Compound useful as fluorescent probe for detection of de-ADP-ribosylating enzyme activity, salt thereof, or solvate thereof

Abstract

To provide a technology that enables easier detection of de-ADP-ribosylating enzyme activity.SOLUTION: There are provided a compound represented by general formula (1), a salt thereof, or a solvate thereof. [In the formula, R1 and R2 are such that R1 represents -NH2 and R2 represents =N-, or R1 represents =O and R2 represents -NH-, R3 and R4 are the same or different and represent a hydroxy group or a hydrogen atom, R5 to R12 are the same or different and represent a hydrogen atom, a halogen atom, or an alkoxy group, and a double line consisting of a solid line and a dotted line represents a single bond or a double bond.]SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a compound, a salt thereof, or a solvate thereof, which is useful as a fluorescent probe for detecting ADP-ribosylase activity. [Background technology]

[0002] ADP-ribosyltransferases (ADPs) remove ADP-ribose (ADPr) from proteins and DNA by hydrolyzing the glycosidic bond. These enzymes are widely conserved in organisms ranging from viruses to bacteria, plants, and humans. They are broadly divided into two families: ADP-ribose glycohydrolases (ARHs) and macrodomains (Macs). ARHs have been reported to play important roles in defense against Vibrio cholerae infection and DNA damage response in humans. Meanwhile, Macs have recently been shown to play important roles in viral replication in pathogenic viruses. Therefore, pathogenic virus-derived Macs are attracting attention as molecular targets for the development of novel antiviral therapeutics. Therefore, the development of a highly sensitive and simple method for detecting ADP-ribosyltransferase activity is anticipated, which will enable the discovery of enzyme inhibitors that could serve as potential antiviral therapeutics. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Cell Chemical Biology, 2018 Dec 20;25(12):1562-1570. [Non-patent document 2] Bioorganic & Medicinal Chemistry Letters, 2013 Feb 4;23(7):2245-2249. Summary of the Invention [Problem to be solved by the invention]

[0004] Conventional techniques for detecting ADP-ribosylation deactivation enzyme activity use probes in which paranitrophenol (pNP) or 4-trifluoromethylumbelliferone (TFMU) has been introduced into the reducing end of ADPr (Non-Patent Document 1). However, problems exist with detecting enzyme activity using these probes, such as difficulty in detecting enzyme activity under acidic conditions, difficulty in detecting pNP and TFMU (Stokes shift: 120 nm) produced in the enzyme reaction at low probe concentrations because they are non-fluorescent dyes or fluorescent dyes with a small Stokes shift, and the need for analytical equipment such as a high performance liquid chromatograph (HPLC) or a microplate reader.

[0005] An objective of the present invention is to provide a technique for more simply detecting ADP-ribosylase deactivation activity. [Means for solving the problem]

[0006] In view of the above problems, the present inventors have conducted extensive research and have found that a compound represented by the general formula (1):

[0007] [ka]

[0008] It has been found that the above-mentioned problems can be solved by using a compound represented by the formula:

[0009] Section 1. General formula (1):

[0010] [ka]

[0011] [In the formula: R 1 and R 2 is R 1 represents -NH2 and R 2 indicates =N- or R 1indicates =O and R 2 represents -NH-. R 3 and R 4 are the same or different and represent a hydroxy group or a hydrogen atom. 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are the same or different and represent a hydrogen atom, a halogen atom, or an alkoxy group. A double line consisting of a solid line and a dotted line represents a single bond or a double bond.] A compound represented by the formula (I), a salt thereof, or a solvate thereof.

[0012] Section 2.R 1 is -NH2 and R 2 Item 2. The compound according to item 1, a salt thereof, or a solvate thereof, wherein is =N-.

[0013] Section 3.R 3 and R 4 Item 3. The compound, salt thereof, or solvate thereof according to Item 1 or 2, wherein: is a hydroxy group.

[0014] Section 4.R 6 , R 7 , and R 11 are the same or different and are a hydrogen atom, a halogen atom, or an alkoxy group, and R 5 , R 8 , R 9 , R 10 , and R 12 Item 4. The compound, salt thereof, or solvate thereof according to any one of Items 1 to 3, wherein is a hydrogen atom.

[0015] Section 5.R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 5. The compound, salt thereof, or solvate thereof according to any one of items 1 to 4, wherein is a hydrogen atom.

[0016] Item 6. The compound represented by formula (1A):

[0017] [ka]

[0018] 6. The compound according to any one of items 1 to 5, a salt thereof, or a solvate thereof, which is a compound represented by the following formula:

[0019] Item 7. A fluorescent probe comprising the compound according to any one of items 1 to 6, a salt thereof, or a solvate thereof.

[0020] Item 8. The fluorescent probe according to Item 7, which is an agent for detecting ADP-ribosylase deactivation activity.

[0021] Item 9. The fluorescent probe according to Item 8, wherein the ADP-ribosylating enzyme is derived from a coronavirus.

[0022] Item 10. A reagent comprising the compound according to any one of items 1 to 6, a salt thereof, or a solvate thereof.

[0023] Item 11. A medicine comprising the compound according to any one of items 1 to 6, a salt thereof, or a solvate thereof.

[0024] Item 12. A method for detecting ADP-ribosylase activity, comprising contacting the compound according to any one of items 1 to 6, a salt thereof, or a solvate thereof with an ADP-ribosylase and measuring the fluorescence intensity. Item 13. A screening method for a deADP-ribosylation activity regulator, comprising contacting the compound according to any one of items 1 to 6, a salt thereof, or a solvate thereof with a deADP-ribosylation enzyme and a test substance, and measuring the fluorescence intensity as an indicator.

[0025] Item 14. (a) A step of contacting the compound according to any one of Items 1 to 6, a salt thereof, or a solvate thereof with an ADP-ribosylating enzyme and a test substance; (b) measuring the fluorescence intensity (test fluorescence intensity) in the step (a) and comparing the test fluorescence intensity with the fluorescence intensity in the absence of contact with the test substance (control fluorescence intensity); and (c) selecting the test substance as an ADP-ribosylation deactivation activity regulator when the test fluorescence intensity is different from the control fluorescence intensity; Item 14. The screening method according to Item 13, comprising: [Effects of the Invention]

[0026] The present invention provides a technique for more simply detecting ADP-ribosylase activity, specifically, a compound, a salt thereof, or a solvate thereof that is useful as a fluorescent probe for detecting ADP-ribosylase activity. [Brief explanation of the drawings]

[0027] [Figure 1] 1 is a photographic image showing the results of Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0028] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0029] 1.Compound In one aspect, the present invention provides a compound represented by general formula (1):

[0030] [ka]

[0031] The present invention relates to a compound represented by the formula (hereinafter, sometimes referred to as "the compound of the present invention"), a salt thereof, or a solvate thereof (hereinafter, sometimes referred to as "the probe compound of the present invention").

[0032] R1 and R 2 is R 1 represents -NH2 and R 2 indicates =N- or R 1 indicates =O and R 2 represents -NH-. The double line consisting of a solid line and a dotted line in general formula (1) represents a single bond or a double bond. That is, the double line represents R 1 and R 2 Depending on the choice of , it represents a single bond or a double bond. Specifically, it is as follows.

[0033] R in general formula (1) 1 and R 2 Substructure (1X) containing:

[0034] [ka]

[0035] is R 1 represents -NH2 and R 2 If =N-, the partial structure (1Xa):

[0036] [ka]

[0037] and R 1 indicates =O and R 2 When represents -NH-, the partial structure (1Xb) is:

[0038] [ka]

[0039] is.

[0040] The partial structure (1Xa) is the adenine structure of intracellular ADP-ribose. On the other hand, it has been reported that even if a part of the adenine is modified, as in the partial structure (1Xb), it can be recognized by ADP-ribosylase deactivation enzymes (Non-Patent Document 1).

[0041] From the viewpoints of detection sensitivity and recognition by the macrodomain, which is an ADP-ribosylating enzyme, R 1 is -NH2 and R 2 is particularly preferably =N- (partial structure (1Xa)).

[0042] R 3 and R 4 are the same or different and represent a hydroxy group or a hydrogen atom.

