Improved method for preparing nucleoside compound through Mitsunobu reaction

Abstract

The invention belongs to the technical field of preparation of nucleoside compounds, and particularly relates to a method for preparing nucleoside compounds through an improved Mitsunobu reaction. According to the method, N-bromosuccinimide (NBS) partially or completely replaces a traditional azo reagent to serve as an oxidizing reagent for the first time, monosaccharide hydroxyl is efficiently activated in a solvent in the presence of a phosphine ligand and alkali, and a nucleoside compound is prepared through coupling of monosaccharide hydroxyl and alkaloid. According to the method, the problems that a large amount of waste is easily generated, DEAD is toxic and has potential explosiveness and the like when diethyl azo dicarboxylate (DEAD) is used as an oxidizing agent in the traditional Mitsunobu reaction are solved, the use of azo compounds is reduced, the environmental influence is reduced, and the sustainability of the reaction is improved. The method is high in target compound yield, wide in substrate application range, simple and easy to operate, and capable of realizing green and economical preparation of nucleoside compounds.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of nucleoside compounds, and particularly relates to a method for preparing nucleoside compounds by an improved Mitsunobu reaction. Background Art

[0002] The Mitsunobu reaction is a common bimolecular nucleophilic substitution reaction in organic synthesis, which is used for the functional group transformation of hydroxyl groups and the stereoconfiguration inversion of chiral centers. It was discovered by the Japanese chemist Mitsunobu Oyo in 1967. It refers to the reaction in which an alcohol and a nucleophile react under the action of a phosphine ligand and an azo compound to generate an intermediate phosphonium salt, and primary alcohols and secondary alcohols can be converted into corresponding esters, phenyl ethers, thioethers, etc. It is often used to construct chemical bonds such as C-O, C-N, C-S, C-C and C-X. It can be used for the preparation of nucleoside compounds. For example, Yan Lianhai et al. disclosed in "Simple Synthesis of Furanosylbenzimidazole C-Nucleosides by the Mitsunobu Reaction" that an intermediate polyhydroxy chain benzimidazole was obtained by reacting an unprotected five- or six-membered monosaccharide with o-phenylenediamine, and further, by using the Mitsunobu reaction, intramolecular dehydration was carried out to synthesize furanosylbenzimidazole C-nucleosides with inverted and retained configurations. Diao Guolong disclosed in "Synthesis of 1,5-Anhydrohexitol Nucleosides and Cyclohexene Nucleoside Analogs" that 1,5-anhydrohexitol thymidine, adenine and guanine nucleoside analogs were synthesized using 1,5-anhydrohexitol as a raw material. In the synthesis of thymidine and guanine nucleoside analogs, the Mitsunobu reaction method was used to achieve the coupling of 1,5-anhydrohexitol and a base, and in the synthesis of adenine nucleoside, the method of nucleophilic substitution reaction between the product of hydroxyacylation and the base was used for coupling.

[0003] In the above-disclosed technologies, the Mitsunobu reaction conditions all adopted traditional catalytic oxidation systems, that is, using azo compounds DEAD or DIAD as oxidants and phosphine ligands as reducing agents for catalytic oxidation. These compounds are toxic and unstable, and at the same time, there are problems such as the generation of a large amount of waste and potential explosiveness in the reaction. In large-scale synthesis, due to the use of stoichiometric amounts of azo compounds, there will be problems such as increased costs, difficult treatment of by-products, and operational safety, which limit its application in actual synthesis. Exploring more environmentally friendly oxidants or developing green chemical methods such as electrochemical reduction to improve the sustainability of the reaction and reduce the impact on the environment will be the development direction of future synthetic chemistry.

