WO2019221024A1 - Guanosine derivative and production method therefor - Google Patents

Abstract

[Problem] To develop a purine base derivative. [Solution] A guanosine derivative compound characterized by being represented by the formula, wherein one of R₁ and R₂ is H and the other is any of H, OH, OCH₃, and F, R₃ is any of a compound having a double bond or triple bond, a compound having an azido group, and a cyclic compound, and R₄ is 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.

Description

Guanosine derivative and process for producing the same

The present invention relates to a guanosine derivative and a production method thereof. 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.

Regarding nucleic acids such as RNA and DNA, a technique for binding a derivative to nucleic acid in a cell and fluorescently labeling it has been studied. Since this technology can visualize the behavior and localization of nucleic acids, it is considered to reflect cell proliferation and protein synthesis, and is expected to be useful for nucleic acid analysis and medical diagnosis.

For such nucleic acid labeling technology, derivatives corresponding to nucleobases constituting RNA and DNA are required.
That is, RNA requires adenine, guanosine, cytosine, uridine, while DNA requires adenine, guanosine, cytosine, thymine, and derivatives related to these.

Of these, several derivatives related to pyrimidine bases have been developed.
For example, 5-EdU (5-deoxy-ethynil-uridine, 5-ethynyl-deoxy-uridine, non-patent documents 1, 2, 4, 5) has been developed as a DNA type uridine derivative. Further, 5-EU (5-ethynil-uridine, 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 each incorporated into intracellular nucleic acids and can be labeled with fluorescence.
As described above, many derivatives related to pyrimidine bases have been developed, but purine base derivatives have not yet been developed.

One reason that purine base derivatives have not been developed yet is thought to be the difficulty of synthesis.
That is, purine bases are poorly handled as compounds, cause aggregation, and tend to be viscous. Under such circumstances, it is considered that the purine base derivative has not been developed yet.
Against the background of the above circumstances, an object of the present invention is to develop a purine base derivative.

As a result of diligent research, the inventors found that guanosine was used as a starting material for bromination, protection of the hydroxyl group at the sugar site and protection of the nucleobase moiety, coupling reaction using Pd catalyst (introduction of ethynyl group), Through the process, the inventors succeeded in synthesizing a guanosine derivative having an ethynyl group introduced at the 8-position of guanosine and completed the invention.
The inventors have also completed the invention by confirming that the synthesized guanosine derivative is incorporated into RNA and that cell labeling is possible by a click reaction.
Similarly, the inventors used deoxyguanosine as a starting material to synthesize deoxyguanosine derivatives in which an ethynyl group was introduced at the 8-position of deoxyguanosine by bromination and coupling reaction using Pd catalyst (introduction of ethynyl group). It has been successful and the invention has been completed.
Furthermore, the inventors have conceived a compound developed on the basis of an ethynyl group-introduced guanosine derivative or a vinyl group-introduced guanosine derivative by clarifying the usefulness by completing a vinyl group-introduced guanosine derivative having a vinyl group introduced, The invention has been completed.

The present invention has the following configuration.
A first configuration of the present invention is a guanosine derivative compound represented by the following formula,
One of R ₁ and R ₂ is H and the other is represented by one of H, OH, OCH ₃ and F;
R ₃ is represented by either a compound having a double bond or a triple bond, a compound having an azide group, or a cyclic compound,
R ₄ is represented by any of H, monophosphate, diphosphate, and triphosphate,
This is a guanosine derivative compound.

According to a second configuration of the present invention, _one of R ₁ and R ₂ of the guanosine derivative is ^{one of 1} H, ² H, and ³ H, and the other is any of ¹ H, ² H, and ³ H. Or a guanosine derivative according to the first configuration represented by ¹⁸ F or ¹⁹ F.
According to a third configuration of the present invention, there is provided a compound having an azido group, wherein R ₃ of the guanosine derivative is any one of ¹¹ C, ¹² C, ¹³ C, and ¹⁴ C in which C of a double bond or a triple bond is N is a guanosine derivative according to the first or second configuration represented by any one of ¹³ N, ¹⁴ N, and ¹⁵ N.
According to a fourth configuration of the present invention, R ₁ and R ₂ are any one of the first to third configurations in which one of them is H and the other is represented by either H or OH. It is a guanosine derivative compound.
A fifth configuration of the present invention is the guanosine derivative compound according to any one of the first to fourth configurations in which R ₄ is represented by H.
A sixth configuration of the present invention is the guanosine derivative according to any one of the first to fifth configurations, in which R ₃ is further represented by any of the following substituents.