[0043] R 3 , R 4 is preferably a hydroxy group from the viewpoints of detection sensitivity, recognition by the macrodomain, which is an ADP-ribosylating enzyme, and the like. 3 and R 4 It is particularly preferred that both of are hydroxy groups.

[0044] R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are the same or different and represent a hydrogen atom, a halogen atom, or an alkoxy group.

[0045] Examples of halogen atoms include fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, etc. Among these, chlorine atoms, bromine atoms, etc. are preferred from the viewpoint of detection sensitivity, etc.

[0046] The alkoxy group may be a lower alkoxy group, such as a linear or branched (preferably linear) alkoxy group having 1 to 8 carbon atoms. From the viewpoint of detection sensitivity and the like, the number of carbon atoms in the alkoxy group is preferably 1 to 6, more preferably 1 to 4, even more preferably 1 or 2, and particularly preferably 1.

[0047] The fluorescent compound (R5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 It has been previously reported that a compound obtained by hydrolyzing the moiety to which the substituent is attached, which is represented by the following general formula (Y), has fluorescent activity even when substituted with a halogen atom or an alkoxy group.

[0048] [ka]

[0049] From the viewpoint of the fluorescent activity of the above fluorescent compounds, R 6 , R 7 , and R 11 are the same or different and are a hydrogen atom, a halogen atom, or an alkoxy group, and R 5 , R 8 , R 9 , R 10 , and R 12 is preferably a hydrogen atom, and R 6 is a hydrogen atom or an alkoxy group, and R 7 is a hydrogen atom, a halogen atom, or an alkoxy group, and R 11 is a hydrogen atom or a halogen atom, and R 5 , R 8 , R 9 , R 10 , and R 12 is more preferably a hydrogen atom.

[0050] R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are preferably all hydrogen atoms from the viewpoints of the fluorescent activity, detection sensitivity, and recognizability by ADP-ribosylating decarboxylase (particularly, recognizability by the macrodomain) of the fluorescent compound.

[0051] The compound of the present invention is, from the viewpoints of the fluorescent activity, detection sensitivity, recognizability from ADP-ribosylating enzymes (particularly, recognizability from macrodomains) of the above fluorescent compound, a compound represented by the formula (1A):

[0052] [ka]

[0053] It is particularly preferable that the compound is represented by the following formula:

[0054] The compound of the present invention is in a state where the hydroxy group of the pyrophosphate moiety is ionized (-O - ), that is, the case of the following general formula (1B).

[0055] [ka]

[0056] The salt of the compound of the present invention is not particularly limited, but is preferably a pharmaceutically acceptable salt when intended for use as an in vivo probe. The salt of the compound of the present invention can be either an acid salt (e.g., a salt with the amino group moiety of the compound of the present invention) or a basic salt (e.g., a salt with the phosphate moiety of the compound of the present invention). Examples of acid salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, trifluoroacetic acid, methanesulfonate, benzenesulfonate, and paratoluenesulfonate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; salts with ammonia; and salts with organic amines such as morpholine, piperidine, pyrrolidine, monoalkylamines, dialkylamines, trialkylamines, mono(hydroxyalkyl)amines, di(hydroxyalkyl)amines, and tri(hydroxyalkyl)amines.

[0057] The compounds of the present invention can also be solvated, such as hydrates. When considering use as an in vivo probe, pharmaceutically acceptable solvents are preferred. Examples of solvents include ethanol, glycerol, acetic acid, methanol, propanol, acetone, N,N-dimethylformamide, and benzene. Hydrates of the compounds of the present invention also include those formed by absorbing moisture from the air.

[0058] The probe compound of the present invention can be synthesized by various methods. Methods for synthesizing ADP-ribose derivatives such as the probe compound of the present invention are known, for example, a synthesis method using the phosphoramidite method, which is a common method for nucleic acid synthesis. However, this synthesis method requires multi-step synthesis, and in addition to the formation of pyrophosphate bonds using a large excess of adenosine phosphoramidite building blocks, there are synthetic challenges with final deprotection, which significantly reduces the overall yield. Therefore, a more efficient synthesis method is desirable.

[0059] From the above viewpoint, it is desirable to synthesize the probe compound according to the method described later in Example 1. For example, the probe compound of the present invention can be synthesized according to a method including the following Scheme Z.

[0060] [ka]

[0061] The compound represented by general formula (2) and the compound represented by general formula (3) can be synthesized according to or in accordance with a known method, or according to the method in Example 1 described below.

[0062] From the viewpoints of yield, ease of synthesis, etc., the amount of the compound represented by general formula (2) used is usually preferably 0.5 to 2.0 mol, more preferably 0.7 to 1.5 mol, per 1 mol of the compound represented by general formula (3).

[0063] This reaction is usually carried out in the presence of a reaction solvent. The reaction solvent is not particularly limited, but an example thereof is N,N-dimethylformamide. The solvent may be used alone or in combination.

[0064] In this reaction, it is preferable to use magnesium chloride as an additive, which can improve the yield. From the viewpoints of yield, ease of synthesis, etc., the amount of magnesium chloride used is preferably 1.0 to 4.0 mol, more preferably 2.0 to 3.0 mol, per 1 mol of the compound represented by general formula (2).

[0065] The reaction can be carried out under heating, at room temperature, or under cooling, and is usually carried out at a temperature of 0 to 100° C. The reaction temperature is particularly preferably 15 to 35° C. The reaction time is not particularly limited, and is usually 1 to 12 hours.

[0066] The progress of the reaction can be monitored by conventional methods such as chromatography. After the reaction is complete, the solvent is removed by distillation, and the product can be isolated and purified by conventional methods such as chromatography and recrystallization. The structure of the product can also be determined by elemental analysis, MS (ESI-MS), IR analysis, and the like. 1 H-NMR, 13 It can be identified by C-NMR or the like.

[0067] 2.Applications The probe compound of the present invention reacts with a deADP-ribosylase to produce the above-mentioned fluorescent compound (a compound represented by general formula (Y)). Therefore, the probe compound of the present invention can be used as a fluorescent probe, more specifically, as an agent for detecting the activity of a deADP-ribosylase. The probe compound of the present invention can also be used as a reagent or a pharmaceutical (e.g., a diagnostic drug).

[0068] The above-mentioned fluorescent compound (compound represented by general formula (Y)) has a wide Stokes shift of approximately 150 nm due to excited-state intramolecular proton transfer (Non-Patent Document 2). Furthermore, fluorescence can be easily confirmed (for example, visually). Furthermore, the fluorescent compound can emit fluorescence regardless of pH conditions. Additionally, the probe compound of the present invention is water-soluble due to its sugar moiety, making it easy to construct a reaction system with ADP-ribosyltransferase. Furthermore, the above-mentioned fluorescent compound (compound represented by general formula (Y)) released by the enzymatic reaction with ADP-ribosyltransferase is poorly soluble in water and is therefore expected to aggregate and form bright spots in aqueous solvents. Therefore, by identifying the fluorescent sites in vivo, cells, or containers, it is possible to attempt to identify the location of ADP-ribosyltransferase or the site of its activity.

[0069] ADP-ribosylating enzymes are enzymes that have the activity of removing ADP-ribose (ADPr) present on proteins or DNA by hydrolyzing the glycosidic bond, and are not particularly limited thereto. They are broadly divided into two families, ADP-ribose glycohydrolase (ARH) and macrodomain (Mac), and both families can be targeted in the present invention.

[0070] Examples of sources of ADP-ribosylating enzymes include mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer.

[0071] The source of the ADP-ribosylating enzyme can be, for example, a virus, a bacterium, a fungus, etc. Furthermore, the source of the ADP-ribosylating enzyme can be a pathogenic microorganism.

[0072] Examples of viruses include influenza viruses (e.g., types A and B), rubella viruses, Ebola viruses, coronaviruses, measles viruses, varicella-zoster viruses, mumps viruses, arboviruses, respiratory syncytial viruses, SARS viruses, hepatitis viruses (e.g., hepatitis B viruses, hepatitis C viruses), yellow fever viruses, AIDS viruses, rabies viruses, hantaviruses, dengue viruses, Nipah viruses, and lyssaviruses; and non-enveloped viruses (viruses without an envelope), such as adenoviruses, noroviruses, rotaviruses, human papillomaviruses, polioviruses, enteroviruses, coxsackieviruses, human parvoviruses, encephalomyocarditis viruses, polioviruses, and rhinoviruses. Among these, coronaviruses are particularly preferred.

