Guanosine derivatives and their production method

Abstract

To develop a purine base derivative.SOLUTION: A guanosine derivative compound is represented by the following formula, where one of R1 and R2 represents H and the other represents any of H, OH, OCH3, and F; R3 represents a compound having a double bond; and R4 represents any of H, monophosphoric acid, diphosphoric acid, and triphosphoric acid. Thus, a guanosine derivative having a substituent at the 8-position is provided. Use of the guanosine derivative renders nucleic-acid labeling possible.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a guanosine derivative and a method for producing the same. More specifically, the present invention relates to a guanosine derivative having a substituent such as an ethynyl group or a vinyl group at the 8-position. [Background technology]

[0002] Research is being conducted into the technology of fluorescently labeling nucleic acids such as RNA and DNA by binding derivatives to the nucleic acids within cells. This technology is expected to be useful for nucleic acid analysis and medical diagnosis, as it can visualize the behavior and localization of nucleic acids and is thought to reflect cell proliferation and protein synthesis.

[0003] For such nucleic acid labeling techniques, derivatives corresponding to the nucleic acid bases that make up RNA and DNA are required. That is, RNA requires adenine, guanosine, cytosine, and uridine, whereas DNA requires adenine, guanosine, cytosine, thymine, and their derivatives.

[0004] Among these, several derivatives related to pyrimidine bases have been developed. For example, 5-EdU (5-deoxy-ethynyl-uridine, Non-Patent Documents 1, 2, 4, 5) has been developed as a DNA-type uridine derivative, and 5-EU (5-ethynyl-uridine, Non-Patent Document 3) has been developed as an RNA-type uridine derivative. It has been disclosed that these pyrimidine base derivatives are incorporated into intracellular nucleic acids and can label the nucleic acids by fluorescence. Thus, although many derivatives of pyrimidine bases have been developed, no derivatives of purine bases have yet been developed. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] T. Ishizuka, HS Liu, K. Ito, Y. Xu, Fluorescence Imaging of Chromosomal DNA using Click Chemistry. Sci. Rep. 6, 33217 (2016) [Non-Patent Document 2] Salic, A. & Mitchison, TJ A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc. Natl. Acad. Sci. USA, 105, 2415-2420 (2008). [Non-Patent Document 3] Jao, CY & Salic, A. Exploring RNA transcription and turnover in vivo by using click chemistry. Proc. Natl. Acad. Sci. USA 105, 15779-15784 (2008). [Non-Patent Document 4] Neef, AB & Luedtke, NW Dynamic metabolic labeling of DNA in vivo with arabinosyl nucleosides. Proc. Natl. Acad. Sci. USA, 108, 20404-20409 (2011). [Non-Patent Document 5] Yamakoshi, H. et al. Imaging of EdU, an alkyne-tagged cell proliferation probe, by Raman microscopy. J. Am. Chem. Soc. 133, 6102-6105 (2011). DISCLOSURE OF THEINVENTION [Problem to be solved by the invention]

[0006] One of the reasons why purine base derivatives have not yet been developed is thought to be the difficulty of synthesis. In other words, purine bases are difficult to handle as compounds, and tend to aggregate and become viscous. For these reasons, it is believed that purine base derivatives have not yet been developed. In light of the above circumstances, an object of the present invention is to develop a purine base derivative. [Means for solving the problem]

[0007] As a result of extensive research, the inventors have succeeded in synthesizing a guanosine derivative in which an ethynyl group has been introduced at the 8th position of guanosine through a series of steps using guanosine as the starting material, including bromination, protection of the hydroxyl group at the sugar moiety and protection of the nucleic acid base portion, and a coupling reaction using a Pd catalyst (introduction of an ethynyl group), thereby completing the invention. The inventors also confirmed that the synthesized guanosine derivative can be incorporated into RNA and that cell labeling is possible through a click reaction, thereby completing the invention. Similarly, the inventors used deoxyguanosine as a starting material, brominated it, and through a Pd-catalyzed coupling reaction (introduction of an ethynyl group) successfully synthesized a deoxyguanosine derivative in which an ethynyl group was introduced at the 8th position of deoxyguanosine, thereby completing the invention. Furthermore, the inventors have completed vinyl-group-introduced guanosine derivatives by introducing a vinyl group and demonstrating their usefulness, thereby conceiving of compounds developed based on ethynyl-group-introduced guanosine derivatives or vinyl-group-introduced guanosine derivatives, and have completed the invention.

