Nucleosides Z-form and E-form 6 Process for the synthesis of hydroxyisopentenyladenosine and uses thereof

Abstract

The application belongs to the field of nucleic acid epigenetics and pharmaceutical research, and discloses a nucleoside Z Formula and E Formula N 6 A synthesis method and application of hydroxyisopentenyladenosine, wherein the nucleoside E Formula N 6 The synthesis route of the synthesis method of hydroxyisopentenyladenosine is as follows. The application is a nucleoside Z Formula and E Formula N 6 The synthesis of hydroxyisopentenyladenosine provides a new way; the synthesized nucleoside E Formula N 6 Hydroxyisopentenyladenosine or a nucleoside Z Formula N 6 Hydroxyisopentenyladenosine can be used for screening potential markers for treating cerebral infarction.

Description

Technical Field

[0001] This invention belongs to the fields of nucleic acid epigenetics and pharmaceutical research, and more specifically, relates to a nucleoside. Z - formula and E -Mode N 6 Synthesis and application of 1-hydroxyisopentene adenosine N 6 -Hydroxyisoprene adenosine, i.e., io 6 A) provides a novel chemical synthesis method for nucleic acid lipid modification, using which the following is synthesized: Z - formula and E -Mode N 6 -Hydroxyisoprene adenosine can further label, locate, and enrich nucleic acids in vivo and in vitro, promoting subsequent molecular mechanism and drug development research. Background Technology

[0002] Epigenetics and other research areas focus on modifying groups on nucleic acids, which have extremely important physiological significance. Current research mainly concentrates on modifying groups such as methylation. Among the more than 150 nucleic acid modifying groups, research methods are very limited except for methylation. Lipid modification is an important type of chemical modification, but its molecular mechanisms are still in their early stages, and research methods are very limited, resulting in very slow progress. Developing a specific method for the localization, labeling, and enrichment of lipid modifications will have practical scientific research significance. Especially for... N 6 The synthesis and application of hydroxyisoprene-modified nucleosides are currently well-established, but there is a significant lack of high-efficiency chemical synthesis, highly selective transformation, and potential biological applications.

[0003] Taking five synthetic routes reported in existing technologies as examples: (1) Synthesis Route 1: E - formula and Z - Hybrid io 6 A Nucleoside Chemical Synthesis

[0004] Document 1 ( Biochemistry 2013, 52 (36), 6182) reported the reaction of a mixed configuration of E / Z type 1-hydroxyisoamylamine (A2) with a 6-chloroadenosine base derivative (A1') to obtain a diastereomer mixture A3 / A3'. Subsequently, this mixture was condensed with a glycosyl donor A4 under Vorbruggen reaction conditions to obtain a nucleoside product mixture A5 / A5'.

[0005] This route is relatively simple and achieves the synthesis of the target structure to a certain extent. However, due to the ongoing issue of configurational mixing during the reaction, the resulting product is a mixture of different stereoisomers, thus limiting its application value in structurally well-defined bioactivity studies or drug development. Currently, efficient and stereoselective synthetic methods for single-configuration products still need to be established.

[0006] (2) Synthesis Route Two: E - formula and Z - Hybrid io 6 A Nucleoside Chemical Synthesis

[0007] Document 2 ( ACS Med. Chem. Lett. 2017, 9 (2), 89–93) reported a synthetic route starting from a mixture of isomer alcohols (B1 / B1'). Under typical Mitsunob reaction conditions (using DIAD, triphenylphosphine, and THF as solvent), the mixture was reacted with potassium phthalimide to give the corresponding mixed-configuration product B2 / B2'. Subsequently, the silane protecting group was removed by tetrabutylammonium fluoride (TBAF) to give the intermediate mixture B3 / B3'.

[0008] This route has well-defined steps and mature conditions, making it highly feasible for laboratory synthesis. However, because the entire reaction process lacks stereochemical control, the final product is always a mixture of stereoisomers, which greatly limits its application value in studies requiring clear structure-activity relationships or the development of single optically pure drugs. Therefore, developing synthetic methods that can efficiently and selectively obtain products with a single configuration remains of great significance.

[0009] (3) Synthesis Route 3: E -io 6 A Nucleoside Chemical Synthesis

[0010] Document 3 ( Synlett 1991, 11 , 795-6; J. Am. Chem. Soc. 2006, 128 (7025-7035) reported a stereoselective synthetic route starting with an alcohol substrate C1 of a specific configuration. First, in the presence of N-chlorosuccinimide (NCS) and triphenylphosphine, C1 undergoes chlorination, converting to the key intermediate chloride C2 with configuration retention. Subsequently, C2 undergoes nucleophilic substitution with potassium phthalimide to give the substituted product C3. Finally, tetrabutylammonium fluoride (TBAF) is used to remove the silyl protecting group from C3, efficiently yielding the single-configuration final product alcohol C4.