The seventh configuration of the present invention is the guanosine derivative according to any one of the first to fifth configurations, wherein R ₃ is represented by any of the following substituents.

The eighth configuration of the present invention is the guanosine derivative according to any one of the first to fifth configurations, in which R ₃ is represented by the following substituent.

A ninth configuration of the present invention is the guanosine derivative according to the sixth configuration, wherein R ₃ is a substituent represented by an ethynyl group (A1).
A tenth configuration of the present invention is the guanosine derivative according to the seventh configuration, in which R ₃ is a substituent represented by a vinyl group (B5).

The eleventh configuration of the present invention is a click reaction method in which a click reaction is performed using R ₃ of the guanosine derivative of any one of the first to tenth configurations and a reporter compound that reacts therewith.
A twelfth configuration of the present invention is the click reaction method according to the eleventh configuration, wherein the reporter compound is a compound having an azide group.
The thirteenth configuration of the present invention is the click reaction method according to the twelfth configuration, wherein the compound having an azide group is selected from any one of Azidebenzene, Coumarin Azide, Tetramethylrhodamine (TAMRA) azide, and Biotin azide. is there.
In a fourteenth configuration of the present invention, the click reaction is a label for labeling a cell or a biological tissue by performing the click reaction method according to any one of the eleventh to thirteenth configurations on the cell or the biological tissue. Is the method.
A fifteenth configuration of the present invention is the label according to the fourteenth configuration, wherein the label is selected from a fluorescent label, a label with a luminescent material, a radiolabel, a nuclear magnetic resonance active label, or any one or a plurality thereof Is the method.

The sixteenth configuration of the present invention is a cancer imaging agent comprising the guanosine derivative of any of the first to tenth configurations as an active ingredient.
A seventeenth configuration of the present invention is the cancer imaging agent according to the sixteenth configuration, wherein the cancer is a cancer in a solid cancer including brain, large intestine, and stomach cancer.

The eighteenth configuration of the present invention is a method for producing a guanosine derivative compound represented by the following formula,
Bromination step of bromination at the 8-position starting from guanosine,
A hydroxyl group protecting step for protecting hydroxyl groups in the sugar skeleton;
A carbonyl group protecting step for protecting the carbonyl group of the nucleobase moiety;
R ₃ introduction step for introducing substituent R ₃ by coupling reaction at the 8-position;
This is a method for producing a guanosine derivative compound comprising an elimination step for eliminating all protecting groups.

(In the formula, R ₃ is represented by a substituent represented by either a compound having a double bond or a triple bond, a compound having an azide group, or a cyclic compound)

According to the present invention, a purine base derivative can be provided.
That is, a method for synthesizing a guanosine derivative having a substituent at the 8-position is provided by the 8-ethynylguanosine derivative or 8-vinylguanosine derivative completed in the present invention, and nucleic acid labeling is performed using this method. It becomes possible.

The figure which showed the synthetic pathway of 8-ethynyl guanosine. The figure which showed the NMR chart of 8-ethynyl guanosine. The figure which showed the synthetic pathway of 8-ethynyl-2'-guanosine. The figure which showed the NMR chart of 8-ethynyl-2'-guanosine. The figure which showed the LC-MS analysis result of the reaction product with 8-ethynyl guanosine and azido zensen. The figure which showed the NMR chart of the reaction product with 8-ethynyl guanosine and azido zensen. The figure which showed the mode of fluorescence of the reaction product with 8-ethynyl guanosine and azido sensen. The figure which showed a mode that 8-ethynyl guanosine and azide zensen were made to react in a cell. The figure which showed the result in the distribution and click reaction in the brain at the time of administering 8-ethynyl guanosine in vivo. The figure which showed the result of the distribution and click reaction in the intestine when 8-ethynylguanosine was administered in vivo. The figure which showed the result in the distribution and click reaction in the kidney at the time of administering 8-ethynyl guanosine in vivo. The figure which showed the result of the cytotoxicity evaluation by the alamar blue assay of 8-ethynyl guanosine (8EG) and ethynyl uridine (EU). The figure which showed the synthetic | combination route of 8-vinyl guanosine (8-VG), and the NMR chart of 8-VG. The figure which showed a mode that 8-vinyl guanosine and FAM-Tetrazine were made to react in a cell. The figure which showed the method of the in vivo experiment. The figure which showed the result of the in vivo experiment. The figure which showed the result of having investigated stability of 8-EG or 8-VG.