[0008] The present invention comprises the following configuration. [1] A guanosine derivative compound represented by the following formula: Either R1 or R2 is H, and the other is H, OH, OCH3, or F. R 3is represented by a compound having a double bond, R4 is represented by H, monophosphate, diphosphate, or triphosphate. A guanosine derivative compound characterized in that: [ka] [2] In the above guanosine derivative, R1 and R2 are One side 1 H, 2 H, 3 H, The other one, 1 H, 2 H, 3 Either H or 18 F or 19 F, The guanosine derivative according to [1], [3] In the R3 of the guanosine derivative, The double bond C is 11 C, 12 C, 13 C, 14 Either C The guanosine derivative according to [1] or [2], [4] A guanosine derivative compound according to any one of [1] to [3], wherein one of R1 and R2 is H and the other is either H or OH. [5] The guanosine derivative compound according to any one of [1] to [4], wherein R4 is represented by H.

[0009] [6] Furthermore, the guanosine derivative according to any one of [1] to [5], wherein R3 is represented by any one of the following substituents: [ka] [7] The guanosine derivative according to [6], wherein R3 is a substituent represented by a vinyl group (B5).

[0010] [8] A click reaction method in which a click reaction is performed using R3 of the guanosine derivative [1] to [7] and a reporter compound that reacts therewith. [9] The click reaction method according to [8], wherein the reporter compound is a compound having an azide group.

[10] The click reaction method according to [9], wherein the compound having an azide group is selected from the group consisting of azidebenzene, coumarin azide, tetramethylrhodamine (TAMRA) azide, and biotin azide.

[11] [1] to [7] are used as active ingredients of the guanosine derivatives, The click reaction method according to any one of [8] to

[10] is carried out in cells or biological tissue to label cells or biological tissue in animals. Imaging agents.

[12] The compound according to

[11] , wherein the label is selected from any one or more of a fluorescent label, a label with a luminescent substance, a radioactive label, and a nuclear magnetic resonance active label. Imaging agents.

[0011]

[13] A cancer imaging agent containing the guanosine derivatives [1] to [7] as active ingredients.

[14] The cancer imaging agent according to

[13] , wherein the cancer is a solid cancer such as brain, colon or stomach cancer.

[15] A method for producing a guanosine derivative compound represented by the following formula: a bromination step in which guanosine is used as a starting material to brominate the 8-position; a hydroxyl group protection step for protecting the hydroxyl groups in the sugar skeleton; a carbonyl group protection step for protecting the carbonyl group of the nucleic acid base portion; an R3 introduction step in which the substituent R3 is introduced at the 8-position by a coupling reaction; and a removal step of removing all of the protecting groups. [ka] (Wherein, R3 is Represented by compounds with double bonds (represented by the substituent) Effect of the Invention

[0012] The present invention makes it possible to provide purine base derivatives. In other words, the 8-ethynylguanosine derivatives and 8-vinylguanosine derivatives completed by the present invention provide a method for synthesizing guanosine derivatives having a substituent at the 8th position, which can be used to label nucleic acids. [Brief description of the drawings]

[0013] [Figure 1] A diagram showing the synthetic route for 8-vinylguanosine (8-VG) and the NMR chart of 8-VG. [Diagram 2] A diagram showing the reaction of 8-vinylguanosine and FAM tetraazine in cells. [Diagram 3] FIG. 1 shows the in vivo experimental method. [Figure 4] FIG. 1 shows the results of an in vivo experiment. [Diagram 5] FIG. 1 shows the results of investigating the stability of 8-EG or 8-VG. [Figure 6] Diagram showing the synthetic pathway for 8-ethynylguanosine. [Figure 7] NMR chart of 8-ethynylguanosine. [Figure 8] Diagram showing the synthetic pathway for 8-ethynyl-2'-guanosine. [Figure 9] NMR chart of 8-ethynyl-2'-guanosine. [Figure 10] FIG. 1 shows the results of LC-MS analysis of the reaction products with 8-ethynylguanosine and azidobenzene. [Figure 11] NMR charts of 8-ethynylguanosine and its reaction products with azidobenzene. [Figure 12] A graph showing the fluorescence of the reaction products with 8-ethynylguanosine and azidobenzene. [Figure 13] A diagram showing the reaction of 8-ethynylguanosine and azidobenzene in cells. [Figure 14] A diagram showing the distribution in the brain and the click reaction results when 8-ethynylguanosine was administered in vivo. [Figure 15] A diagram showing the distribution of 8-ethynylguanosine in the intestine and the results of the click reaction when administered in vivo. [Figure 16] A diagram showing the distribution of 8-ethynylguanosine in the kidney after in vivo administration and the results of the click reaction. [Figure 17] A diagram showing the results of cytotoxicity evaluation of 8-ethynylguanosine (8EG) and ethynyluridine (EU) by Alamar Blue assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The guanosine derivative compound of the present invention will be explained below.