[0004] The catalytic Mitsunobu reaction using iodobenzenediacetate (PIDA) as an external oxidant to achieve the recycling of azo reagents (such as DEAD) was first reported by Toy et al. in 2006 (BUT T Y S, TOY P H. Organocatalytic Mitsunobu Reactions[J]. Journal of the American Chemical Society, 2006, 128(30):9636-7). The advantage of PIDA is that its oxidation products (iodobenzene and acetic acid) can be easily separated from the reaction system. This method uses 0.1 equivalent of diethyl azodicarboxylate (DEAD), 2.0 equivalents of PIDA and triphenylphosphine (PPh3) as reaction reagents( Figure 15 ). Through optimized reaction conditions, different carboxylic acids are coupled with primary or secondary alcohols as nucleophiles to form the corresponding esters. This method first realized the recycling of azo reagents, reduced the reagent consumption, and improved the atom economy of the reaction. Although the recycling of azo reagents was achieved, stoichiometric PIDA was still required, and acetic acid by-products generated during the reaction might interfere with the reaction.

[0005] Taniguchi systematically optimized the first-generation catalytic Mitsunobu reaction in 2016( Figure 16) An alternative oxidant system was adopted, using [Fe(Pc)] / O3 and molecular oxygen as terminal oxidants to re-oxidize the reduced azo reagent (such as ethyl hydrazine dicarboxylate) to the active azo reagent (such as DEAD) through a single-electron transfer mechanism (HIROSE D, TANIGUCHI T, ISHIBASHI H. Recyclable Mitsunobu reagents: catalytic Mitsunobu reactions with an iron catalyst and atmospheric oxygen[J]. Angew Chem Int Ed Engl, 2013, 52(17): 4613-7.). In the second-generation reaction, they paid particular attention to the catalyst dosage and type, the substrate equivalent relationship, and the optimization of reaction conditions. When using ethyl 2-(3,4-dichlorophenyl)hydrazinecarboxylate (cat.A) as the catalyst, when the catalyst dosage increased from 10 mol% to 20 mol%, the product yield increased from 75% to 85%; in the coupling reaction of 3-phenylpropanol and 4-nitrobenzoic acid, when the dosage of 4-nitrobenzoic acid increased from 1.2 equivalents to 1.5 equivalents, the product yield increased from 80% to 92%; for different nucleophiles, different arylhydrazine carboxylates were used as catalysts. When the nucleophile was a carboxylic acid, ethyl 2-(3,4-dichlorophenyl)hydrazinecarboxylate (cat.A) had a better reaction effect; when the nucleophile was a non-carboxylic acid substance, ethyl 2-(4-cyanophenyl)hydrazinecarboxylate (cat.B) had a better reaction effect (HIROSE D, GAZVODAM, MRLJ J, et al. Advances and mechanistic insight on the catalytic Mitsunobu reaction using recyclable azo reagents[J]. Chem Sci, 2016, 7(8): 5148-59.). Taniguchi's second-generation catalytic Mitsunobu reaction maintained the characteristics of green chemistry, but the harshness of reaction conditions and the limitation of substrate scope still needed to be solved.

[0006] The silane-mediated catalytic Mitsunobu reaction is a catalytic strategy for the reduction of phosphine oxides to phosphines by an external reducing agent such as silane. This method was first reported by Horner and Mislow over 30 years ago and was first applied to the Wittig reaction by O'Brien et al. in 2009 (ZHANG K, CAI L, YANG Z, et al. Bridged[2.2.1]bicyclic phosphine oxide facilitates catalyticγ-umpolung addition-Wittigolefination[J]. Chemical Science, 2018, 9:1867-72.). Inspired by this, Aldrich et al. further investigated the silane-mediated catalytic Mitsunobu reaction in 2015 and demonstrated two synthetic routes( Figure 17 )(BUONOMO JA, ALDRICH C C. Mitsunobu Reactions Catalytic in Phosphine and a FullyCatalytic System[J]. Angew Chem Int Ed Engl, 2015, 54(44):13041-4.). The silane-mediated catalytic Mitsunobu reaction was first studied in detail, and its application in the coupling reaction of various alcohols and nucleophiles was demonstrated. The core of this method lies in the use of silane as a reducing agent to reduce phosphine oxides to phosphines, thereby realizing the recycling of phosphorus reagents, which is applicable to the coupling reaction of various primary and secondary alcohols with nucleophiles, including substrates containing sensitive functional groups such as esters, amides, phthalimides, etc. However, a stoichiometric amount of silane needs to be used as a reducing agent, which may increase the reaction cost and handling difficulty. Since the reduction reaction may require specific reaction conditions such as temperature and solvent selection, this also limits the applicability of the reaction. On the basis of the first-generation reaction, Aldrich et al. further attempted to combine Taniguchi's [Fe(Pc)] / O3 strategy with the silane-mediated catalytic Mitsunobu reaction to achieve the dual recycling of phosphorus reagents and azo reagents, further reducing the amount of stoichiometric reagents used, but the reaction conditions are relatively complex and multiple parameters need to be optimized simultaneously.