[0011] The advantage of this synthetic route lies in the complete stereochemical control achieved, successfully obtaining a final product with a single configuration, which provides an important foundation for subsequent structure-activity relationship studies and drug development. However, this strategy is highly dependent on the starting material C1, which has a specific absolute configuration and high optical purity. The literature does not elaborate on the synthetic efficiency of C1 itself, the feasibility of large-scale preparation, or the synthetic challenges involved, which makes the practical application value and universality of this route somewhat uncertain.

[0012] (4) Synthesis Route Four: E- io 6 A Nucleoside Chemical Synthesis

[0013] Document 4 ( J. Am. Chem. Soc. 2020, 142 (49), 20788–20795, reported an improved route based on the core strategy of the Gabriel amine synthesis method. This method uses the inexpensive and readily available isopentenyl derivative D1 as a starting material, directly chlorinating it with hypochlorous acid (in a disinfectant system) to obtain intermediate D2. However, the regioselectivity and stereoselectivity of this step, as well as the specific reaction efficiency, were not clearly reported in the original paper. The obtained chlorinated product was directly subjected to nucleophilic substitution with potassium acetate without separation and purification, converting it into alcohol D3. Subsequently, D3 reacted with phosphorus tribromide to generate brominated product D4, which reacted with potassium phthalimide without purification to obtain the protected amine intermediate D5. Finally, the protecting group was removed by hydrolysis with hydrazine hydrate, successfully yielding the target amine product D6.

[0014] The advantages of this synthetic route lie in its use of inexpensive and readily available starting materials, its adoption of the classic Gabriel synthesis strategy, its logical overall design, and the fact that some intermediates do not require purification, making the operation relatively simple. However, the transformation steps from D1 to D3 (i.e., the chlorination and nucleophilic substitution steps) have been found to have low synthetic efficiency, which directly affects the overall yield and practical application prospects of the route, potentially limiting its feasibility for large-scale preparation. Therefore, while this route has certain reference value for laboratory synthesis, further optimization of the efficiency and selectivity of the earlier steps is still needed before actual production applications.

[0015] (5) Synthesis Route Five: E- io 6 A Nucleoside Chemical Synthesis

[0016] literature( Org Lett 2025; 27The synthetic route reported in (10), 2284-2288, uses 1-hydroxyacetone as the starting material. First, the starting material reacts with the Wittig reagent to generate product E3 in 62% yield, but the stereoselectivity of this step is not clearly defined in the literature. Subsequently, the terminal hydroxyl group of E3 is protected by TBS to obtain E4 in 96% yield; then, the ethyl ester group in compound 4 is reduced using lithium aluminum hydride (LiAlH4, abbreviated as LAH) to obtain the alcohol intermediate E5 in 98% yield. Although the subsequent steps are all well-established and conventional methods in the literature, and the overall route is feasible, this strategy has a significant bottleneck: the Wittig reaction cannot achieve complete stereospecificity, resulting in the formation of non-equilibrium compounds. E / Z The two isomers are a mixture of configurations and are difficult to separate. Summary of the Invention

[0017] In view of the above-mentioned defects or improvement needs of the prior art, the purpose of this invention is to provide nucleosides. Z - formula and E -Mode N 6 Synthesis and applications of hydroxyisoprene adenosine, a nucleoside Z - formula and E -Mode N 6 A new pathway was provided for the synthesis of hydroxyisoprene adenosine; the synthesized nucleosides E -Mode N 6 -Hydroxyisopentene adenosine, nucleoside Z -Mode N 6 -Hydroxyisoprene adenosine can be used to screen potential biomarkers for the treatment of cerebral infarction.

[0018] To achieve the above objectives, according to one aspect of the present invention, a nucleoside is provided. E -Mode N 6 The method for synthesizing -hydroxyisopentene adenosine is characterized by the following synthetic route: .

[0019] As a further preferred embodiment of the present invention, the synthetic route of compound B3 is as follows: .