[0015] The guanosine derivative compound of the present invention is defined as a compound represented by the following formula 6 or a salt thereof.

[0016] [ka]

[0017] In the above formula 6, one of R1 and R2 is H, and the other is H, OH, OCH3, or F. An example of such a compound is the compound shown in the following formula 7. [ka] That is, when both R1 and R2 are H (D1), it is expected to be incorporated into DNA as a deoxyguanosine derivative. When either R1 or R2 is OH (D2, D3), it is expected to be incorporated into RNA as a guanosine derivative (non-deoxyguanosine derivative). In addition, when either R1 or R2 is OCH3 (D6), it is expected to be possible to detect the behavior of a methylguanosine derivative with 2' methylation. Furthermore, when either R1 or R2 is F (D4, D5), it is expected that F can be detected. 18 By using F, it is expected that the behavior of the guanosine derivative as a label can be detected, as described below.

[0018] In the above formula 6, R4 represents either H, monophosphate, diphosphate, or triphosphate. That is, when R4 is H, it is expected to provide a nucleic acid derivative, and when R4 is monophosphate, diphosphate, or triphosphate, it is expected to provide a phosphorylated nucleic acid derivative, in which case it can be incorporated into nucleic acids and the behavior of each derivative can be detected.

[0019] In the above formula 6, R3 is a substituent represented by any of a compound having a double bond or a triple bond, a compound having an azide group, and a cyclic compound. In other words, R3 undergoes a click reaction with a reporter compound described later, thereby enabling labeling such as fluorescent labeling. In order to perform such function, R3 is a substituent represented by any of a compound having a double bond or a triple bond, a compound having an azide group, or a cyclic compound, and its structure can be appropriately changed depending on the structure of the reporter compound for the click reaction.

[0020] For R3, for example, the substituent shown in Chemical Formula 8 below can be used. [ka] That is, the substituents shown in A1 to A8 are a group of substituents that react with a reporter compound having an azide group. R3 is preferably a substituent represented by A1 (ethynyl group), which allows the structure of the guanosine derivative to be compact, and has the effect of improving the stability of the compound or its uptake into nucleic acids.

[0021] As another embodiment of R3, the substituent shown in Chemical formula 9 below can be used. [ka] That is, the substituents shown in B1 to B8 are a group of substituents that react with a reporter compound having a tetrazine group. R3 is preferably a substituent represented by B5 (vinyl group), which allows the structure of the guanosine derivative to be compact, and has the effect of improving the stability of the compound or its uptake into nucleic acids.

[0022] As still another embodiment of R3, the substituent shown in Chemical formula 10 below can be used. [ka] That is, such substituents are a group of substituents that react with a reporter compound having a triple bond, such as an ethynyl group.

[0023] A so-called click reaction can be carried out using R3 of the guanosine derivative of the present invention and a reporter compound that reacts with R3. In other words, the click reaction causes the guanosine derivative R3 and the reporter compound to bind to each other to form a single compound (e.g., Figure 12a ) In addition, by using the click reaction, Figure 12 As shown in the example, the guanosine derivative itself and the reporter compound (azidobenzene) itself do not emit fluorescence, but when these are combined into a single compound through a click reaction, they become fluorescent and enable labeling of nucleic acids.

[0024] The structure of the reporter compound can be appropriately changed in consideration of the reactivity with R3 in the guanosine derivative, the fluorescence when reacted, the labeling method, etc. For example, combinations of R3 and the reporter compound include an alkyne (linear and cyclic) having a triple bond and a compound having an azide group, an alkene (linear and cyclic) having a double bond and a compound having a tetrazine group, a carbonyl compound and a compound having a heteroatom, etc. As the reporter compound, preferably, a compound having an azide group can be used, and examples of such compounds that can be used include azidebenzene, coumarin azide, tetramethylrhodamine (TAMRA) azide, and biotin azide.

[0025] In the present invention, the click reaction method can be used in various environments without any particular limitation as long as the click reaction can occur, but it is most preferably used in cells or biological tissues. This makes it possible to visualize the behavior of nucleic acids in cells and biological tissues, and is expected to be applicable to nucleic acid analysis and medical diagnosis.

[0026] The labeling method of the present invention is not particularly limited as long as it is possible to label cells or biological tissues, and can be carried out using a variety of techniques. For example, this includes in vitro labeling using cells or pathological sections, and in vivo labeling of humans or animals.