[0007] In 2019, Denton et al. developed a redox-neutral catalytic system based on a phosphine oxide catalyst( Figure 18)(BEDDOE R H, ANDREWS K G, MAGNé V, et al. Redox-neutral organocatalytic Mitsunobu reactions[J]. Science, 2019, 365(6456): 910-4.) In this system, a cyclic oxophosphonium salt is formed by the reaction of a catalyst with a nucleophile. Subsequently, the alcohol reacts with the phosphonium salt to form a new P-O bond, and finally the catalyst is released and the product is formed. This method does not require the use of external oxidants or reductants, the reaction conditions are mild, and the by-product is only water, thus significantly improving the atom economy and environmental friendliness of the reaction. It is applicable to the coupling reactions of a variety of primary and secondary alcohols with nucleophiles (such as carboxylic acids, phenols, etc.), including substrates containing sensitive functional groups (such as esters, amides, nitriles, etc.). This method is suitable for industrial applications because it simplifies the reaction steps and reduces the generation of waste. However, despite the relatively mild reaction conditions, the high temperature requirement and the limitation of the substrate scope still need to be further addressed.

[0008] It can be seen that although some progress has been made in the field of catalytic Mitsunobu reactions, there are still problems such as high reaction condition requirements, limited applicability of the substrate scope, interference from by-products, high reaction costs and processing difficulties. Therefore, there is an urgent need to further optimize the catalytic system, explore new catalysts and reaction mechanisms to achieve wider applications, and then discover a preparation method of nucleoside compounds with high yield, green, economical, wide substrate scope and simple operation. Summary of the Invention

[0009] In view of the above problems existing in the prior art, the present invention provides an improved method for preparing nucleoside compounds by Mitsunobu reaction. The method has a high yield of the target compound, a wide substrate scope, and a simple and easy-to-operate preparation method, and can achieve the green and economical preparation of nucleoside compounds.

[0010] In a first aspect, the present invention provides an improved method for preparing nucleoside compounds by Mitsunobu reaction, comprising: reacting alkaloids and monosaccharides as reaction substrates in the presence of a phosphine ligand, an oxidation reagent, a base and a solvent to prepare nucleoside compounds; the oxidation reagent contains N-bromosuccinimide.

[0011] In some embodiments, the oxidation reagent is: i) N-bromosuccinimide; or ii) a combination of N-bromosuccinimide and N,N,N',N'-tetramethylazodicarboxamide.

[0012] In some embodiments, the molar ratio of N-bromosuccinimide to N,N,N',N'-tetramethylazodicarboxamide in the combination is 0.12-2:0.11-0.55, preferably 2:0.22.

[0013] In some embodiments, the alkaloid is selected from substituted or unsubstituted purines, theophylline, pyrazoles or imidazoles, wherein the substitution is such that at least one hydrogen atom is replaced by a halogen, an alkyl group or -NO2.

[0014] In some embodiments, the alkaloid is selected from the compounds shown below:

[0015]

[0016] In some embodiments, the monosaccharide is selected from pentoses, methyl pentoses, hexoses or ketohexoses.

[0017] In some embodiments, the monosaccharide is selected from the compounds shown below:

[0018]

[0019] In some embodiments, the phosphine ligand is selected from triphenylphosphine, tri-n-octylphosphine, tri-n-butylphosphine or diphenyl-2-pyridylphosphine, preferably tri-n-octylphosphine.

[0020] In some embodiments, the base is selected from triethylamine, N,N-diisopropylethylamine, potassium carbonate or 1,8-diazabicyclo(5,4,0)-7-undecene, preferably 1,8-diazabicyclo(5,4,0)-7-undecene.