[0073] Coronaviruses belong to the Orthocoronavirus subfamily. Examples of coronaviruses include the Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus genera, with Betacoronavirus being preferred. Examples of Betacoronavirus include SARS-related coronavirus (SARSr-CoV), circulating coronavirus HKU1, and MERS coronavirus, with SARSr-CoV being preferred. Examples of SARSr-CoV include SARS-CoV-2 and SARS-CoV-1, with SARS-CoV-2 being preferred.

[0074] Examples of bacteria and fungi include Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, Salmonella enterica, Helicobacter pylori, Clostridium perfringens, Clostridium botulinum, Campylobacter, Escherichia coli, Staphylococcus aureus, Streptococcus staphylococcus, Bacillus cereus, Vibrio parahaemolyticus, Propionibacterium acnes, Clostridium faecalis, Clostridium difficile, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella pneumoniae, Klebsiella pneumoniae, Koinebacterium hemolyticum, Streptococcus hemolyticus, Pseudomonas aeruginosa, Staphylococcus aureus, Mycoplasma, Candida, and Aspergillus.

[0075] When the probe compound of the present invention is used as a reagent or a medicine, the content of the probe compound of the present invention depends on the mode of use, the target of application, the state of the target of application, etc., and is not limited thereto, but can be, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.

[0076] The reagents and medicines may contain other ingredients, such as bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0077] The mode of use of the probe compound of the present invention is not particularly limited. The probe compound of the present invention can be used, for example, in vitro (e.g., by contacting with an ADP-ribosylase in a test tube) or in vivo (e.g., by administering to / ingesting to an animal). The animal in question is not particularly limited, and examples thereof include the above-mentioned mammals.

[0078] When the probe compound of the present invention is administered to / ingested by an animal, any dosage form can be used, for example, oral preparations such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), and jellies; and parenteral preparations such as injectable preparations (e.g., drip injections (e.g., intravenous drip preparations), intravenous injections, intramuscular injections, subcutaneous injections, and intradermal injections), topical preparations (e.g., ointments, poultices, and lotions), suppositories, inhalants, eye preparations, eye ointments, nasal drops, ear drops, and liposomes. The route of administration is not particularly limited as long as the desired effect is obtained, and examples include enteral administration such as oral administration, tube feeding, and enema administration; and parenteral administration such as intravenous administration, intraarterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration, and intraperitoneal administration.

[0079] The dosage of the probe compound of the present invention when administered to an animal is not particularly limited as long as it is an effective amount for functioning as a fluorescent probe, and is usually 0.1 to 1000 mg / kg body weight per day in the case of oral administration, and 0.01 to 100 mg / kg body weight per day in the case of parenteral administration. The dosage can be increased or decreased as appropriate depending on the age, purpose, condition, etc.

[0080] One aspect of the present invention relates to a method for detecting ADP-ribosylase activity, which comprises contacting a probe compound of the present invention with a deADP-ribosylase and measuring the fluorescence intensity.

[0081] The contact is not particularly limited as long as the enzymatic activity of the ADP-ribosylating enzyme is exerted, and is usually carried out in an aqueous solution. The contact time and temperature are also not particularly limited as long as the enzymatic activity of the ADP-ribosylating enzyme is exerted, and are, for example, 10 to 40°C and 1 to 1000 hours.

[0082] The aqueous solution may contain, if necessary, a buffer, a divalent cation (e.g., Mg 2+ The aqueous solution may contain a reducing agent (e.g., DTT, etc.), a soluble ion, etc., and a reducing agent (e.g., DTT, etc.). The pH of the aqueous solution is not particularly limited and may be in the acidic, neutral, or alkaline range. The pH may be, for example, 4.0 or more, 5.0 or more, 6.0 or more, 6.5 or more, or 7.0 or more, or 10.0 or less, 9.0 or less, 8.0 or less, or 7.5 or less.

[0083] The fluorescence intensity can be measured by irradiating the fluorescent compound (compound represented by general formula (Y)) with light having an excitation wavelength and detecting the resulting fluorescence. The irradiated light is preferably light having a wavelength of 260 to 430 nm, more preferably light having a wavelength of 300 to 400 nm, and even more preferably light having a wavelength of 330 to 380 nm.

[0084] Detection can be carried out in vitro or in vivo. For example, a biological sample containing the deADP-ribosylating enzyme (e.g., tissue or cell extract, body fluid, purified body fluid sample, etc.) can be used as the deADP-ribosylating enzyme. Alternatively, the probe compound of the present invention can be administered to an animal to bring it into contact with the deADP-ribosylating enzyme in vivo. Alternatively, the probe compound of the present invention can be brought into contact with the deADP-ribosylating enzyme in a microorganism or cell by contacting the probe compound with the microorganism or cell.

[0085] In one aspect, the present invention relates to a method for screening for a deADP-ribosylation activity regulator, which uses as an indicator the fluorescence intensity when the probe compound of the present invention, a deADP-ribosylation enzyme, and a test substance are contacted.

[0086] A wide range of test substances can be used, regardless of whether they are naturally occurring or artificially produced. Furthermore, not only purified compounds but also compositions containing a variety of compounds and animal and plant extracts can be used. Compounds are not limited to low-molecular-weight compounds, but also include high-molecular-weight compounds such as proteins, nucleic acids, and polysaccharides.

[0087] More specifically, the screening method includes the following steps (a) and (b), and preferably further includes step (c): (a) contacting the probe compound of the present invention with an ADP-ribosylase and a test substance; (b) measuring the fluorescence intensity (test fluorescence intensity) in the step (a) and comparing the test fluorescence intensity with the fluorescence intensity in the absence of contact with the test substance (control fluorescence intensity); (c) selecting the test substance as an ADP-ribosylation deactivation activity regulator when the test fluorescence intensity is different from the control fluorescence intensity.

[0088] The control fluorescence intensity is the fluorescence intensity when the same treatment as in step (a) is carried out except that the test substance is not used.

[0089] If the test fluorescence intensity is higher than the control fluorescence intensity (e.g., 1.1-fold, 1.3-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold higher than the control fluorescence intensity), the test substance can be selected as an ADP-ribosylation deactivation activity enhancer. On the other hand, if the control fluorescence intensity is higher than the test fluorescence intensity (e.g., 1.1-fold, 1.3-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold higher than the test fluorescence intensity), the test substance can be selected as an ADP-ribosylation deactivation activity inhibitor. By selecting the ADP-ribosylation enzyme and microorganisms / cells to be contacted, deactivation ADP-ribosylation activity inhibitors can be used as candidates for antimicrobial drugs, such as antiviral drugs. [Example]

[0090] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0091] The abbreviations used in the examples are as follows. Ac: Acetyl, ADP: Adenosine diphosphate, All: Allyl, AMP: Adenosine monophosphate, Ar: Aryl, ARH: Adenosine diphosphate ribosylhydrolase, ART: Adenosine diphosphate ribosyltransferase, ATP: Adenosine triphosphate, Bu: Butyl, Bn: Benzyl, BTP: 2-Benzothiazol-2-yl-phenol, DAST: (Diethylamino)sulfur trifluoride, DCM: Dichloromethane, DDQ: 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone, DMAP: N,N-dimethyl-4-aminopyridine, DMBA: Barbituric Acid, DMC: 2-Chloro-1,3-dimethylimidazolinium chloride, DMF: N,N-dimethylformamide, DNA: Deoxyribonucleic acid, DTT: l-Dithiothreitol solution, Et: Ethyl, Im: Imidazole, 2-MeImIm-Cl: 2-(2-Methylimidazolyl)-1,3-dimethylimidazolinium chloride, Me: Methyl, mRNA: messenger-Ribonucleic acid, MS: Molecular sieves, NAD: Nicotinamide adenine dinucleotide, Nap: 2-Naphthylmethyl, nsp: Non-structural protein, Pr: Propyl, pro: Protease, Ph: Phenyl, RNA: Ribonucleic acid, TBS: tert-Butyldimethylsilyl, TBAF: Tetra-n-butylammonium fluoride, TBDPS: tert-Butyldiphenylsilyl, TBSOTf: tert-Butyldimethylsilyl trifluoromethanesulfonate, TEA: Triethylamine, TFA: Trifluoroacetic acid, THF: Tetrahydrofuran, TIPDS: 1,1,3,3-Tetraisopropyldisiloxane, TLC: Thin-layer chromatography. ,