[0027] The labeling method of the present invention does not need to be particularly limited as long as it is possible to label cells or biological tissues, and various techniques can be used, for example, fluorescent labeling, labeling with a luminescent substance, radiolabeling, and nuclear magnetic resonance active labeling, any of which can be used alone or in combination.

[0028] As the labeling method, various techniques can be used. For example, the H (hydrogen atom) used in guanosine derivatives is 1 Not only H, 2 H and3 For example, H is used. 2 When H is used, it can be used for diagnosis etc. by detecting it as deuterium using MRI etc. 3 When H is used, β - Since it emits radiation, it can be used as a radiolabeled compound, for example as a reagent. In such a case, either one of R1 and R2 of the guanosine derivative is 1 H, 2 H, 3 For example, one atom may be H and the other may be H, OH, OCH3, or F.

[0029] In addition, as another method for labeling, F (fluorine atom) used in guanosine derivatives, 18 F and 19 For example, use F. 19 F is almost nonexistent in the body. 19 The F-containing guanosine derivative compound can be used as a diagnostic compound for MRI with high spatial and temporal resolution and low background noise. 18 F is a positron isotope + Since they emit X-rays, they can be used as radiolabeled compounds in diagnostic agents for in vitro detection. That is, as shown in animal experiments such as Experiment 5 and Experiment 9, the metabolism of purine base derivatives is enhanced in cancer, so purine base derivatives can be used in PET cancer diagnosis. In particular, brain tumors consume large amounts of purine base derivatives, so information about the disease can be obtained with high resolution. In such a case, either one of R1 and R2 of the guanosine derivative is 18 F and 19 One can be called F and the other H, etc.

[0030] In addition, as another method for labeling, the C (carbon atom) used in guanosine derivatives is 11C, 12 C, 13 C, 14 For example, use C. 11 C is, 18 Like F, it is a positron isotope of β + Since it emits radiation, it can be used as a radiolabeled compound for in vitro detection in diagnostic agents. 13 C is, 12 Since its abundance is smaller than that of C, 13 By making it into a guanosine derivative compound containing C, it can be used as a reagent for NMR and the like. 14 C is, 3 Same as H, β - Since it emits radiation, it can be used as a radiolabeled compound, for example as a reagent. In such a case, the C of the double or triple bond in R3 of the guanosine derivative is 11 C, 12 C, 13 C, 14 C or the like. In particular, 8-vinylguanosine derivatives are relatively easy to synthesize, making them readily available. 11 C and 18 Positron-labeled substances such as F are considered to be highly desirable in the medical field. In addition, their usage is no different from conventional positron-labeled substances, so excellent diagnostic effects can be expected.

[0031] In addition, as another method for labeling, N (nitrogen atom) used in guanosine derivatives, 13 N, 14 N, 15 For example, use N. 13 N is, 18 Like F, it is a positron isotope of β + Since it emits radiation, it can be used as a radiolabeled compound for in vitro detection in diagnostic agents. 15 N is, 14Since its abundance is smaller than that of N, 13 By making it into a guanosine derivative compound containing C, it can be used as a reagent for NMR and the like. In such a case, the N of the compound having an azide group in R3 of the guanosine derivative is 13 N, 14 N, 15 N.

[0032] The guanosine derivative of the present invention can be used as a cancer imaging agent containing the same as an active ingredient. That is, the guanosine derivative of the present invention can be injected intravenously or locally, followed by administration of a reporter compound, to perform cancer imaging. In such cases, as in Experiment 9, imaging can be performed in vivo, such as in humans or animals (in vivo use). Also, as in Experiment 5, the guanosine derivative can be administered in vivo, and then tissue can be removed and reacted with a reporter compound (ex vivo use). In this way, when used in vivo, it serves as a diagnostic agent for evaluating the localization of cancer and protein synthesis ability, and when used ex vivo, it serves as a reagent for animal experiments, etc. The cancer to be imaged does not need to be particularly limited as long as imaging is possible, but it is preferable to use solid cancer such as brain, colon, and stomach cancer, which has the effect of improving the diagnostic effect of the guanosine derivative of the present invention.

[0033] Among the guanosine derivatives of the present invention, the guanosine derivative compound represented by the following formula 11 can be produced by the following series of steps. (1) A bromination step in which guanosine is used as the starting material to brominate the 8-position. (2) A hydroxyl group protection step for protecting the hydroxyl groups in the sugar skeleton (3) A carbonyl group protection step for protecting the carbonyl group of the nucleic acid base portion (4) R3 introduction step to introduce the substituent R3 at the 8-position by coupling reaction (5) A removal step for removing all protecting groups [ka] (In the formula, R3 is represented by a substituent that is a compound having a double bond or triple bond, a compound having an azide group, or a cyclic compound.)