[0020] As a further preferred embodiment of the present invention, the synthesis method includes the following steps: (1) Compound A1 and compound B1 are dissolved together in N,N-dimethylformamide (DMF) solvent and stirred at 20°C to 50°C to generate compound A2; preferably, the stirring reaction is carried out at room temperature; (2) Dissolve compound A2 in dichloromethane (DCM), add salicylic acid, tert-butyl hydroperoxide (t-BuOOH) and selenium dioxide, and stir at 20℃~40℃ to generate compound A3; preferably, the stirring reaction is carried out at room temperature; (3) Dissolve compound A3 in pyridine, add 4,4'-bismethoxytriphenylmethyl chloride (DMTrCl), and stir the reaction at 10℃~40℃ to generate compound A4; preferably, the stirring reaction is carried out at room temperature; (4) Dissolve compound A4 in ethanol (EtOH), add hydrazine hydrate, and stir at 50℃~70℃ to generate compound A5; preferably, the stirring reaction is carried out at 55℃; (5) Dissolve compound A5 and compound B3 in n-butanol solvent and heat them under reflux at 90°C to 120°C with stirring to generate compound A6; preferably, the reaction is carried out at 110°C. (6) Dissolve compound A6 in tetrahydrofuran (THF) solvent, add tetrabutylammonium fluoride (TBAF) reagent, and stir the reaction at 0℃~50℃ to generate compound A7; preferably, the stirring reaction is carried out at room temperature.

[0021] As a further preferred embodiment of the present invention, in step (1), the equivalent ratio of compound A1 to compound B1 is 1:(1~1.2). In step (2), the equivalent ratio of compound A2, salicylic acid, tert-butyl hydroperoxide, and selenium dioxide is 1:(0.1~0.2):(2~4):(0.05~0.1). In step (3), the equivalent ratio of compound A3 to 4,4'-bismethoxytriphenylmethyl chloride is 1:(1~2). In step (4), the equivalent ratio of compound A4 to hydrazine hydrate is 1:(1~2). In step (5), the equivalent ratio of compound A5 to compound B3 is 1:(1~1.2). In step (6), the equivalent ratio of compound A6 to tetrabutylammonium fluoride is 1:(3~5).

[0022] As a further preferred embodiment of the present invention, the method for synthesizing compound B3 is as follows: Compound B2 was dissolved in tetrahydrofuran, and imidazole and tert-butyldimethylchlorosilane (TBSCl) were added. The mixture was stirred at 0°C to 50°C to generate compound B3. Preferably, the stirring reaction was carried out at room temperature. More preferably, the equivalent ratio of compound B2, imidazole, and tert-butyldimethylchlorosilane is 1:(1~3):(1~3).

[0023] According to another aspect of the invention, the present invention provides a nucleoside Z -Mode N 6 The method for synthesizing -hydroxyisopentene adenosine is characterized by the following synthetic route: .

[0024] As a further preferred embodiment of the present invention, the synthesis method includes the following steps: S1: Dissolve compound C1 in ethanol, and slowly add an aqueous sodium hydroxide solution at 10℃~40℃ to react and generate compound C2; preferably, the reaction is carried out at room temperature; S2: Dissolve compound C2 in tetrahydrofuran, add imidazole and tert-butyldimethylchlorosilane (TBSCl), and stir the reaction at 10°C to 40°C to generate compound C3; preferably, the stirring reaction is carried out at room temperature. S3: Dissolve compound C3 in methanol, slowly add sulfuric acid, react at 40~70℃, wash the product with saturated brine after rotary drying, dissolve in dichloromethane, slowly add diisopropylaluminum hydride (DIBAL-H), and react at -40℃~0℃ to generate compound C4. S4: Dissolve compound C4 in a dichloromethane solution, add triphenylphosphine and carbon tetrabromide, and react at 10°C to 40°C to generate compound C5; preferably, the reaction is carried out at room temperature; S5: Compound C5 and compound B1 are dissolved together in N,N-dimethylformamide (DMF) solvent and reacted by stirring at 10°C to 50°C to generate compound C6; preferably, the stirring reaction is carried out at room temperature; S6: Dissolve compound C6 in ethanol, add hydrazine hydrate, and stir at 50°C to 70°C to generate compound C7; preferably, the stirring is carried out at 55°C; S7: Compound C7 and compound B3 are dissolved in n-butanol solvent and heated under reflux at 90°C~120°C with stirring to generate compound C8; preferably, the reaction is carried out at 110°C. S8: Dissolve compound C8 in tetrahydrofuran (THF) solvent, add tetrabutylammonium fluoride (TBAF) solvent, and stir the reaction at 0℃~40℃ to generate compound C9; preferably, the stirring reaction is carried out at room temperature.

[0025] As a further preferred embodiment of the present invention, in step S1, the equivalent ratio of compound C1 to NaOH is 1:(2~10). In step S2, the equivalent ratio of compound C2: imidazole and tert-butyldimethylchlorosilane is 1:(1~3):(1~3); In step S3, the equivalence ratio of compound C3 to diisopropylaluminum hydride is 1:(2.5~4). In step S4, the equivalent ratio of compound C4, triphenylphosphine, and carbon tetrabromide is 1:(1~4):(1~2). In step S5, the equivalent ratio of compound C5 to compound B1 is 1:(1~1.2). In step S6, the equivalence ratio of compound C6 to hydrazine hydrate is 1:(1~2). In step S7, the equivalent ratio of compound C7 to compound B3 is 1:(1~1.2). In step S8, the equivalent ratio of compound C8 to tetrabutylammonium fluoride is 1:(3~6).