[0092] The following procedures, instruments, and equipment were used in the examples. The following descriptions are also consistent. For TLC analysis, a TLC plate silica gel 60F254 (Merck) was used. Detection was by UV absorption (254 nm) and a color-developing reagent (10% H2SO4 in EtOH, 12MoO3·H3PO4-H2O / H2SO4 / 85% aqueous H3PO4 mixed solvent). Concentration was performed under reduced pressure while heating in a water bath or by lyophilization. For column chromatography, silica gel (Fuji Silysia, 80 mesh), silica gel 120 (spherical) (Kanto Chemical, RP-18), DEAE Sephacel (GE Healthcare), and Sephadex (GE Healthcare, LH-20) were used. The developing solvent for TLC and the elution solvent for column chromatography are shown (v / v).1 H-NMR spectrum and 13 C-NMR spectra were obtained using a Bruker BipSpin AVANCE III 500 (500 MHz). Chemical shift values ​​are shown as σ values ​​(ppm) relative to an internal standard. 31 P-NMR spectra are expressed as σ values ​​(ppm) relative to an external standard. Spectral data are denoted according to the signal shape as follows: (Legend: d = doublet, t = triplet, q = qultet, dd = double doublet, m = multiplet / multiple resonances.) Optical rotations were measured using a Horiba High Sensitive Polarimeter SEPA-300. HRMS spectra (ESI-TOF) were measured using a Bruker Daltonics microTOF (ESI-TOF) mass spectrometer in either positive or negative ion mode.

[0093] Example 1. Synthesis of Compound 1 (ADP-ribose BTP glycoside) To achieve a highly efficient synthesis of the target molecule 1, this example employed pyrophosphate bond formation, a key reaction that chemically bonds unprotected phosphate groups (Scheme 1). For the unprotected phosphate-phosphate coupling reaction, we employed a phosphate activator (2-MeImIm-Cl) capable of selectively introducing an imidazole leaving group into the phosphate group. This activator is a chemical species in which a methyl group has been introduced into the imidazole moiety of the existing phosphate activator (ImIm-Cl), improving the hydrolysis resistance that had been a problem with ImIm-Cl. Specifically, the phosphate group of AMP was chemoselectively activated using 2-MeImIm-Cl, followed by unprotected phosphate-phosphate coupling reaction with ribose-5-phosphate derivative 3 to derive 1.

[0094] [ka]

[0095] We planned to synthesize a ribose 5-phosphate building block bearing a BTP solid-state fluorescent dye at the anomeric position by utilizing a highly α-selective ribofuranosylation reaction using a fluoroglycosyl donor (Scheme 2). Based on the design of known ribofuranose fluoroglycosyl donors, we selected All groups as the 2- and 3-position protecting groups and NaP as the 5-position protecting group. After introducing the BTP solid-state fluorescent dye at the anomeric position by α-selective ribofuranosylation, we performed a protecting group conversion, phosphorylation of the 5-hydroxyl group, and final deprotection to synthesize the desired ribose 5-phosphate derivative 3.

[0096] [ka]

[0097] Example 1-1. Synthesis of ribose 5-phosphate derivatives Example 1-1-1. Synthesis of ribose donor precursor To synthesize a ribose 5-phosphate derivative bearing a BTP solid-state luminescent dye via an α-glycosidic bond, we synthesized 5-Nap-protected ribose 4 (Scheme 3). Based on a previous report (J. Am. Chem. Soc. 2015, 137, 3558-3564), d-ribose 7 was reacted with hydrochloric acid in a mixed solvent of methanol and acetone. The reaction proceeded smoothly, affording the isopropylidene-protected methyl glycoside 8 in 66% yield. Subsequently, a Nap group was introduced at the 5-hydroxyl group by Williamson ether synthesis, successfully affording the desired 5-Nap-protected derivative 9 in 42% yield.

[0098] [ka]

[0099] Next, we attempted to remove the isopropylidene in the presence of sulfuric acid to liberate the 2- and 3-hydroxyl groups (Scheme 4). TLC showed that 9 was completely consumed, but no spot corresponding to the dihydroxyl derivative 10 was observed. Instead, several highly polar compounds were observed. The use of highly acidic sulfuric acid likely led to the simultaneous acid hydrolysis of the undesired methyl glycoside or NaP group. Therefore, we decided to abandon this synthetic route.

[0100] [ka]

[0101] Based on the above results, we modified the synthetic route to one that does not require the introduction and removal of the isopropylidene group (Scheme 5). Specifically, we decided to selectively introduce a TBDPS group at the 5-hydroxyl group, and then convert the remaining 2- and 3-hydroxyl groups to Allyl groups. First, commercially available methyl ribofuranoside 2 was reacted with TBDPSCl to obtain TBDPS derivative 11 in 99% yield. Next, the 2- and 3-hydroxyl groups were converted to Allyl groups using the Williamson ether synthesis method, yielding Allyl derivative 12 in 49% yield.

[0102] Specifically, it is as follows:

[0103] Methyl β-d-ribofuranoside (500 mg, 3.05 mmol) was dissolved in DMF (30.5 mL) under an Ar atmosphere, and imidazole (498 mg, 7.32 mmol) and TBDPSCl (0.95 mL, 3.66 mmol) were added. The mixture was stirred at room temperature for 2 h. After completion of the reaction was confirmed by TLC (n-hexane / EtOAc = 1 / 1), the reaction mixture was extracted three times with CHCl3, washed with water and saturated brine, dried over anhydrous sodium sulfate, and evaporated. The resulting residue was purified by flash silica gel chromatography (n-hexane / EtOAc = 11 / 9) to give compound 11 (1216 mg, 3.02 mmol, 99%) as a colorless, clear, viscous liquid. [α]D -28.8° (c 1.0, CHCl3). 1 H NMR (500 MHz, CDCl3) δ 7.70-7.37 (m, 10 H, Ar), 4.85 (s, 1 H, H-1), 4.34 (near t, 1 H, H-4), 4.03 (m, 2 H, H-3, H-2), 3.83 (dd, 1 H, J 4,5a = 4.9 Hz, J gem = 10.6 Hz, H-5 a ), 4.03 (dd, 1 H, J 4,5b = 5.8 Hz, H-5 b ), 3.31 (s, 3 H, OCH3), 2.64 (s, 1 H, OH), 2.32 (s, 1 H, OH), 1.07 (s, 9 H, 3 CH3). 13 C NMR (125 MHz, CDCl3) δ 135.7, 133.4, 129.8, 129.8, 127.8, 127.8, 108.2, 83.5, 75.3, 72.1, 65.0, 55.3, 26.9, 19.3. HRMS (ESI) m / z: found [M+Na] + 425.1754, C 22 H 30 O5Si calcd. for [M+Na] + 425.1755.。

[0104] Under an Ar atmosphere, compound 11 (1.22 g, 3.02 mmol) was dissolved in DMF (12.1 mL) at 4 °C. NaH (362 mg, 9.06 mmol) was added at 4 °C and the mixture was stirred for 30 min. Allyl bromide (0.770 mL, 9.06 mmol) was then added at 4 °C and the mixture was stirred for 5 h. After confirming the completion of the reaction by TLC (n-hexane / EtOAc = 4 / 1), MeOH was added at 4 °C to quench the reaction. The reaction mixture was then extracted three times with CHCl3, washed with water and saturated brine, dried over anhydrous sodium sulfate, and evaporated. The resulting residue was purified by flash silica gel chromatography (n-hexane / EtOAc = 21 / 4) to give compound 12 (717 mg, 1.49 mmol, 49%) as a colorless, transparent, viscous liquid. [α] D -7.1° (c 1.0, CHCl3). 1 H NMR (500 MHz, CDCl3) δ 7.71-7.35 (m, 10 H, Ar), 5.98-5.86 (m, 2 H, 2 CH2CHCH2), 5.33-5.15 (m, 4 H, 2 CH2CHCH2), 4.91 (d, 1 H, J 1,2 = 1.2, H-1), 4.19-3.99 (m, 6 H, H-3, H-4, 2 CH2CHCH2), 3.83 (m, 2 H, H-2, H-5 a ), 3.73 (dd, 1 H, J 4,5b = 4.2 Hz, J gem = 11.2 Hz, H-5 b ), 3.33 (s, 3 H, OCH3), 1.06 (s, 9 H, 3 CH3). 13 C NMR (125 MHz, CDCl3) δ 135.6, 134.6, 134.5, 133.5, 129.7, 129.7, 127.8, 127.7, 127.7, 117.6, 117.4, 106.4, 81.9, 80.1, 77.6, 71.5, 71.5, 64.2, 55.3, 26.9, 19.3. HRMS (ESI) m / z: found [M+Na] + 505.2382, C 28 H 38 O5Si calcd. for [M+Na] + 505.2381.