[0034] The bromination step is a step in which the 8th position of guanosine is brominated ( Figure 6 a) The bromination step does not need to be particularly limited as long as the 8-position of guanosine can be brominated, and can be carried out under various conditions. An example of the bromination step is to react guanosine with NBS in acetonitrile / water.

[0035] The hydroxyl group protection step is a step of protecting the hydroxyl group in the sugar skeleton. The hydroxyl group protection step does not need to be particularly limited as long as the hydroxyl group in the sugar skeleton can be protected, and can be carried out under various conditions. For example, the hydroxyl group protection step is carried out using TBS in the presence of imidazole using DMF as a solvent. Figure 6 ,b).

[0036] The carbonyl group protection step is a step of protecting the carbonyl group of the nucleic acid base portion. The carbonyl group protection step does not need to be particularly limited as long as the carbonyl group of the nucleic acid base portion can be protected, and can be carried out under various conditions. For example, the carbonyl group protection step is carried out by reacting with trimethylsilylethanol in the presence of triphenylphosphine and diisopropyl azodicarboxylate using dioxane as a solvent. Figure 6 ,c).

[0037] The R3 introduction step is a step of introducing a substituent R3 at the 8-position by a coupling reaction. The R3 introduction step does not need to be particularly limited as long as the substituent R3 can be introduced at the 8-position by a coupling reaction, and can be carried out under various conditions. In addition, the substituent R3 may be introduced in the form of a protecting group attached to the substituent R3, taking into consideration the subsequent removal step, etc. For example, the R3 introduction step is carried out in toluene as a solvent in the presence of tetrakis(triphenylphosphine)palladium, by introducing a substituent R3 with a protecting group ( Figure 6 ,d).

[0038] The elimination step is a step in which all of the protecting groups are eliminated. The elimination step does not need to be particularly limited as long as the protecting groups can be eliminated, and can be carried out under various conditions. For example, the removal step involves removing all protecting groups in the presence of tetra-n-butylammonium fluoride using THF as a solvent ( Figure 6 ,e). EXAMPLES

[0039] Here, further details are provided using examples.

[0040] <<Experiment: Synthesis of 1,8-ethynylguanosine>> 1. About 8-ethynylguanosine, Figure 6 The synthesis was carried out according to the scheme. (1) Guanosine was used as the starting material and reacted with NBS in acetonitrile / water as a solvent to brominate the 8-position (a, bromination step). (2) The hydroxyl groups of the sugar skeleton were protected with TBS in the presence of imidazole using DMF as a solvent (b, hydroxyl group protection step). (3) The carbonyl group of the base moiety was protected with trimethylsilylethanol in the presence of triphenylphosphine and diisopropyl azodicarboxylate using dioxane as a solvent (c, carbonyl group protection step). (4) Using toluene as a solvent, the compound was reacted with trimethylsilylacetylene in the presence of tetrakis(triphenylphosphine)palladium to introduce an ethylene group at the 8-position (d, R3 introduction step). (5) All protecting groups were removed in the presence of tetra-n-butylammonium fluoride using THF as a solvent to obtain 8-ethynylguanosine (e, removal step). 2. NMR chart of the synthesized compound Figure 7 As shown in. 1 H-NMR (400 MHz, DMSO-d6) δ 10.85 (s, 1H), 6.56 (s, 2H), 5.78 (d, J = 6.5 Hz, 1H), 5.45 (d, J = 6.2 Hz, 1H), 5.10 (d, J = 5.0 Hz, 1H), 4.96-4.92 (m, 2H), 4.78 (s, 1H), 4.14-4.10 (m, 1H), 3.87-3.84 (m, 1H), 3.66-3.61 (m, 1H), 3.56-3.48 (m, 1H).

[0041] <<Experimental Synthesis of 2,8-ethynyl-2'-deoxyguanosine>> 1. About 8-ethynyl-2'-deoxyguanosine, Figure 8 The synthesis was carried out according to the scheme. (1) 2'-Deoxyguanosine was used as the starting material and reacted with NBS in acetonitrile / water to brominate the 8-position (a). (2) Using DMF as a solvent, the compound was reacted with trimethylsilylacetylene in the presence of tetrakis(triphenylphosphine)palladium, copper iodide, and triethylamine to introduce an ethylene group at the 8-position. The compound was then reacted further in the presence of tetra-n-butylammonium fluoride in THF as a solvent to obtain 8-ethynyl-2'-deoxyguanosine. 2. NMR chart of the synthesized compound Figure 9 As shown in. 1H-NMR (400 MHz, DMSO-d6) δ 10.84 (s, 1H), 6.56 (s, 2H), 6.25 (dd, J = 6.5, 8.2 Hz, 1H), 5.27 (d, J = 4.2 Hz, 1H), 4.88 (t, J = 6.0 Hz, 1H), 4.78 (s, 1H), 4.39-4.35 (m, 1H), 3.82-3.78 (m, 1H), 3.64-3.58 (m, 1H), 3.53-3.44 (m, 1H), 3.10-3.03 (m, 1H), 2.10 (ddd, J = 2.7, 6.5, 13.2 Hz, 1H).