[0021] In some embodiments, the solvent is selected from tetrahydrofuran, acetonitrile, toluene or 1,2-dichloroethane, preferably 1,2-dichloroethane.

[0022] In some embodiments, the reaction temperature is 40 - 60 °C, preferably 40 °C, and the reaction time is 12 - 24 h, preferably 12 h.

[0023] In a second aspect, the present invention provides the use of the nucleoside compounds prepared by the aforementioned method. Specifically, the novel nucleoside compounds 3da, 3aa, 3ea, 3fa prepared by the method of the present invention have a killing effect on gastric cancer cells at 80 μMol / L, 100 μMol / L, 100 μMol / L, and 120 μMol / L respectively, and the IC 50 values are 138.4 μMol / L, 132.1 μMol / L, 121.0 μMol / L, and 135.6 μMol / L respectively, indicating that they have a certain inhibitory and killing effect on the gastric cancer HGC27 cell line and can be used for the development of drugs for the treatment of gastric cancer.

[0024] The beneficial effects of the present invention are as follows:

[0025] 1. The method provided by the present invention, through an improved Mitsunobu reaction, that is, for the first time, N-bromosuccinimide (NBS) is used in part or completely to replace the traditional azo reagent as an oxidizing reagent. In the presence of a phosphine ligand and a base, the hydroxyl groups of monosaccharides are efficiently activated in a solvent, and nucleoside compounds are prepared by coupling with alkaloids. This solves the problems of the traditional Mitsunobu reaction using diethyl azodicarboxylate (DEAD) as an oxidant, which is prone to produce a large amount of waste, DEAD is toxic and potentially explosive, reduces the use of azo compounds, reduces the environmental impact and improves the sustainability of the reaction.

[0026] 2. The method provided by the present invention uses NBS, or a combination of NBS and the azo compound TMAD as an oxidizing reagent. Compared with using a single TMAD, it has the following advantages: 1) significantly simplifies the post-purification. Specifically, as Figure 3 shown, in the column chromatography purification stage, the dihydroazo by-product TMAD has serious tailing in the chromatographic column and is likely to come out together with the target compound, making it more difficult to obtain a high-purity product; 2) reduces the usage amount of azo compounds, thereby reducing the explosion safety risk when azo compounds are used in large quantities, and greatly improving the safety of the reaction; 3) when the yields of the target compounds are basically equivalent, the alternative of NBS greatly reduces the economic cost (TMAD reagent 100g / 880 yuan, NBS reagent 100g / 38 yuan).

[0027] 3. The method provided by the present invention, the alkaloid substrate can be selected from purine, pyrazole, imidazole, etc., and the monosaccharide substrate can be selected from pentose aldose, methyl pentose, hexose aldose, hexose ketose, etc., with the characteristic of a wide range of substrate applicability.

[0028] 4. The method provided by the present invention, all raw materials can be commercially purchased without prior preparation, making the reaction implementation easier and the reaction operation more convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the reaction route of Example 1 of the present invention;

[0030] Figure 2 is the speculated reaction mechanism diagram;

[0031] Figure 3 is the TLC plate analysis comparison of different reaction systems; where: A: UV chromogenic diagram of the reaction solution at 254 nm; B: chromogenic diagram of the reaction solution after potassium permanganate staining; 1: ribose; 2: hexachloropurine; 3: hexachloropurine-ribose derivative; 4: TMAD (2.1 equiv), TOP (2.0 equiv); 5: TMAD (0.22 equiv), TOP (2.0 equiv), NBS (2.0 equiv);

[0032] Figure 4 1H NMR spectrum of compound 3aa;

[0033] Figure 5 1H NMR spectrum of compound 3ab;

[0034] Figure 6 1H NMR spectrum of compound 3ac;

[0035] Figure 7 1H NMR spectrum of compound 3ad;

[0036] Figure 8 1H NMR spectrum of compound 3ae;

[0037] Figure 9 1H NMR spectrum of compound 3af;

[0038] Figure 10 1H NMR spectrum of compound 3ba;