The guanosine derivative compound of the present invention will be described.

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

In the chemical formula 6, one of R ₁ and R ₂ is H, and the other is represented by any one of H, OH, OCH ₃ and F. As such a compound, the compound shown in the following chemical formula 7 is exemplified.

That is, when both R ₁ and R ₂ are H (D1), it is expected to be incorporated into DNA as a deoxyguanosine derivative. When either R ₁ or R ₂ is OH (D2, D3), it is expected to be incorporated into RNA as a guanosine derivative (non-deoxyguanosine derivative). In addition, when either R ₁ or R ₂ is OCH ₃ (D6), it is expected that the behavior of methyl guanosine derivatives with 2 ′ methylated can be detected. Furthermore, when either R ₁ or R ₂ is F (D4, D5), it is expected that the behavior of the guanosine derivative can be detected as a label as described later by setting F to ¹⁸ F or the like.

In the chemical formula 6, R ₄ is represented by any one of H, monophosphate, diphosphate, and triphosphate.
That is, when R ₄ is H, it is a nucleic acid derivative, and when R ₄ is a monophosphate, diphosphate, or triphosphate, it is a phosphorylated nucleic acid derivative. Etc. can be expected.

In the chemical formula 6, R ₃ is a compound having a double bond or a triple bond, a compound having an azide group, a cyclic compound, or a substituent represented by any of these. That is, R ₃ causes a click reaction with a reporter compound described later, thereby enabling a label such as a fluorescent label.
R ₃ is a compound having a double bond or a triple bond, a compound having an azide group, a cyclic compound, or a substituent represented by any one of these, and a reporter compound for a click reaction. Depending on the structure, the structure can be changed as appropriate.

As R ₃ , for example, a substituent shown in the following chemical formula 8 can be used.

That is, the substituents represented by A1 to A8 are a group of substituents that react with a reporter compound having an azide group.
R ₃ is preferably a substituent represented by A1 (ethynyl group). Thereby, the structure of a guanosine derivative can be made compact, and it has the effect of improving the stability as a compound or nucleic acid uptake.

As other embodiments of R ₃ , substituents shown in the following chemical formula 9 can be used.

That is, the substituents represented by B1 to B8 are a group of substituents that react with a reporter compound having a tetrazine group.
R ₃ is preferably a substituent (vinyl group) represented by B5. Thereby, the structure of a guanosine derivative can be made compact, and it has the effect of improving the stability as a compound or nucleic acid uptake.

As still another embodiment of R ₃ , substituents shown in the following chemical formula 10 can be used.

That is, such a substituent is a group of substituents that react with a reporter compound having a triple bond such as an ethynyl group.

A so-called click reaction can be performed with R ₃ of the guanosine derivative of the present invention and a reporter compound that reacts with R ₃ .
That is, by the click reaction, R ₃ of the guanosine derivative and the reporter compound bind each other's molecules to form one compound (for example, FIG. 7a). In addition, by using the click reaction, as illustrated in FIG. 7, the guanosine derivative itself or the reporter compound (azidobenzene) itself does not emit fluorescence. The nucleic acid can be labeled.

The structure of the reporter compound can be appropriately changed in consideration of the reactivity with R ₃ in the guanosine derivative, the fluorescence when reacted, the labeling technique, and the like. For example, as a combination of R ₃ and a reporter compound, an alkyne having a triple bond (straight and cyclic types) and a compound having an azide group, an alkene having a double bond (linear and cyclic types) and a compound having a tetrazine group, A carbonyl compound and a compound having a hetero atom.
As the reporter compound, a compound having an azide group can be preferably used, and examples of such a compound include Azidebenzene, Coumarin Azide, Tetramethylrhodamine (TAMRA) azide, and Biotin azide.

In the present invention, the click reaction method is not particularly limited as long as it can cause a click reaction and can be used in various environments, but most preferably, it can be used in cells or living tissues. As a result, the behavior of nucleic acids in cells and living tissues can be visualized, and application to nucleic acid analysis and medical diagnosis can be expected.

The labeling method of the present invention is not particularly limited as long as cells or biological tissues can be labeled, and various methods can be used.
For example, in vitro labeling using cells or pathological sections, or in vivo labeling for humans or animals.