[0105] [ka]

[0106] To obtain the ribofuranosyl donor, the methyl glycoside was hydrolyzed with aqueous TFA (Scheme 6). Contrary to expectations, the TBDPS group was removed, rather than the methyl glycoside, to give the TBDPS-free derivative 14 in 88% yield. This result demonstrated that the presence of the TBDPS group makes acid hydrolysis of the methyl glycoside difficult.

[0107] [ka]

[0108] To ensure acid hydrolysis of the methyl glycoside, the protecting group at the 5-hydroxyl group was changed to a Bn group, which is stable under acidic conditions. Therefore, to derive the 5-Bn protected form, the TBDPS group of 12 was first removed (Scheme 7). 12 was reacted with TBAF in THF solvent to give the 5-hydroxyl free form 14 in 95% yield.

[0109] Specifically, it is as follows:

[0110] Compound 12 (1.87 g, 3.88 mmol) was dissolved in THF (77.7 mL) under an Ar atmosphere, and TBAF (3.31 mL, 11.7 mmol) was added at 4 °C. The mixture was stirred at room temperature for 2 h. After confirming the completion of the reaction by TLC (n-hexane / EtOAc = 1 / 1), the reaction mixture was extracted three times with CHCl3, washed with water and saturated brine, dried over anhydrous sodium sulfate, and evaporated. The resulting residue was purified by flash silica gel chromatography (CHCl3 / MeOH = 87 / 13) to give compound 14 (900 mg, 3.68 mmol, 95%) as a colorless, clear, viscous liquid. [α] D +1.7° (c 1.0, CHCl3). 1 H NMR (500 MHz, CDCl3) δ 5.98-5.88 (m, 2 H, 2 OCH2CHCH2), 5.34-5.20 (m, 4 H, 2 OCH2CHCH2), 4.89 (s, 1 H, H-1), 4.22-4.01 (m, 6 H, H-3, H-4, 2 OCH2CHCH2), 3.83 (m, 2 H, H-2, H-5 a ), 3.61 (dd, 1 H, J 4,5b = 3.1 Hz, J gem = 11.9 Hz, H-5 b ), 3.41 (s, 3 H, OCH3). 13 C NMR (125 MHz, CDCl3) δ 135.6, 134.6, 134.5, 133.5, 129.7, 129.7, 127.8, 127.7, 127.7, 117.6, 117.4, 106.4, 81.9, 80.1, 77.1, 71.5, 71.5, 64.2, 55.3, 26.9, 19.3. HRMS (ESI) m / z: found [M+Na] + 267.1205, C 12 H 20 O5calcd. for [M+Na] + 267.1203.

[0111] [ka]

[0112] Next, we attempted to convert the 5-hydroxyl group to a hemiacetal by introducing a Bn group and acid hydrolysis of the methyl glycoside (Scheme 8). The Williamson ether synthesis method was used to introduce a Bn group to the 5-hydroxyl group of 14, yielding the Bn derivative 15 in 68% yield. Subsequently, acid hydrolysis of the methyl glycoside with aqueous TFA successfully yielded the desired hemiacetal derivative 16 in 83% yield.

[0113] Specifically, it is as follows:

[0114] Compound 14 (868 mg, 3.55 mmol) was dissolved in DMF (14.2 mL) at 4 °C under an Ar atmosphere. NaH (213 mg, 5.33 mmol) was added at 4 °C and the mixture was stirred for 30 min. Benzyl bromide (0.63 mL, 5.33 mmol) was then added at 4 °C and the mixture was stirred for 3 h. After confirming the completion of the reaction by TLC (n-hexane / EtOAc = 2 / 1), MeOH was added at 4 °C to quench the reaction. The reaction mixture was then extracted three times with CHCl3, washed with water and saturated brine, dried over anhydrous sodium sulfate, and evaporated. The resulting residue was purified by flash silica gel chromatography (n-hexane / EtOAc = 13 / 7) to give compound 15 (807 mg, 2.41 mmol, 68%) as a pale yellow viscous liquid. [α] D +9.6° (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) δ 7.35-7.23 (m, 5 H, Ar), 5.96-5.85 (m, 2 H, 2 OCH2CHCH2), 5.32-5.15 (m, 4 H, 2 OCH2CHCH2), 4.89 (s, 1 H, H-1), 4.59 (m, 2 H, CH2Ar), 4.26 (m, 1 H, H-4), 4.17-3.97 (m, 5 H, H-3, 2 OCH2CHCH2), 3.82 (d, 1 H, J 2,3 = 4.7 Hz, H-2), 3.64 (dd, 1 H, J 4,5a = 3.8 Hz,J gem = 10.6 Hz, H-5 a ), 3.55 (dd, 1 H, J 4,5b = 5.8 Hz, H-5 b ), 3.33 (s, 3 H, OCH3). 13 C NMR (125 MHz, CDCl3) δ 138.4, 134.5, 134.5, 128.3, 127.6, 127.5, 117.5, 117.4, 106.4, 80.4, 79.8, 78.4, 73.2, 71.5, 71.4, 71.4, 55.0. HRMS (ESI) m / z: found [M+Na] + 357.1673, C 19 H 26 O5calcd. for [M+Na] + 357.1672.。

[0115] Compound 15 (807 mg, 2.41 mmol) was dissolved in 90% TFA (24.1 mL) in HCl (aq) at 0 °C under an Ar atmosphere and stirred at 0 °C for 17 h. After confirming the completion of the reaction by TLC (n-hexane / EtOAc = 1 / 1), MeOH was added at 4 °C to quench the reaction. The reaction mixture was then extracted three times with CHCl3, washed (saturated aqueous sodium hydroxide, saturated brine), dried (anhydrous sodium sulfate), and evaporated. The resulting residue was purified by flash silica gel chromatography (n-hexane / EtOAc = 21 / 29) to give compound 16 (647 mg, 2.02 mmol, 83%) as a pale yellow viscous liquid. 1 H NMR (500 MHz, CDCl3) δ 7.33-7.24 (m, 10 H, Ar), 5.96-5.81 (m, 4 H, 2 OCH2CHCH2), 5.32-5.14 (m, 10 H, H-1, 2 OCH2CHCH2), 4.60-4.46 (m, 4 H, CH2Ar), 4.30 (m, 2 H, H-4), 4.23-4.03 (m, 10 H, H-3, 2 OCH2CHCH2), 3.95 (m, 2 H, H-2, 1-OH), 3.90 (t, 1 H, J 1,2 = 4.3 Hz, J 2,3 = 4.9 Hz, H-2), 3.81 (d, 1 H, J 1, 1-OH = 4.7 Hz, 1-OH), 3.66 (dd, 1 H, J 4,5a = 3.2 Hz, J gem = 10.5 Hz, H-5 a ), 3.57 (dd, 1 H, J 4,5b = 4.4 Hz, H-5 b ), 3.53-3.46 (m, 2 H, H-5 a , H-5 b ). 13C NMR (125 MHz, CDCl3) δ 138.0, 137.8, 134.6, 134.5, 134.3, 134.2, 128.4, 128.4, 127.8, 127.7, 127.6, 117.6, 117.5, 117.4, 100.3, 96.1, 80.9, 80.9, 80.4, 77.8, 77.8, 77.7, 73.5, 73.3, 71.8, 71.5, 71.5, 71.4, 70.6, 70.1. HRMS (ESI) m / z: found [M+Na] + 343.1512, C 18 H 24 O5calcd. for [M+Na] + 343.1516.