[0042] Experiments 1 and 2 enabled the synthesis of 8-ethynylguanosine and 8-ethynyl-2'-deoxyguanosine. Based on these compounds, it is expected that phosphorylated derivatives can be synthesized. For example, 8-ethynyl-2'-deoxyguanosine or 8-ethynylguanosine (0.1 mmol) is added to 1,8-bis(dimethylamino)naphthalene (0.15 mmol) and dissolved in 500 μL of trimethyl phosphate. Phosphoryl chloride (0.13 mmol) is added to the solution on ice and stirred on ice for 2 hours. n-Butylamine (0.5 mmol) is added to the reaction solution, followed by a 0.5 M solution of tributylammonium pyrophosphate in DMF (0.5 mmol). After 5 minutes, 500 μL of 0.5 M triethylammonium bicarbonate (TEAB) solution is added to stop the reaction. The reaction product is purified by DEAE Sephadex A-25 column chromatography. The eluent is a linear gradient of 50 mM to 1 M TEAB. If necessary, the product is further purified by C18 reverse-phase HPLC to obtain the purity required for cell and animal experiments, using a linear gradient of 10% to 50% acetonitrile in water containing 100 mM triethylamine acetate as the eluent.

[0043] <<Experiment: Confirmation of the click reaction of 3,8-ethynylguanosine>> The click reaction between 1,8-ethynylguanosine and azidobenzene was confirmed.

[0044] 2.LC-MS analysis results Figure 10 In the figure, a shows the results of LC-MS analysis of 8-ethynylguanosine, b shows azidobenzene, and c shows the results of the reaction solution. (1) 8-Ethynylguanosine and azidobenzene were reacted in the presence of copper sulfate at 37°C overnight. (2) When the reaction solution was analyzed by LC-MS, the 8-ethynylguanosine peak almost disappeared, and the azidobenzene peak was low. In addition, the component eluted at 20.84 minutes (hereinafter referred to as the "click reaction compound") had a molecular weight that matched that of the predicted compound.

[0045] 3.NMR analysis results Figure 11 As shown in. Figure 10 The peak at 20.84 minutes (click reaction compound) obtained in was separated and purified, and NMR analysis was performed to identify each proton of the click reaction compound, confirming that it was the desired compound. 1 H-NMR (400 MHz, DMSO-d6) δ 10.81 (s, 1H), 9.38 (s, 1H), 8.03-8.00 (m, 2H), 7.66-7.62 (m, 2H), 7.57-7.52 (m, 1H), 7.02 (dd, J = 6.7, 7.9 Hz, 1H), 6.42 (s, 2H), 5.20 (s, 1H), 5.00 (s, 1H), 4.44-4.41 (m, 1H), 3.83-3.80 (m, 1H), 3.69-3.62 (m, 1H), 3.53-3.49 (m,1H), 3.23-3.14 (m, 1H), 2.16 (ddd, J = 2.6, 6.6, 13.0 Hz, 1H).

[0046] 4. Observe the fluorescence of the click reaction compound Figure 12 As shown in. (1) The click reaction compound was confirmed to emit fluorescence at 470 nm (λex = 342 nm) (c). (2) In addition, it was confirmed that fluorescence detection by UV irradiation was possible (b).

[0047] 5. These results demonstrated that 8-ethynylguanosine could undergo a click reaction with azidobenzene, that the reaction product emitted fluorescence, and that the fluorescence could be detected by UV irradiation.

[0048] <<Experiment: Confirmation of RNA incorporation and click reaction of 4,8-ethynylguanosine>> Studies were carried out to determine whether 1,8-ethynylguanosine could be incorporated into RNA and whether it could undergo a click reaction after incorporation.