The labeling method of the present invention is not particularly limited as long as cells or biological tissues can be labeled, and various methods can be used. Resonance active labels, any of these can be used alone or in combination.

Various methods can be used as the labeling method.
For example, for H (hydrogen atom) used in guanosine derivatives, not only ¹ H but also ² H and ³ H are used.
^{When 2} H is used, it can be used for diagnosis etc. by detecting MRI as deuterium.
^{When 3} H is used, β ^- rays are released as tritium, so it can be used as a radiolabeled compound in reagents.
In such a case, _one of R ₁ and R ₂ of the guanosine derivative may be ¹ H, ² H, ³ H, and the other may be H, OH, OCH ₃ , F, or the like.

As another method for labeling, ¹⁸ F or ¹⁹ F is used for F (fluorine atom) used in guanosine derivatives.
^{Since 19} F hardly exists in the body, a guanosine derivative compound containing ¹⁹ F can be used as a diagnostic compound for MRI having high background and temporal resolution with little background noise.
^{Since 18} F emits β ⁺ rays as a positron nuclide, it can be used as a diagnostic compound for in vitro detection as a radiolabeled compound. That is, as shown in animal experiments such as Experiment 5 and Experiment 9, since the metabolism of purine base derivatives is accelerated in cancer, the purine base derivatives can be used for PET cancer diagnosis. In particular, brain tumors consume a large amount of purine base derivatives, so information on diseases can be obtained with high resolution.
In such a case, _one of R ₁ and R ₂ of the guanosine derivative may be ¹⁸ F or ¹⁹ F and the other may be H.

In addition, as another method for labeling, ¹¹ C, ¹² C, ¹³ C, and ¹⁴ C are used for C (carbon atom) used in guanosine derivatives.
¹¹ C, like ¹⁸ F, emits β ⁺ rays as a positron nuclide, so it can be used as a radiolabeled compound for diagnostics for in vitro detection.
^{Since 13} C has a small abundance ratio compared to ¹² C, it can be used as a reagent for NMR and the like by forming a guanosine derivative compound containing ¹³ C.
¹⁴ C, like ³ H, emits β ^- rays, so it can be used as a radiolabeled compound in reagents.
In such a case, in R ₃ of the guanosine derivative, the double bond or triple bond C may be any of ¹¹ C, ¹² C, ¹³ C, and ¹⁴ C.
In particular, since the synthesis of 8-vinylguanosine derivatives is relatively easy, it is easy to obtain, and positron-labeled compounds such as ¹¹ C and ¹⁸ F are considered very desirable in the medical field. In addition, since the usage is the same as the conventional positron label, an excellent diagnostic effect can be expected.

In addition, as another method of labeling, ¹³ N, ¹⁴ N, and ¹⁵ N are used for N (nitrogen atom) used in guanosine derivatives.
¹³ N, like ¹⁸ F, emits β ⁺ rays as a positron nuclide, so it can be used as a radiolabeled compound for diagnostics for in vitro detection.
^{Since 15} N has a lower abundance ratio than ¹⁴ N, a guanosine derivative compound containing ¹³ C can be used as a reagent for NMR and the like.
In such a case, in R ₃ of the guanosine derivative, N of the compound having an azide group may be any of ¹³ N, ¹⁴ N, and ¹⁵ N.

The guanosine derivative of the present invention can be used as a cancer imaging agent containing this as an active ingredient.
That is, cancer imaging can be performed by intravenously or locally injecting the guanosine derivative of the present invention and subsequently administering the reporter compound. In such a case, as in Experiment 9, it is possible to perform in vivo imaging of humans and animals (in vivo use). Further, as in Experiment 5, etc., the guanosine derivative can be administered in vivo, and then the tissue can be taken out and reacted with the reporter compound (ex vivo use). Thus, in vivo use, it plays a role as a diagnostic agent for evaluating cancer localization and protein synthesis ability. In ex vivo use, it serves as a reagent for animal experiments. It plays a role.
The cancer at the time of imaging is not particularly limited as long as imaging is possible, but it is preferably a cancer in solid cancer including brain, large intestine and stomach cancer. This has the effect of improving the diagnostic effect of the guanosine derivative of the present invention.