[0116] [ka]

[0117] Example 1-1-2. α-Selective Ribosylation Reaction with Solid-State Luminescent Dyes After completing the synthesis of hemiacetal 16, the precursor of the donor, we decided to convert it into a fluorinated sugar donor and an imidate donor, and then to carry out a ribofuranosylation reaction with a solid-state luminescent dye to examine the effect of the leaving group on the stereoselectivity.

[0118] First, we synthesized a fluoroglycosyl donor (Scheme 9). The reaction of 16 with DAST in THF solvent afforded fluoroglycosyl donor 17 in 73% yield.

[0119] Specifically, it is as follows:

[0120] Under an Ar atmosphere, compound 16 (521 mg, 1.63 mmol) was dissolved in THF (8.15 mL) at -50 °C. Diethylaminosulfurtrifluoride (0.260 mL, 1.96 mmol) was added and the mixture was stirred at -50 °C for 1 h. After completion of the reaction was confirmed by TLC (n-hexane / EtOAc = 4 / 1), the reaction was quenched by adding saturated aqueous sodium hydroxide at -50 °C. The reaction mixture was then extracted three times with CHCl3, washed (saturated aqueous sodium hydroxide, saturated brine), dried (anhydrous sodium sulfate), and evaporated. The resulting residue was purified by flash silica gel chromatography (n-hexane / EtOAc = 79 / 21) to give compound 17 (460 mg, 1.43 mmol, 88%) as a pale yellow viscous liquid. [α] D +58.0° (c 1.0, CHCl3). 1 H NMR (500 MHz, CDCl3) δ 7.36-7.26 (m, 5 H, Ar), 5.96-5.85 (m, 2 H, 2 OCH2CHCH2), 5.76-5.64 (d, 1 H, J 1,2 = 63.5 Hz, H-1), 5.35-5.19 (m, 4 H, 2 OCH2CHCH2), 4.65 (d, 1 H, J gem = 12.1 Hz, CH2Ar), 4.59 (d, 1 H, CH2Ar), 4.35 (m, 1 H, H-4), 4.18-3.99 (m, 6 H, H-2, H-3, 2 OCH2CHCH2), 3.73 (dd, 1 H, J 4,5a = 3.4 Hz, J gem = 11.1 Hz, H-5 a ), 3.63 (dd, 1 H, J 4,5b = 5.4 Hz, H-5 b ). 13C NMR (125 MHz, CDCl3) δ 138.2, 134.2, 134.0, 128.4, 127.6, 127.6, 118.1, 117.8, 113.6, 111.8, 82.4, 82.3, 79.2, 78.9, 73.4, 71.9, 71.8, 70.3. HRMS (ESI) m / z: found [M+Na] + 345.1475, C 18 H 23 FO4calcd. for [M+Na] + 345.1473.

[0121] [ka]

[0122] The ribofuranosylation of the BTP solid-state luminescent dye 18 was carried out using the glycofluoride 17 (Scheme 10). When a dichloromethane solution of 17 and 18 was reacted with boron trifluoride diethyl etherate in the presence of molecular sieves, the desired glycoside product 19 was successfully obtained in moderate yield but with high α-stereoselectivity (α:β = 11.5:1). The high α-stereoselectivity can be explained by the reaction mechanism of 1,2-cis ribofuranosylation via a 1,3-cis envelope conformation reported by Woerpel et al. Specifically, the presence of ether protecting groups at the 2- and 3-positions stabilizes the 1,3-cis envelope conformational oxocarbenium ion intermediate, allowing subsequent nucleophilic attack from the inside of the glycosyl acceptor to selectively afford the α-glycoside.

[0123] Specifically, it is as follows:

[0124] Compound 17 (436 mg, 1.35 mmol) and 2-(benzothiazole-2-yl)-phenol 18 (307 mg, 1.35 mmol) were dissolved in CHCl (27.0 mL) under an Ar atmosphere. The solution was cannulated into a recovery flask containing MS4Å (2.70 g) and stirred at -80 °C for 1 h. Boron trifluoride (0.170 mL, 1.35 mmol) was then added and stirred at -80 °C for 6 h. After completion of the reaction was confirmed by TLC (n-hexane / EtOAc = 3 / 1), the reaction was quenched with saturated aqueous sodium hydroxide at 4 °C. The reaction mixture was then filtered through Celite. The filtrate was extracted three times with CHCl, washed with saturated aqueous sodium hydroxide and saturated brine, dried over anhydrous sodium sulfate, and evaporated. The resulting residue was purified by flash silica gel chromatography (n-hexane / EtOAc = 61 / 39) to give compound 19 (422 mg, 0.798 mmol, 59%) as a pale yellow viscous liquid. [α] D +64.5° (c 1.0, CHCl3). 1 H NMR (500 MHz, CDCl3) δ 8.57-7.15 (m, 13 H, Ar), 5.99-5.85 (m, 3 H, H-1, 2 OCH2CHCH2), 5.33-5.11 (m, 4 H, 4 OCH2CHCH2), 4.64 (d, 1 H, J gem = 12.1 Hz, CH2Ar), 4.55 (d, 1 H, CH2Ar), 4.45 (m, 1 H, H-4), 4.19-4.09 (m, 6 H, H-2, H-3, 2 OCH2CHCH2), 3.65 (near d, 2 H, H-5 a , H-5 b ). 13C NMR (125 MHz, CDCl3) δ 164.0, 155.3, 152.3, 138.0, 137.4, 135.1, 134.4, 131.4, 129.7, 128.5, 127.8, 127.6, 125.5, 124.2, 123.4, 122.7, 122.1, 121.3, 117.2, 116.7, 115.6, 100.4, 83.7, 79.4, 76.6, 73.6, 72.1, 71.7, 70.1. HRMS (ESI) m / z: found [M+Na] + 552.1817, C 31 H 31 NO5S calculation for [M+Na] + 552.1815.

[0125] [ka]

[0126] To investigate the effect of leaving groups on the ribofuranosylation reaction, an imidate donor was prepared from the hemiacetal 16 (Scheme 11). The reaction of 16 with N-phenylacetimidoyl chloride in the presence of cesium carbonate afforded the imidate 20 in 71% yield.

[0127] [ka]

[0128] The imidate 20 was used to carry out the ribofuranosylation of the BTP solid-state luminescent dye 18 (Scheme 12). When the condensation reaction was carried out in the presence of TBSOTf, the reaction proceeded with complete α-selectivity, but the desired glycoside product 19 was obtained in only 14% yield. The low yield may be due to the overreactivity of 20, which may have been hydrolyzed during the preparatory step of the condensation reaction.

[0129] [ka]

[0130] From these results, we concluded that fluorine is the most suitable leaving group for the donor in the ribofuranosylation reaction of BTP solid-state luminescent dye 18, taking into account the yield and reproducibility.

[0131] Example 1-1-3. Phosphate esterification of hydroxyl group at 5-position To convert the 5-hydroxyl group to a phosphate ester, we attempted to derive the free 5-hydroxyl group by protecting group conversion (Scheme 13). First, the All group was removed from BTP glycoside 18 by treating it with Pd(PPh3)4 and DMBA in methanol, and then the free hydroxyl group was converted to Ac by the standard method, affording the diacetylated derivative 21 in a two-step yield of 54%. The conversion of the All group to an Ac group was performed to prevent anomerization, which is a concern during phosphate conversion under acidic conditions.