[0049] 2. HeLa cells were stained as follows. (1) 5 × 10 HaLa cells were grown at 37°C in DMEM containing 10% FBS in the presence of 5% CO2. 5 Cultured. (2) 8-ethynylguanosine (stock solution: 10 mM DMSO solution) was added to the medium at a final concentration of 100 μM, and the medium was incubated for 24 hours. (3) If RNase A was used, it was added at this point to a final concentration of 200 μg / mL, and the mixture was incubated at room temperature for 45 minutes. (4) Wash three times with PBS. (5) The components of the cell staining solution are as follows (amount for one 3.5 cm dish). The figures in parentheses indicate the final concentrations. 100 μL (100 mM) of 1.0 M Tris (pH 8.5), 20 μL (1.0 mM) of 50 mM CuSO4, 2.5 μL (25 μM) of 10 mM Alexa Fluor 488 azide, 627.5 μL of Milli Q water, and 200 μL (100 mM) of 0.5 M Ascorbic acid. (6) After incubation at room temperature for 1 hour, the plate was washed three times with PBS. (7) Hoechst staining was performed. (8) Observations were made using a fluorescence microscope.

[0050] 3. Results Figure 13 As shown in. (1) Hoechst staining revealed that nuclei were stained in both the presence and absence of RNase A (a, d). (2) No staining was observed with Alexa Fluor 488 azide in the presence of RNaseA (e), but staining was observed in the absence of RNaseA (b). (3) In these merged images, the RNA staining and the nuclear staining were consistent (c). (4) These results confirmed that 8-ethynylguanosine was incorporated into RNA and that it stained the RNA through the click reaction.

[0051] <<Experiment: In vivo RNA labeling with 5,8-ethynylguanosine>> We investigated whether 1,8-ethynylguanosine can be incorporated into RNA in vivo and whether it undergoes a click reaction after incorporation.

[0052] 2.The experiment was conducted as follows. (1) 0.5 mL of 8-ethynylguanosine solution (4 mg / mL, 4% DMSO / PBS solution) was administered to each mouse via the tail vein (2 mg / body of 8-EG). (2) After anesthesia with isoflurane, the rats were subjected to cervical dislocation and tissue extraction. (3) The extracted tissue was fixed in 10% formalin for 24 hours, embedded in paraffin, and then sliced ​​into 1 μm-thick sections. (4) Thin sections were subjected to click reaction using TMR-N3, nuclei were stained using DAPI mounting medium, and observed under a fluorescent microscope.

[0053] 3. Results Figure 14 from Figure 16 As shown in the figure, Figure 14 The brain, Figure 15 Intestines, Figure 16 are the renal results. (1) In the brain, cell nuclei were stained, and TMR-azide was found to produce fluorescence at the same location as the cell nuclei. On the other hand, in the control, cell nuclei were stained, but no fluorescence was observed due to TMR-azide. This indicates that after administration, 8-EG passes through the BBB, migrates to brain tissue, and is incorporated into RNA. In addition, it was confirmed that the click reaction using TMR-azide is possible even in living tissue. (2) In the intestine and kidney, although the fluorescence of the cell nuclei was confirmed, the fluorescence of TMR-azide was not confirmed, which suggested that 8-EG was not taken up in the intestine or kidney.

[0054] <<Experimental evaluation of cytotoxicity of 6,8-ethynylguanosine>> A study was conducted to evaluate the cytotoxicity of 1,8-ethynylguanosine.

[0055] 2. Evaluation was performed using the Alamar Blue assay. (1) HeLa cells were used, and 1×10 3 The cells were seeded onto a 96-well plate at 100 cells / well. (2) 8-EG or ethynyluridine (EU) as a control was prepared at various concentrations and incubated at 37°C for 48 or 72 hours, after which the fluorescence at 580 / 610 nm was measured.

[0056] 3. Results Figure 17 In the graph, the vertical axis indicates the fluorescence intensity at 580 / 610 nm, and the horizontal axis indicates the respective substrate concentrations (μM). (1) After 48 hours, 8EG had higher fluorescence and cell viability than EU at all concentrations ( Figure 17 ,left). (2) After 72 hours, the levels of EU were higher at 1000 μM, but at lower concentrations the levels were almost the same or higher for 8EG. (3) These results suggest that 8EG is less toxic than EU.

[0057] <<Experimental Synthesis of 7,8-Vinylguanosine>> 1. About 8-vinylguanosine, Figure 1 The synthesis was carried out according to the scheme. (1) Guanosine was used as the starting material and reacted with NBS in acetonitrile / water as a solvent to brominate the 8-position (a). (2) In the presence of tetrakis(triphenylphosphine)palladium in N-methylpyrrolidone as a solvent, the compound was reacted with tributylvinyltin to introduce a vinyl group at the 8-position (b). 2. NMR chart of the synthesized compound Figure 1 As shown in. 1 H-NMR (400 MHz, DMSO-d6) δ 10.72 (s, 1H), 6.97 (dd, J = 11.0, 17.0 Hz, 1H), 6.46 (br s, 2H), 6.12 (dd, J = 2.1, 17.0 Hz, 1H), 5.80 (d, J = 7.2 Hz, 1H), 5.42 (dd, J = 2.1, 11.0 Hz, 1H), 5.33 (d, J = 6.2 Hz, 1H), 5.12 (t, J = 5.5 Hz, 1H), 5.06 (d, J = 4.5 Hz, 1H), 4.52 (q, J = 6.5 Hz, 1H), 4.08 (m, 1H), 3.85 (q, J = 3.4 Hz, 1H), 3.64 (m, 1H), 3.59 (m, 1H).