Of 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) Bromination step of bromination at position 8 using guanosine as a starting material (2) Hydroxyl protection step for protecting the hydroxyl group in the sugar skeleton (3) Carbonyl group protection step for protecting the carbonyl group of the nucleobase moiety (4) 8 R ₃ introduction process to introduce substituent R ₃ by coupling reaction at the position (5) Elimination process to remove all protecting groups

(In the formula, R ₃ is represented by a substituent represented by either a compound having a double bond or a triple bond, a compound having an azide group, or a cyclic compound)

The bromination step is a step of brominating the 8-position of guanosine (FIG. 1, a). The bromination step is not particularly limited as long as the 8-position of guanosine can be brominated, and can be performed under various conditions.
In the bromination step, for example, guanosine is reacted with NBS in a solvent of acetonitrile / water.

The hydroxyl group protecting step is a step for protecting the hydroxyl group in the sugar skeleton. The hydroxyl group protecting step is not particularly limited as long as the hydroxyl group in the sugar skeleton can be protected, and can be performed under various conditions.
In the hydroxyl group protection step, for example, DMF is used as a solvent and TBS is protected in the presence of imidazole (FIG. 1, b).

The carbonyl group protecting step is a step of protecting the carbonyl group of the nucleobase moiety. The carbonyl group protecting step is not particularly limited as long as the carbonyl group of the nucleobase moiety can be protected, and can be performed under various conditions.
In the carbonyl group protecting step, for example, protection is carried out by reacting with trimethylsilylethanol in the presence of triphenylphosphine and diisopropyl azodicarboxylate using dioxane as a solvent (FIG. 1, c).

The R ₃ introduction step is a step of introducing the substituent R ₃ at the 8-position by a coupling reaction. The R ₃ introduction step is not particularly limited as long as the substituent R ₃ can be introduced by coupling reaction at the 8-position, and can be performed under various conditions. In addition, the substituent R ₃ may be introduced in a form in which a protecting group is added to the substituent R ₃ in consideration of a subsequent elimination step.
In the R ₃ introduction step, for example, a substituent R ₃ with a protecting group is introduced in the presence of tetrakis (triphenylphosphine) palladium using toluene as a solvent (FIG. 1, d).

The desorption step is a step of removing all protecting groups. The elimination step is not particularly limited as long as the protecting group can be eliminated, and can be performed under various conditions.
An example of the desorption step is desorption of all protecting groups in the presence of tetra-n-butylammonium fluoride using THF as a solvent (FIG. 1, e).

Here, further detailed description will be made using examples.

<< Experiment 1,8-Ethynylguanosine Synthesis >>
1. 8-Ethynylguanosine was synthesized according to the scheme of FIG.
(1) Bromination at position 8 was performed by reacting with NBS using guanosine as a starting material and acetonitrile / water as a solvent (a, bromination step).
(2) The hydroxyl group of the sugar skeleton was 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, it was reacted with trimethylsilylacetylene in the presence of tetrakis (triphenylphosphine) palladium to introduce an ethylene group at the 8-position (d, R ₃ introduction step).
(5) Using THF as a solvent, all protecting groups were removed in the presence of tetra-n-butylammonium fluoride to obtain 8-ethynylguanosine (e, elimination step).
2. An NMR chart of the synthesized compound is shown in FIG.
¹ H-NMR (400 MHz, DMSO-d ₆ ) 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).

<< Experiment 2,8-Ethynyl-2'-deoxyguanosine synthesis >>
1. 8-Ethynyl-2′-deoxyguanosine was synthesized according to the scheme of FIG.
(1) Bromination at position 8 was performed by reacting with NBS using 2'-deoxyguanosine as a starting material and acetonitrile / water as a solvent (a).
(2) Reaction with trimethylsilylacetylene in the presence of tetrakis (triphenylphosphine) palladium, copper iodide and bird ethylamine using DMF as a solvent, introducing an ethylene group at the 8-position, and further using tetrafluoro (tetrafluoride) as a solvent using THF. The reaction was carried out under -n-butylammonium to obtain 8-ethynyl-2'-deoxyguanosine.
2. An NMR chart of the synthesized compound is shown in FIG.
¹ H-NMR (400 MHz, DMSO-d ₆ ) 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).