[0132] Specifically, it is as follows:

[0133] Compound 19 (411 mg, 0.776 mmol) was dissolved in MeOH (15.5 mL) under an Ar atmosphere. DMBA (242 mg, 0.155 mmol) and Tetrakis(triphenylphosphine)palladium (8.97 mg, 7.76 μmol) were added at 4 °C, and the mixture was stirred at 80 °C for 6 h. After confirming the completion of the reaction by TLC (n-hexane / EtOAc = 1 / 1), SiliMets DMT (155 mg) was added at room temperature to adsorb Pd. The reaction mixture was then filtered through Celite. The filtrate was extracted three times with CHCl3, washed (saturated ammonium chloride solution, saturated sodium hydroxide solution, saturated brine), dried (anhydrous sodium sulfate), and evaporated. The resulting residue was dissolved in pyridine (7.76 mL) under an Ar atmosphere. Acetic anhydride (0.290 mL, 3.10 mmol) was added at 4 °C and the mixture was stirred at room temperature for 30 min. DMAP (19.0 mg, 0.155 mmol) was then added and the mixture was stirred at room temperature for 1.5 h. After confirming the completion of the reaction by TLC (n-hexane / EtOAc = 3 / 1), the reaction mixture was extracted three times with CHCl3, washed (1 M hydrochloric acid, saturated aqueous sodium hydroxide, saturated brine), dried (anhydrous sodium sulfate), and evaporated. The resulting residue was purified by flash silica gel chromatography (n-hexane / EtOAc = 4 / 1) to give compound 21 (226 mg, 0.432 mmol, 54%) as a white solid. [α] D +70.0° (c 1.0, CHCl3). 1 H NMR (500 MHz, CDCl3) δ 8.45-7.07 (m, 13 H, Ar), 6.08 (d, 1 H, J 1,2 = 4.6 Hz, H-1) , 5.46 (dd, 1 H, J 3,4 = 1.9 Hz, J 2,3= 6.6 Hz, H-3), 5.25 (dd, 1 H, H-2), 4.51 (m, 2 H, 2 CH2Ar), 4.36 (near d, 1 H, H-4), 3.66-3.58 (m, 2 H, H-5 a , H-5 b ), 1.98 (2 s, 6 H, 2 COCH3). 13 C NMR (125 MHz, CDCl3) δ 169.4, 168.8, 162.1, 153.6, 151.3, 136.6, 135.6, 130.8, 128.9, 128.7, 127.9, 127.5, 127.3, 127.0, 126.8, 126.6, 124.9, 123.7, 122.1, 121.8, 121.3, 119.5, 114.0, 98.2, 82.1, 72.7, 70.5, 69.8, 68.5, 20.0, 19.6. HRMS (ESI) m / z: found [M+Na] + 556.1397, C 29 H 27 NO7S calculation for [M+Na] + 556.1400.

[0134] Compound 21 (81.4 mg, 0.153 mmol) was dissolved in CHCl / H0 (20 / 1, 3.1 mL) under an Ar atmosphere. DDQ (724 mg, 3.06 mmol) was added at 4 °C and the mixture was stirred at room temperature for 24 h. DDQ (362 mg, 1.53 mmol) was added again 6 h after the start of the reaction. After monitoring the progress of the reaction by TLC (n-hexane / EtOAc = 1 / 1), the reaction mixture was extracted three times with CHCl, washed (saturated aqueous sodium hydroxide, saturated brine), dried (anhydrous sodium sulfate), and evaporated. The resulting residue was purified by flash silica gel chromatography (n-hexane / EtOAc = 9 / 16) to give compound 5 (28.2 mg, 0.0636 mmol, 41%) as a white solid. [α] D+107.3° (c 1.0, CHCl3). 1 1H NMR (500 MHz, CDCl3) δ 8.54 - 7.18 (m, 8 H, Ar), 6.18 (d, 1 H, J 1,2 = 4.5 Hz, H-1), 5.50 (dd, 1 H, J 3,4 = 2.6 Hz, J 2,3 = 7.0 Hz, H-3), 5.25 (dd, 1 H, H-2), 4.40 (dd, 1 H, J 4,5 = 5.5 Hz, H-4), 3.91 - 3.85 (m, 2 H, H-5 a , H-5 b ), 2.12 (s, 3 H, COCH3), 2.06 (s, 3 H, COCH3). 13 13C NMR (125 MHz, CDCl3) δ 170.6, 169.9, 163.1, 154.5, 152.3, 136.5, 131.8, 130.1, 126.0, 124.8, 123.1, 122.9, 122.5, 120.6, 115.0, 99.2, 84.3, 71.5, 70.4, 62.2, 21.0, 20.6. HRMS (ESI) m / z: found [M+Na] + 466.0930, C 22 H 21 NO7S calcd. for [M+Na] + 466.0931. <~

[0135]

Chemistry

[0136] Next, we attempted to remove the Bn group at the 5-position of 21 (Table 1). We investigated reaction conditions that would allow the removal of only the Bn group without damaging the BTP glycoside. In Entry 1, a large excess of DDQ was used, successfully obtaining the desired 5-hydroxyl group free product 5 in 35% yield. Furthermore, 50% of 21 was recovered. In Entry 2, the Lewis acid boron trichloride was applied to 21, but the reaction did not proceed at all. In Entry 3, catalytic hydrogenation was performed in the presence of a Pd / C catalyst, but TLC confirmed that only trace amounts of 5 were produced. In Entry 4, the catalyst for catalytic hydrogenation was changed to Pd(OH)2 / C, but TLC confirmed the production of numerous byproducts in addition to 5. Based on these results, we concluded that the DDQ oxidation in Entry 1 is optimal for the removal of the Bn group at the 5-position.

[0137] [Table 1]

[0138] Next, following the synthetic strategy for ribose 5-phosphate derivatives, we attempted to convert 5 to phosphoryl ester using an amidite reagent (Scheme 14). To suppress acidity, we reacted 5 with the amidite reagent 22 in the presence of imidazole and imidazole hydrochloride, but the conversion to phosphorite did not proceed at all. This is likely due to the sterically bulky and poorly reactive 22 used.

[0139] [ka]

[0140] Based on the above results, the amidite reagent was changed to the highly reactive Bn form 23, and 5 was phosphorylated (Scheme 15). When 5 was reacted with the Bn amidite reagent 23 in the presence of 1H-tetrazole, the desired phosphite formation proceeded smoothly. tAfter oxidation to phosphorus(V) with BuOOH, removal of the Bn group, and subsequent hydrolysis of the Ac group, we successfully obtained the ribose 5-phosphate derivative 3 in 26% yield. Finally, to increase the solubility in DMF used in the unprotected phosphate-phosphate coupling, we performed cation exchange by treatment with aqueous TEA to prepare the triethylammonium salt of the ribose 5-phosphate derivative 3.

[0141] Specifically, it is as follows:

[0142] Compound 5 (57.7 mg, 0.130 mmol) and dibenzyl N,N-diisopropylphosphoramidite (130 μL, 0.390 mmol) were dissolved in CHCl (5.20 mL) under an Ar atmosphere. 1H-Tetrazole (27.3 mg, 0.390 mmol) was added at -4 °C and the mixture was stirred at room temperature for 3 h. After confirming the reaction by TLC (n-hexane / EtOAc = 1 / 1), tert-butyl hydroperoxide (5.5 mg, 70.0 μL, 0.390 mmol) was added at -4 °C and the mixture was stirred at room temperature for 1 h. Completion of the reaction was confirmed by TLC (n-hexane / EtOAc = 1 / 1). The reaction was quenched by adding saturated aqueous sodium thiosulfate at -50 °C. The reaction mixture was then extracted three times with CHCl, washed with saturated aqueous sodium hydroxide and saturated brine, dried over anhydrous sodium sulfate, and evaporated. The resulting residue was dissolved in MeOH (1.13 mL) under a H atmosphere. Palladium-activated carbon (Pd 10%, 12.2 mg, 0.0113 mmol) was added at room temperature and stirred for 5 hours. Hydrogenation was then performed. Completion of the reaction was confirmed by TLC (n-hexane / EtOAc = 1 / 1), followed by filtration through Celite and evaporation of the solvent. The resulting residue was dissolved in TEA / MeOH / HO (1 / 3 / 1, 2.82 mL) and stirred at room temperature for 5 hours. Completion of the reaction was confirmed by TLC (n-hexane / EtOAc = 1 / 1), followed by evaporation of the solvent. The resulting residue was purified by size exclusion chromatography (MeOH / HO = 1 / 1) to give compound 3 (15.0 mg, 0.0343 mmol, 26%) as a pale yellow-white solid. [α] D +403.3° (c 1.0, H2O). 1 H NMR (500 MHz, D2O) δ 7.67-6.85 (m, 8 H, Ar), 5.64 (d, 1 H, J 1,2 = 4.1 Hz, H-1) , 4.30 (dd, 1 H, J 2,3 = 5.0 Hz, H-2), 4.15 (m, 2 H, H-3, H-4), 3.91-3.85 (m, 2 H, H-5 a , H-5 b ). 13 C NMR (125 MHz, D2O) δ 165.5, 153.8, 151.1, 134.1, 132.7, 130.0, 126.6, 125.4, 122.9, 121.6, 121.5, 116.3, 101.7, 85.1, 85.0, 71.6, 69.7, 64.7, 64.7. 31 P NMR (200 MHz, DO) δ 2.58 HRMS (ESI) m / z: found [M+H]- 438.0419, C 22 H 21 NO7S calcd. for [M+H]- 438.0418.