[0039] Figure 11 1H NMR spectrum of compound 3ca;

[0040] Figure 12 1H NMR spectrum of compound 3da;

[0041] Figure 13 1H NMR spectrum of compound 3ea;

[0042] Figure 14 1H NMR spectrum of compound 3fa;

[0043] Figure 15 Catalytic photoreaction route of Toy;

[0044] Figure 16 Catalytic photoreaction route of Taniguchi;

[0045] Figure 17 Catalytic photoreaction route of Aldrich;

[0046] Figure 18 Catalytic photoreaction route of Denton;

[0047] Figure 19Effect of nucleoside compounds prepared for this invention on the proliferation of gastric cancer cells and breast cancer cells; wherein: A: Effect of different concentrations of 3da on the proliferation rate of gastric cancer HGC27 cell line; B: Effect of different concentrations of 3aa on the proliferation rate of gastric cancer HGC27 cell line; C: Effect of different concentrations of 3ea on the proliferation rate of gastric cancer HGC27 cell line; D: Effect of different concentrations of 3fa on the proliferation rate of MDA-MB-231 cell line; E: Effect of different concentrations of 3da on the proliferation rate of MDA-MB-2317 cell line; F.: Effect of different concentrations of 3aa on the proliferation rate of MDA-MB-231 cell line; G: Effect of different concentrations of 3ea on the proliferation rate of MDA-MB-231 cell line; H: Effect of different concentrations of 3fa on the proliferation rate of MDA-MB-231 cell line. Detailed implementation manners

[0048] To make the objectives, technical solutions and advantages of the embodiments of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below. Apparently, the described embodiments are some but not all of the embodiments of this invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of this invention without creative efforts shall fall within the scope of protection of this invention.

[0049] In the following embodiments:

[0050]

[0051]

[0052] Embodiment 1 A preparation method of a nucleoside compound

[0053] In a dry two-necked flask, 6-chloropurine (310 mg, 2.00 mmol, 2.0 equiv) and 1,2-dichloroethane (10 mL) were added, and it was sonicated for 15 min to suspend it. DBU (300 μL, 2.00 mmol, 2.0 equiv) was slowly added dropwise, and the mixture was stirred at room temperature for 15 min. The flask was placed in an ice-water bath and cooled to 0 °C, and TOP (990 μL, 2.00 mmol, 2.0 equiv) was slowly added. TMAD (38 mg, 0.22 mmol, 0.22 equiv) and NBS (354 mg, 2.00 mmol, 2.0 equiv) were added successively within 5 min. Finally, D-ribose (150 mg, 1.00 mmol, 1.0 equiv) was added at once, and the vacuum-nitrogen filling operation was carried out 3 times. The ice bath was removed, and the reaction system was heated to 40 °C and stirred for 12 h. After the reaction was completed, 1 M hydrochloric acid was added dropwise until pH = 7, and the solvent was removed by concentration under reduced pressure. The residue was dissolved in a mixed solvent of methanol-dichloromethane (2 mL, v / v = 1:1), and purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10:1, v / v). The target component was collected and concentrated under reduced pressure to obtain a pure white solid powder product. The reaction route and the speculated reaction mechanism are respectively as Figure 1 and 2 shown.

[0054] Experimental study on the screening of the preparation process of nucleoside compounds in Example 2

[0055] The present invention screened and optimized parameters such as the reaction solvent (such as tetrahydrofuran, acetonitrile, toluene, 1,2-dichloroethane, etc.), azo compounds (diisopropyl azodicarboxylate, N,N,N',N'-tetramethylazodicarboxamide, diethyl azodicarboxylate, dipiperidyl azodicarboxylate, etc.), phosphine ligands (triphenylphosphine, tri-n-octylphosphine, tris(tert-butyl)phosphine, diphenyl-2-pyridylphosphine, etc.), bases (triethylamine, N,N-diisopropylethylamine, potassium carbonate, 1,8-diazabicyclo(5,4,0)-7-undecene, etc.), temperature (RT, 45 °C, 40 °C, 60 °C), oxidants (bis(trifluoroacetyl)iodobenzene, di-tert-butyl peroxide, N-bromosuccinimide, Dess-Martin periodinane, etc.), as follows:

[0056] In a dry two-necked round-bottom flask, 6-chloropurine (310 mg, 2.00 mmol, 2.0 equiv) and acetonitrile (10 mL) were added, and it was sonicated for 15 minutes to fully suspend it. Subsequently, 1,8-diazabicycloundec-7-ene (300 μL, 2.00 mmol, 2.0 equiv) was added, and the mixture was stirred at room temperature for 15 minutes.

