz, 2H), 3.57 (t, J=6.9Hz, 2H). １３ C-NMR (125MHz, CD ３ OD): 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]

[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]

[0200] The target compound was synthesized according to the description in WO 2021 / 187531.

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

[0202]

[0203] The target compound was synthesized according to the description in WO 2021 / 187531.

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

[0205]

[0206] The target compound was synthesized according to the description in WO 2021 / 187531.

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

[0208]

[0209] The target compound was synthesized according to the description in WO 2021 / 187531.

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

[0211]

[0212] The target compound was synthesized according to the description in WO 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]

[0215] The target compound was synthesized according to the description in JP 2012-95649 A.

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

[0217] (Measurement Conditions) 5 μL of protein solution (936 μg / mL) and 45 μL of 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 Optical Plate-96, PerkinElmer), and the signal was immediately measured for 1 minute using a luminescence plate reader (GloMax Explorer Multimode Microplate Reader, Promega) to determine the luminescence intensity ([RLU / min]). (n=3)

[0218] The protein solution used was spike protein derived from SARS-CoV-2 virus (MS Techno Systems, Trimeric SARS-CoV-2 Spike Protein, Full-length, BSV-COV-PR-34; purified protein solution). Coronavirus spike proteins are known to typically form membrane-bound trimers on the viral envelope, and this trimer was used. A solution of human serum-derived IgA (Fujifilm Wako Pure Chemical Industries, Ltd., product number 306-51123) was used as a control protein solution, and the signal-to-noise ratio (S / N ratio) was calculated. The results are shown in Table 1.

[0219]

[0220] 3. Example 2 To identify the luminescence reaction site of native Cypridina luciferin in the spike protein of the SARS-CoV-2 virus, the luminescence activity of each domain of the SARS-CoV-2 spike protein and native Cypridina luciferin was examined in addition to the full-length spike protein. The SARS coronavirus spike protein is divided into three domains: S1, S2, and the receptor binding domain (RBD). The following protein samples were used. All were manufactured by Sino Biological Co., Ltd. and were in the form of monomers. They were also provided in a lyophilized state. SARS-CoV2 Spike S1+S2 (Part Number 40589-V08H04) SARS-CoV2 Spike S1 (Part Number 40591-V08) SARS-CoV2 Spike S2 ​​(Part Number 40590-V08H1) SARS-CoV2 Spike RBD (Part Number 40592-V08H) SARS-CoV2 Spike RBD (Y453F) (Part Number 40592-V08H80) SARS-CoV2 Spike RBD (N501Y) (Part Number 40592-V08H82)

[0221] The luminescence activity of the spike protein of SARS-CoV, a coronavirus associated with SARS, as well as spike proteins of MERS-CoV and human coronaviruses (HCoV), which are coronaviruses other than SARS-associated coronaviruses, and natural Cypridina luciferin was examined. The following monomer protein samples were used. All were manufactured by Sino Biological Co., Ltd. and were in the form of monomers. They were also provided in a lyophilized state. SARS-CoV Spike S1 (Part Number: 40150-V05H1) MERS-CoV Spike S1 (Part Number: HPLC-40069-V08H) HCoV-HKU1 Spike S1 (Part Number: 40021-V08H) HCoV-NL63 Spike S1 (Part Number: 40600-V08H) HCoV-229E Spike S1 (Part Number: 40601-V08H) HCoV-OC43 Spike S1 (Part Number: 40607-V08H1)

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

[0223] Furthermore, as another protein sample, human serum-derived IgA (IgA; Fujifilm Wako Pure Chemical Industries, Ltd., 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 prepare a protein solution. As a control, measurement was also performed using only the solvent (Buffer) instead of the protein sample. The measurement conditions were as follows: (Measurement conditions) 5 μL of protein solution (100 μg / mL of the protein shown in the figure) and 45 μL of luciferin-buffer solution (20 μM native Cypridina luciferin, 1x PBS) were mixed in a 96-well plate (1 / 2 Area Optical Plate-96, PerkinElmer), and the signal was immediately measured for 1 minute using a luminescence plate reader (GloMax Explorer Multimode Microplate Reader, Promega). (n=4) The final concentration of 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 natural Cypridina luciferin induced a luminescent reaction with S1 and S2, but did not react at all with the RBD domain (Figure 1(i)). Furthermore, when the luminescence activity of different coronaviruses was examined, natural Cypridina luciferin reacted with the spike proteins of SARS-CoV-2 and SARS-CoV, but did not react with MERS-CoV (Figure 1(ii)). These results demonstrated that natural Cypridina luciferin can specifically detect SARS-associated coronaviruses. Furthermore, it was found that natural Cypridina luciferin did not induce a luminescent reaction with the immunoglobulin protein IgA, which is abundant in biological samples such as saliva (Figure 2(i)).

[0225] 4. Example 3 The correlation between the concentration of viral spike protein and the luminescence intensity of native Cypridina luciferin was examined. Measurements were conducted in the same manner as in Example 1, except that a trimer of spike protein derived from the SARS-CoV-2 virus (MS Techno Systems, Trimeric SARS-CoV-2 Spike Protein, Full-length, BSV-COV-PR-34) was used as the protein solution, and the concentration was changed. The results are shown in Figure 2(ii). It was revealed that the luminescence intensity of native Cypridina luciferin depends on the concentration of viral spike protein.

[0226] 5. Example 4 Various proteins were used as protein samples to verify that natural Cypridina luciferin and the spike protein of SARS coronavirus specifically induce a luminescent reaction. Trimer of spike protein derived from SARS-CoV-2 virus (3-mer; MS Techno Systems, Trimeric SARS-CoV-2 Spike Protein, Full-length, product number BSV-COV-PR-34), α-amylase (α-amylase; Fujifilm Wako Pure Chemical Industries, Ltd., product number 017-26371), human lactoferrin (Lactferrin; Sigma-Aldrich, product number L4040), human lysozyme, recombinant (expressed in plants) (Lysozyme; Fujifilm Wako Pure Chemical Industries, Ltd., product number 181-02063), and mucin derived from porcine stomach (Mucin; Fujifilm Wako Pure Chemical Industries, Ltd., product number 137-09162). Epidermal growth factor (EGF), human, recombinant (EGF; Fujifilm Wako Pure Chemical Industries, Ltd., product number 059-07873) IgA derived from human serum (IgA; Fujifilm Wako Pure Chemical Industries, Ltd., product number 306-51123) (Measurement conditions) 5 μL of protein solution (100 μg / mL) and 45 μL of luciferin-buffer solution (20 μM native Cypridina luciferin, PBS x 1) were mixed in a 96-well plate (1 / 2 Area Optical Plate-96, PerkinElmer), and the signal was immediately measured for 1 minute using a luminescence plate reader (GloMax Explorer Multimode Microplate Reader, Promega). (n=4) The results are shown in Figure 4.

[0227] Natural Cypridina luciferin did not undergo a luminescence reaction with the above-mentioned various proteins.

Claims

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

2. A method for detecting SARS-associated 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. 【Chemistry 2】 【Chemistry 3】

3. The method for detecting SARS-associated coronavirus described in claim 2, wherein the compound is CLA12, CLA13, CLA14, CLA18, CLA20, CLA21, or natural Cypridina luciferin.

4. 3. The method for detecting a SARS-associated coronavirus according to claim 2, further comprising the step of measuring luminescence after said contacting step.

5. A detection agent for SARS-associated coronavirus, comprising a compound represented by any one of the following formulas or a salt or solvate thereof. 【Chemistry 4】 【Chemistry 5】

Images