Experiments 1 and 2 made it possible to synthesize 8-ethynylguanosine and 8-ethynyl-2′-deoxyguanosine. Based on these compounds, it is expected to synthesize phosphorylated derivatives.
As an example, 1,8-bis (dimethylamino) naphthalene (0.15 mmol) is added to 8-ethynyl-2′-deoxyguanosine (8 mmol) or 8-ethynylguanosine (0.1 mmol) and dissolved with 500 μL of trimethyl phosphate. Add phosphoryl chloride (0.13 mmol) to the solution and stir on ice for 2 hours. Add n-butylamine (0.5 mmol) to the reaction solution, followed by 0.5 M tributylammonium pyrophosphate in DMF (0.5 mmol). After 5 minutes, stop the reaction by adding 500 μL of 0.5 M triethylammonium bicarbonate (TEAB) solution. The reaction is purified by DEAE Sephadex A-25 column chromatography. The eluate should be a linear gradient from 50 mM to 1 M TEAB.
Then, if necessary, further purify by C18 reverse phase HPLC to achieve cell and animal laboratory purity. The eluent is a linear gradient of 10% to 50% aqueous acetonitrile containing 100 mM triethylamine acetate.

<< Experiment 3, Confirmation of Click Reaction of 8-Ethynylguanosine >>
1. The click reaction between 8-ethynylguanosine and azidobenzene was confirmed.

2. The LC-MS analysis results are shown in FIG. In the figure, a is 8-ethynylguanosine, b is azidobenzene, and c is the LC-MS analysis result of the reaction solution.
(1) In the presence of copper sulfate, 8-ethynylguanosine and azidobenzene were reacted overnight at 37 ° C.
(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 “click reaction compound”) had the same molecular weight as the estimated compound.

3. The NMR analysis results are shown in FIG. When the 20.84 minute peak (click reaction compound) obtained in FIG. 5 was separated and purified and subjected to NMR analysis, each proton of the click reaction compound was identified and confirmed to be the target compound.
¹ H-NMR (400 MHz, DMSO-d ₆ ) 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) .

4). The state of fluorescence of the click reaction compound is shown in FIG.
(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).

5. From these results, it was shown that 8-ethynylguanosine can be click-reacted with azidobenzene, and that the reaction product fluoresces and can be detected by UV irradiation.

<< Experiment 4,8-Ethynylguanosine RNA uptake and confirmation of click reaction >>
1. We examined whether 8-ethynylguanosine is incorporated into RNA and whether it causes a click reaction after incorporation.

2. According to the following, HeLa cells were stained.
(1) 5 × 10 ⁵ HaLa cells were cultured in DMEM containing 10% FBS at 37 ° C. in the presence of 5% CO ₂ .
(2) 8-Ethynylguanosine (stock solution is 10 mM DMSO solution) was added to the medium at a final concentration of 100 μM and incubated for 24 hours.
(3) When RNase A was used, it was added to a final concentration of 200 μg / mL and incubated for 45 minutes at room temperature.
(4) Washed 3 times with PBS.
(5) The components of the cell staining solution are as follows (amount for one 3.5 cm dish). The final concentration is shown in parentheses.
1.0 μM Tris (pH 8.5) 100 μL (100 mM), 50 mM CuSO4 20 μL (1.0 mM), 10 mM Alexa Fluor 488 azide 2.5 μL (25 μM), Milli Q water 627.5 μL, 0.5 M Ascorbic 200 μL of acid (100 mM).
(6) After 1 hour incubation at room temperature, the plate was washed 3 times with PBS.
(7) Hoechst staining was performed.
(8) Observation was performed with a fluorescence microscope.

3. The results are shown in FIG.
(1) Nuclei were stained by Hoechst staining in the presence or absence of RNaseA (a, d).
(2) With Alexa Fluor 488 azide, staining was not confirmed in the presence of RNase A (e), but staining was confirmed in the absence of RNase A (b).
(3) In these synthesized images, RNA staining and nuclear staining were consistent (c).
(4) From this result, it was confirmed that 8-ethynylguanosine was incorporated into RNA, and this stained RNA by click reaction.

<< Experimental 5, RNA labeling in vivo with 8-ethynylguanosine >>
1. We examined whether 8-ethynylguanosine is incorporated into RNA in vivo and whether it causes a click reaction after incorporation.

2. The experiment was conducted according to the following.
(1) An 8-ethynylguanosine solution (4 mg / mL, 4% DMSO / PBS solution) was administered to one mouse at 0.5 mL (2 mg / body with 8-EG) from the tail vein.
(2) After anesthesia with isoflurane, cervical dislocation was performed, and each tissue was extracted.
(3) The extracted tissue was fixed in 10% formalin for 24 hours, embedded in paraffin, and then sliced into 1 μm thick sections.
(4) A click reaction was performed on thin sliced sections with TMR-N ₃ and the nuclei were stained with DAPI mounting medium and observed with a fluorescence microscope.