[0057] Low-temperature reaction: Cool the reaction system in an ice-water bath to 0 °C, and sequentially add tributylphosphine (530 μL, 2.00 mmol, 2.0 equiv) and diisopropyl azodicarboxylate (420 μL, 2.10 mmol, 2.1 equiv), both of which are slowly added dropwise via a syringe (the dropping time is 5 minutes).

[0058] Glycosylation reaction: After the dropping is completed, add D-ribose (150 mg, 1.00 mmol, 1.0 equiv) all at once, and immediately conduct a vacuum-nitrogen filling cycle (3 times) on the reaction system to displace air. Subsequently, remove the ice bath, slowly warm the reaction system to 25 °C, and stir the reaction at this temperature for 12 hours.

[0059] Workup: After the reaction is completed, add 1 M hydrochloric acid dropwise to the system (10 - 20 drops) to neutralize to pH = 7, and concentrate under reduced pressure to remove the solvent. The residue is dissolved in a mixed solvent of methanol-dichloromethane (2 mL, v / v = 1:1), and purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10:1, v / v). Collect the target component, and concentrate under reduced pressure to obtain a white solid product. Column chromatography separation gives a pure white solid powder 3aa with a yield of 32.15%. The prepared compound is subjected to NMR analysis, and the results show that it is the target compound 3aa.

[0060] The reaction equation is as follows:

[0061]

[0062] By further optimizing the reaction parameters (see the following table), it was found that the target compound 3aa was finally obtained with a yield of 70.07%.

[0063]

[0064]

[0065]

[0066] [a] The yield was calculated by HPLC; [b] TMAD (1.1 equiv), oxidant 2 (1.2 equiv);

[0067] [c - g] NBS (2.0 equiv), TMAD (0.11 equiv, 0.22 equiv, 0.33 equiv, 0.44 equiv, 0.55 equiv);

[0068] [h] Reaction conditions: 1a (2.0 equiv), 2a (1.0 equiv), phosphine ligand ((2.0 equiv), azo compound (2.1 equiv), solvent (1 mL), base (2.0 equiv).

[0069] Subsequently, we separately conducted substrate expansions for alkaloids and monosaccharides. It was found that in the substrate expansion of alkaloids, purine, pyrazole, and imidazole substrates were all applicable to this reaction, especially for alkaloids with electron-withdrawing groups such as -Cl, -Br, -NO2, C=O, etc. In the substrate expansion of monosaccharides, pentoses (arabinose, ribose), methyl pentoses (fucose), hexoses (glucose, mannose), hexulose (fructose), etc. were all applicable to this reaction.

[0070]

[0071] Conditions: DBU (2.0 equiv), 1a - 1f (2.0 equiv), DCE (10 mL), NBS (2.0 equiv), TMAD (0.22 equiv), TOP (2.0 equiv), 2a (1.0 equiv), 0 - 40 °C, 12 h.

[0072] The 1H NMR spectra of compounds 3aa - 3af are as Figures 4 to 9 shown.

[0073]

[0074] Conditions: DBU (2.0 equiv), 1a (2.0 equiv), DCE (10 mL), NBS (2.0 equiv), TMAD (0.22 equiv), TOP (2.0 equiv), 2a - 2f (1.0 equiv), 0 - 40 °C, 12 h.

[0075] The 1H NMR spectra of compounds 3ba - 3fa are as Figures 10 to 14 shown.