[0143] [ka]

[0144] Example 1-2. Chemical synthesis of ADP-ribose BTP glycoside by unprotected phosphate-phosphate coupling reaction Since the triethylammonium salt of ribose 5-phosphate derivative 3 was successfully prepared, we attempted to synthesize ADP-ribose BTP glycoside 1 by unprotected phosphate-phosphate coupling (Scheme 16). In DMF, triethylammonium AMP salt 25 was converted to activated AMP 27 by the in situ generated phosphate activator 2-MeImIm-Cl. Subsequently, 3 and MgCl2 were added to the reaction mixture, and pyrophosphate bond formation was carried out by phosphate-phosphate coupling. The reaction proceeded smoothly at room temperature, and purification by ion-exchange chromatography (ammonium acetate solution) successfully isolated the target ADP-ribose BTP glycoside 1 in 80% yield.

[0145] Specifically, it is as follows:

[0146] First, a TEA solution (TEA / HO = 1 / 5) was prepared at 4 °C. 150 μL of this solution was added to an Eppendorf tube containing compound 3 (3.60 mg, 0.00561 mmol). The solvent was removed by lyophilization, and MgCl2 (1.30 mg, 0.0140 mmol) was added. Next, 2-Chloro-1,3-dimethylimidazolinium chloride (3.80 mg, 0.0224 mmol) was added to a separate Eppendorf tube and spun down in a centrifuge. AMP (3.10 mg, 0.00561 mmol) and 2-Methylimidazole (3.70 mg, 0.0449 mmol) were added in the same order. The mixture was then dissolved in DMF (0.0560 mL) and incubated at 60 °C for 1 hour. The reaction mixture was then added to the Eppendorf tube containing the compound 3 and MgCl2 mixture and stirred at 25 °C for 5.5 hours. After monitoring the progress of the reaction by ESI, the solvent was removed by lyophilization, and the resulting residue was purified by ion exchange chromatography (MeOH / 500 mM CHCOONH aq. = 3 / 1) to give compound 1 (3.6 mg, 80%) as a white solid. [α] D -30.8° (c 1.0, H2O). 1 H NMR (500 MHz, D2O) δ 8.21-7.11 (m, 10 H, Ar, H-2, H-8), 5.76 (d, 1 H, J 1,2 = 4.4 Hz, H-1′) , 5.55 (d, 1 H, J 1’,2’ = 3.9 Hz, H-1′′) , 4.41 (m, 2 H, H-2′, H-3′′), 4.27 (m, 2 H, H-2′′, H-4′), 4.22 (dd, 1 H, J 2,3 = 3.0 Hz, J 3,4 = 3.3 Hz, H-3′), 4.13 (m, 4 H, H-3′′, H-5′ a , H-5′ b , H-5′′ a ), 4.02 (m, 1 H, H-5′′ b ). 13 C NMR (150 MHz, D2O) δ 166.8, 156.5, 156.0, 153.8, 153.0, 149.6, 136.5, 134.3, 131.25, 128.4, 124.7, 123.6, 123.5, 123.4, 119.9, 104.1, 89.3, 86.4, 84.8, 76.7, 73.4, 71.7., 71.4, 67.7, 66.7. 31 P NMR (200 MHz, D2O) δ -8.88, -8.98, -9.25, -9.36. HRMS (ESI) m / z: found [M+Na]- 789.761, C 28 H 28 NO 14 P2S 2- Calculate for [M+Na]- 789.0763.

[0147] [ka]

[0148] Example 2. Detection of ADP-ribosylase activity using Compound 1 (ADP-ribose BTP glycoside) The reaction was carried out under the following reaction conditions 1 and 2.

[0149] (Reaction Condition 1) A 20 μL reaction solution (50 mM NaHPO4, 10 mM MgCl2, 5 mM DTT, pH 7.4) containing 0.50 μg of human ARH3 (hARH3) and 3.8 nmol of compound 1 was prepared and reacted at 37°C for 12 hours.

[0150] (Reaction condition 2) A 20 μL reaction solution (50 mM NaHPO4, 10 mM MgCl2, 5 mM DTT, pH 7.4) containing 0.050 μg of Macrodomain 1 (Mac1) derived from coronavirus SARS-CoV-2 and 3.8 nmol of compound 1 was prepared and reacted at 25°C for 720 hours.

[0151] After the reaction, the reaction solution was irradiated with a 365 nm UV lamp and observed visually. As shown in Figure 1, fluorescence was specifically confirmed visually when the enzyme was added under both reaction conditions 1 and 2.

Claims

1. General formula (1): 【Chemical 1】 [In the formula: R 1 and R 2 is R 1 Ga-NH 2 and R 2 indicates =N- or R 1 indicates =O and R 2 represents -NH-. R 3 and R 4 are the same or different and represent a hydroxy group or a hydrogen atom. 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 are the same or different and represent a hydrogen atom, a halogen atom, or an alkoxy group. A double line consisting of a solid line and a dotted line represents a single bond or a double bond.] A compound represented by the formula (I), a salt thereof, or a solvate thereof.

2. R 1 Ga-NH 2 and R 2 The compound according to claim 1, a salt thereof, or a solvate thereof, wherein is =N-.

3. R 3 and R 4 The compound according to claim 1 , a salt thereof, or a solvate thereof, wherein

4. R 6 , R 7 , and R 11 are the same or different and are a hydrogen atom, a halogen atom, or an alkoxy group, and R 5 , R 8 , R 9 , R 10 , and R 12 The compound according to claim 1, a salt thereof, or a solvate thereof, wherein is a hydrogen atom.

5. R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , and R 12 The compound according to claim 1, a salt thereof, or a solvate thereof, wherein is a hydrogen atom.

6. The compound has the formula (1A): 【Chemistry 2】 The compound according to claim 1, a salt thereof, or a solvate thereof, which is a compound represented by the formula:

7. A fluorescent probe comprising the compound according to any one of claims 1 to 6, a salt thereof, or a solvate thereof.

8. The fluorescent probe according to claim 7, which is an agent for detecting ADP-ribosylase deactivation activity.

9. The fluorescent probe according to claim 8, wherein the ADP-ribosylase is derived from a coronavirus.

10. A reagent comprising the compound according to any one of claims 1 to 6, a salt thereof, or a solvate thereof.

11. A pharmaceutical comprising the compound according to any one of claims 1 to 6, a salt thereof, or a solvate thereof.

12. A method for detecting ADP-ribosylase activity, comprising contacting the compound according to any one of claims 1 to 6, a salt thereof, or a solvate thereof with an ADP-ribosylase and measuring fluorescence intensity.

13. A method for screening for a deADP-ribosylation activity regulator, comprising contacting the compound according to any one of claims 1 to 6, a salt thereof, or a solvate thereof with a deADP-ribosylation enzyme and a test substance, and using as an indicator the fluorescence intensity.

14. (a) contacting the compound according to any one of claims 1 to 6, a salt thereof, or a solvate thereof with an ADP-ribosylase and a test substance; (b) measuring the fluorescence intensity (test fluorescence intensity) in the step (a) and comparing the test fluorescence intensity with the fluorescence intensity in the absence of contact with the test substance (control fluorescence intensity); and (c) selecting the test substance as an ADP-ribosylation deactivation activity regulator when the test fluorescence intensity is different from the control fluorescence intensity; The screening method according to claim 13, comprising:

Images