3. The results are shown in FIGS. FIG. 9 shows the result of the brain, FIG. 10 shows the result of the intestine, and FIG. 11 shows the result of the kidney.
(1) Staining of cell nuclei was confirmed in the brain, and fluorescence was confirmed by TMR-azide at a position consistent with the cell nuclei. On the other hand, in the control, although fluorescence of the cell nucleus was confirmed, no fluorescence by TMR-azide was confirmed. These results indicate that 8-EG, after administration, passes through the BBB, migrates to brain tissue, and is incorporated into RNA. In addition, it was confirmed that the click reaction by TMR-azide is possible even in living tissue.
(2) In both intestines and kidneys, fluorescence of cell nuclei could be confirmed, but fluorescence from TMR-azide could not be confirmed. This suggests that 8-EG was not taken up in the intestines and kidneys.

<< Experiment 6, cytotoxicity evaluation of 8-ethynylguanosine >>
1. The study was conducted for the purpose of evaluating the cytotoxicity of 8-ethynylguanosine.

2. Evaluation was performed by Alamar Blue assay.
(1) As a cell, HeLa was used and seeded in a 96-well plate at 1 × 10 ³ cell / well.
(2) Ethinyluridine (EU) as 8-EG or control was prepared at various concentrations, incubated at 37 ° C for 48 or 72 hours, and fluorescence at 580/610 nm was measured.

3. The results are shown in FIG. In the graph, the vertical axis represents the fluorescence intensity at 580/610 nm, and the horizontal axis represents the respective substrate concentration (μM).
(1) After 48 hours, at any concentration, 8EG had a higher fluorescence value and higher cell viability than EU (FIG. 12, left).
(2) After 72 hours, EU was higher at 1000 μM, but at lower concentrations, it was almost the same or higher at 8EG.
(3) From these results, 8EG was considered less toxic than the EU.

<< Experiment 7, Synthesis of 8-vinylguanosine >>
1.8-vinylguanosine was synthesized according to the scheme of FIG.
(1) Bromination at position 8 was performed by reacting with NBS using guanosine as a starting material and acetonitrile / water as a solvent (a).
(2) Using N-methylpyrrolidone as a solvent, it was reacted with tributylvinyltin in the presence of tetrakis (triphenylphosphine) palladium to introduce a vinyl group at the 8-position (b).
2. An NMR chart of the synthesized compound is shown in FIG.
¹ H-NMR (400 MHz, DMSO-d ₆ ) 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).

<< Experiment 8, 8-vinylguanosine RNA uptake and confirmation of click reaction >>
1. We examined whether 8-vinyl guanosine is incorporated into RNA and whether or not a click reaction occurs after incorporation.

2. A study was conducted according to Experiment 4. The results are shown in FIG.
(1) Nuclei were stained by Hoechst staining in the presence or absence of 8-vinylguanosine (8-VG).
(2) With FAM Tetrazine, fluorescence was not confirmed in the absence of 8-VG, but fluorescence was confirmed in the presence of 8-VG.
(3) In these synthesized images, RNA staining and nuclear staining were consistent.
(4) From this result, it was confirmed that 8-VG was incorporated into RNA, and this stained RNA by the click reaction.

<< Experiment 9, confirmation of click reaction in vivo using mouse cancer model >>
1. A mouse cancer model was used to confirm whether 8-EG and 8-VG can be clicked in vivo.

2. The experiment was conducted according to FIG.
(1) A brain tumor cell line (U87 cell) was implanted subcutaneously into a nude mouse, and a mouse cancer model was prepared.
(2) Two weeks after transplantation, the 8-EG or 8-VG solution was administered to the mouse cancer model for 3 days at the tumor site at 5 mg per animal.
(3) Tail vein so that BODIPY-Tet and Alexa-azide solutions are 0.1 mg each in mice administered 8-VG or 8-EG 24 hours after the 3-day administration of 8-EG or 8-VG 24 hours later, imaging was performed with a fluorescence detector.