[0058] <<Experiment: Confirmation of RNA incorporation and click reaction of 8,8-vinylguanosine>> We investigated whether 1,8-vinylguanosine could be incorporated into RNA and whether it could undergo a click reaction after incorporation.

[0059] 2. The study was conducted in accordance with Experiment 4. The results are as follows: Figure 2 As shown in. (1) Hoechst staining revealed that nuclei were stained in both the presence and absence of 8-vinylguanosine (8-VG). (2) With FAM Tetrazine, no fluorescence was observed in the absence of 8-VG, but fluorescence was observed in the presence of 8-VG. (3) In these composite images, the RNA staining and the nuclear staining were consistent. (4) These results confirmed that 8-VG was incorporated into RNA and that it stained the RNA through the click reaction.

[0060] <<Experiment 9: Confirmation of in vivo click reaction using mouse cancer model>> 1. Using a mouse cancer model, we investigated whether 8-EG and 8-VG could undergo click reactions in vivo.

[0061] 2. Figure 3 The experiment was conducted in accordance with the above. (1) A mouse cancer model was created by subcutaneously implanting a brain tumor cell line (U87 cells) into nude mice. (2) Two weeks after transplantation, 8-EG or 8-VG solution was administered to the tumor site of the mouse cancer model for three days at a dose of 5 mg per mouse. (3) Twenty-four hours after administration of 8-EG or 8-VG for three days, BODIPY-Tet and Alexa-azide solutions were administered via the tail vein to mice administered 8-VG or 8-EG at 0.1 mg each, and images were taken using a fluorescence detector 24 hours later.

[0062] 3. Results Figure 4 As shown in. (1) In the case of 8-VG, no fluorescence was detected in mice administered PBS alone (a), but it was clearly detectable in mice administered 8-VG (b, c). (2) Similarly, in the case of 8-EG, no fluorescence was detected in mice administered PBS alone (e), but it was clearly detectable in mice administered 8-EG (f, g). (3) These results were consistent with those in Experiment 5, and demonstrated that 8-EG and 8-VG could detect brain tumors.

[0063] <<Experiment 10, Evaluation of the stability of 8-EG or 8-VG>> A study was conducted to investigate the compound stability of 1.8-EG or 8-VG.

[0064] 2.8-EG or 8-VG was dissolved in 5% DMSO / PBS, and this was incubated at 37°C for 72 hours. 3. Results Figure 5 No changes were observed in the HPLC chart after incubation (A) compared to before incubation (B) for any of the compounds, and they maintained a high purity. 4. This suggested that 8-EG and 8-VG were stable.

Claims

1. A guanosine derivative compound represented by the following formula 1, R 1 is OH and R 2 is H; R 3 is a vinyl group, and the carbon atom of the double bond is represented by any one of 11 C, 12 C, 13 C, and 14 C; R 4 is represented by H, monophosphate, diphosphate, or triphosphate, A guanosine derivative compound. 【Chemistry 1】

2. R 4 The guanosine derivative compound according to claim 1, wherein is represented by H.

3. R of the guanosine derivative according to claim 1 or 2 3 and a reporter compound that reacts with said compound, The click reaction method, wherein the reporter compound is a compound having an azide group.

4. The click reaction method according to claim 3, wherein the compound having an azide group is selected from the group consisting of azidebenzene, coumarin azide, tetramethylrhodamine (TAMRA) azide, and biotin azide.

5. An imaging agent which labels cells or biological tissue in an animal by carrying out the click reaction method of either claim 3 or 4 on the cells or biological tissue, wherein the guanosine derivative of claim 1 or 2 is an active ingredient.

6. 6. The imaging agent of claim 5, wherein the label is selected from any one or more of a fluorescent label, a luminescent label, a radiolabel, and a nuclear magnetic resonance active label.

7. A cancer imaging agent comprising the guanosine derivative compound of claim 1 or 2 as an active ingredient.

8. The cancer imaging agent according to claim 7, wherein the cancer is a solid cancer such as brain, colon or stomach cancer.

Images