[0076] Experimental study on the inhibitory effect of nucleoside compounds on the growth of gastric cancer cells and breast cancer cells in Example 3

[0077] CCK-8 Detection and Drug Treatment Experimental Procedures: The human breast cancer cell line MDA-MB-231 and gastric cancer cell line HGC27 were cultured in RPMI-1640 medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin / streptomycin (Beyotime). All cells were routinely cultured in a 37°C incubator with 5% CO2. Cell seeding: Cells were seeded into 96-well plates at a density of 3,000 cells per well, and 100 μL of complete medium was added to each well. The cells were pre-cultured in a 37°C incubator for 24 hours. The test compounds (a total of 6 kinds, 3aa - 3fa) were dissolved in enzyme-free water (final concentration ≤ 0.1%, Sigma) and diluted with complete medium to a concentration gradient of 0 - 140 μM. After removing the original medium, 100 μL of fresh medium containing different concentrations of the drug was added to each well. Three replicate wells were set for each concentration group, and the cells were cultured for another 48 hours. After drug treatment, 10 μL of CCK-8 reagent (G4103, Servicebio) was added to each well. After incubation at 37°C for 24 hours, the absorbance (OD value) at a wavelength of 450 nm was measured using a microplate reader.

[0078] Results: Drugs 3da, 3aa, 3ea, and 3fa had inhibitory effects on the gastric cancer HGC27 cell line. Among them, drug 3da began to have a killing effect on gastric cancer cells at 80 μMol / L, and as the concentration increased, the IC50 was 138.4 μMol / L( Figure 19 A); Drug 3aa had a certain promoting effect on gastric cancer cells at 40 μMol / L, but began to have a killing effect on the gastric cancer cell line at 100 μMol / L, and the IC50 was 132.1 μMol / L( Figure 19 B); Drug 3ea had a killing effect on the gastric cancer cell line at 100 μMol / L, and the IC50 was 121.0 μMol / L( Figure 19 C); Drug 3fa had a certain killing effect on the gastric cancer cell line at 120 μMol / L, and the IC50 was 135.6 μMol / L( Figure 19 D). However, the killing effect of the same concentration of the drug on the breast cancer cell line MDA-MB-231 was relatively low, and the difference was not statistically significant( Figure 19 E-19H).

[0079] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. An improved method for preparing nucleoside compounds by Mitsunobu reaction, characterized in that, The method includes: reacting alkaloid and monosaccharide as reaction substrates in the presence of a phosphine ligand, an oxidizing agent, a base and a solvent to prepare a nucleoside compound; the oxidizing agent contains N-bromosuccinimide.

2. The method according to claim 1, characterized in that, The oxidizing agent is: i) N-bromosuccinimide; or ii) a combination of N-bromosuccinimide and N,N,N',N'-tetramethylazodicarboxamide.

3. The method according to claim 2, wherein In the combination, the molar ratio of N-bromosuccinimide to N,N,N',N'-tetramethylazodicarboxamide is 0.12 - 2:0.11 - 0.

55.

4. The method according to claim 1, characterized in that The alkaloid is selected from substituted or unsubstituted purine, theophylline, pyrazole or imidazole, and the substitution means that at least one hydrogen atom is substituted by a halogen, an alkyl group or -NO2.

5. The method according to claim 4, characterized in that The alkaloid is selected from the following compounds:

6. The method according to claim 1, wherein The monosaccharide is selected from pentose, methyl pentose, hexose or hexulose.

7. The method according to claim 6, characterized in that, The monosaccharide is selected from the following compounds:

8. The method according to claim 1, wherein The phosphine ligand is selected from triphenylphosphine, tri-n-octylphosphine, tri-n-butylphosphine or diphenyl-2-pyridylphosphine; and / or, the base is selected from triethylamine, N,N-diisopropylethylamine, potassium carbonate or 1,8-diazabicyclo(5,4,0)-7-undecene; and / or, the solvent is selected from tetrahydrofuran, acetonitrile, toluene or 1,2-dichloroethane.

9. The method according to claim 1, wherein The reaction temperature is 40 - 60 °C and the reaction time is 12 - 24 h.

10. Use of the nucleoside compound prepared by the method according to any one of claims 1 - 9 in the preparation of a drug for treating gastric cancer.

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