3. The results are shown in FIG.
(1) In 8-VG, fluorescence was not detected by administration of PBS alone (a), but it was clearly detectable in mice administered with 8-VG (b, c).
(2) Similarly, in 8-EG, fluorescence was not detected by administration of PBS alone (e), but it was clearly detectable in mice administered with 8-EG (f, g).
(3) These results are consistent with those in Experiment 5, and it was found that 8-EG and 8-VG can detect brain tumors.

<< Experiment 10, stability evaluation of 8-EG or 8-VG >>
1. The purpose of this study was to investigate the compound stability of 8-EG or 8-VG.

2. 8-EG or 8-VG was dissolved in 5% DMSO / PBS and incubated at 37 ° C. for 72 hours.
3. The results are shown in FIG. None of the compounds remained high in purity after the incubation (A) as compared to that before the incubation (B).
4). This suggests that 8-EG and 8-VG are stable.

Claims (18)

1. A guanosine derivative compound represented by the following formula:
   One of R ₁ and R ₂ is H and the other is represented by one of H, OH, OCH ₃ and F;
   R ₃ is represented by either a compound having a double bond or a triple bond, a compound having an azide group, or a cyclic compound,
   R ₄ is represented by any of H, monophosphate, diphosphate, and triphosphate,
   The guanosine derivative compound characterized by the above-mentioned.
   

   

   
   
2. In R ₁ and R ₂ of the guanosine derivative,
   One is ¹ H, ² H, or ³ H,
   The other is ¹ H, ² H, ³ H, or ¹⁸ F or ¹⁹ F,
   The guanosine derivative | guide_body of Claim 1 represented by these.
   
3. In R ₃ of the guanosine derivative,
   The double bond or triple bond C is either ¹¹ C, ¹² C, ¹³ C, ¹⁴ C,
   N of the compound having an azide group is any one of ¹³ N, ¹⁴ N, and ¹⁵ N,
   The guanosine derivative | guide_body of Claim 1 or 2 represented by these.
   
4. The guanosine derivative compound according to any one of claims 1 to 3, wherein one of R ₁ and R ₂ is H and the other is represented by either H or OH.
   
5. The guanosine derivative compound according to any one of claims 1 to 4, wherein R ₄ is represented by H.
   
6. Furthermore, the guanosine derivative in any one of Claim 1 to 5 by which R < ₃ > is represented by either of the following substituents.
   

   
7. Furthermore, the guanosine derivative in any one of Claim 1 to 5 by which R < ₃ > is represented by either of the following substituents.
   

   
8. Furthermore, the guanosine derivative in any one of Claim 1 to 5 by which R < ₃ > is represented by the following substituent.
   

   
9. The guanosine derivative according to claim 6, wherein R ₃ is a substituent represented by an ethynyl group (A1).
   
10. The guanosine derivative according to claim 7, wherein R ₃ is a substituent represented by a vinyl group (B5).
    
11. 11. A click reaction method in which a click reaction is carried out using R ₃ of the guanosine derivative according to claim 1 and a reporter compound that reacts therewith.
    
12. The click reaction method according to claim 11, wherein the reporter compound is a compound having an azide group.
    
13. The click reaction method according to claim 12, wherein the compound having an azide group is selected from any of Azidebenzene, Coumarin Azide, Tetramethylrhodamine (TAMRA) azide, and Biotin azide.
    
14. A labeling method for labeling a cell or a living tissue by performing the click reaction method according to claim 11 on the cell or the living tissue.
    
15. The labeling method according to claim 14, wherein the label is selected from one or more of a fluorescent label, a label with a luminescent material, a radiolabel, a nuclear magnetic resonance active label.
    
16. A cancer imaging agent comprising the guanosine derivative according to claim 1 as an active ingredient.
    
17. The cancer imaging agent according to claim 16, wherein the cancer is a solid cancer including brain, large intestine, and stomach cancer.
    
18. A method for producing a guanosine derivative compound represented by the following formula:
    Bromination step of bromination at the 8-position starting from guanosine,
    A hydroxyl group protecting step for protecting hydroxyl groups in the sugar skeleton;
    A carbonyl group protecting step for protecting the carbonyl group of the nucleobase moiety;
    R ₃ introduction step for introducing substituent R ₃ by coupling reaction at the 8-position;
    A method for producing a guanosine derivative compound comprising an elimination step for eliminating all protecting groups.
    

    

    (In the formula, R ₃ is represented by a substituent represented by either a compound having a double bond or a triple bond, a compound having an azide group, or a cyclic compound)
    

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