[0026] As a further preferred embodiment of the present invention, the synthetic route of compound B3 is as follows: .

[0027] According to another aspect of the invention, the invention provides nucleosides E -Mode N 6 -Hydroxyisopentene adenosine or nucleoside Z -Mode N 6 Application of 1,5-hydroxyisoprene adenosine in screening potential biomarkers for the treatment of cerebral infarction.

[0028] Compared with the prior art, the present invention provides a highly efficient and selective synthetic route for Z-type and... E -io 6 A chemical synthesis method overcomes the bottlenecks of existing methods, such as being lengthy, complex, and having low selectivity and low efficiency.

[0029] (1) First, regarding E -io 6 A. The synthetic route is as follows:

[0030] The synthetic route of this application uses inexpensive and readily available isopentenyl bromide derivatives as starting materials. First, the starting materials are reacted with potassium phthalimide salt ( N The intermediate undergoes a nucleophilic substitution reaction with phthalimide potassium salt. After the reaction is complete, a high-purity intermediate can be obtained efficiently through simple post-processing steps such as water washing and extraction. This step has a quantitative yield, is easy to operate, does not require column chromatography purification, and has good potential for large-scale production.

[0031] Subsequently, the intermediate undergoes a key transformation step in this route: under the catalysis of selenium dioxide (SeO2), the terminal methyl group is selectively oxidized to an allyl alcohol structure, specifically generating... E Configuration product A3. This step successfully overcomes the bottlenecks commonly found in existing methods, such as poor stereoselectivity and mixed product configurations, achieving single-product optimization. E Efficient construction of the configuration product. Although the reaction yield in this step is about 40%, the unreacted raw materials can be recycled with a recovery rate of up to 90% through a simple recycling process, which significantly reduces material costs and waste generation.

[0032] After obtaining A3, first compare it with 4 and 4'. Dimethoxytriphenylchloromethane (DMTr) The phthalimide protecting group of DMTr is removed by a hydroxyl protection reaction (preferably carried out under pyridine as a base) to generate the protected product A4. Subsequently, the phthalimide protecting group of A4 is efficiently hydrolyzed by hydrazine hydrate. After simple filtration and washing, the free amine intermediate A5 can be obtained in high yield.

[0033] Finally, A5 and 6 The chloroadenosine reaction constructs a nucleoside bond (this reaction can be particularly carried out under reflux at 90°C–120°C, e.g., reflux at 110°C; and especially under conditions where n-butanol is used as solvent and triethylamine as base), yielding compound A6. Compound A6 is dissolved in a solvent, and the tert-butyldimethylsilyl group is deprotected by tetrabutylammonium fluoride solution. The crude product is then purified by rapid column chromatography, successfully obtaining the target product in a single configuration— E Type Isopentenyl adenosine ( E io 6 A). The reaction conditions for this step are mature, and the post-processing is simple. As illustrated in Examples 6 and 7 below, the final product is confirmed by spectroscopic analysis to be a pure product with a single configuration.

[0034] In summary, the core innovation and key advantage of this route lies in: utilizing a SeO2-mediated highly selective oxidation reaction to achieve the conversion of the terminal methyl group of isopentenyl groups to... E The efficient and specific conversion of allyl alcohol configurations fundamentally solves the long-standing technical problems in existing synthetic methods, such as mixed product configurations, low selectivity, and difficulty in purification.

[0035] (2) In addition, regarding Z -io 6 A. The synthetic route is as follows:

[0036] The synthetic route of this application starts with the commercially available lactone C1, which is first hydrolyzed with sodium hydroxide to obtain the ring-opening carboxylate product C2. Based on this, the terminal hydroxyl group is further reacted to obtain a crude silane-protected TBS product (this reaction can be carried out, for example, with TBS-Cl), which is then purified to C3 after simple washing. For the terminal carboxyl group, through screening a series of reducing reagents, it was finally confirmed that methanol esterification followed by diisopropylaluminum hydride can effectively reduce it to an alcohol, which is then purified to crude product C4 after simple washing. Alcohol C4 reacts to give a brominated product C5 (this reaction can preferably be carried out with the participation of phosphorus tribromide and carbon tetrabromide), which is then reacted with N-phthalimide to give crude product C6. C6 is separated by silica gel column chromatography to obtain the target product C6. C6 is hydrolyzed with hydrazine hydrate and reacted with 6-chloroadenosine to obtain the target product nucleoside TBS-Z-io. 6 A (This reaction can be carried out, especially under n-butanol-triethylamine reflux conditions). Finally, the silane protecting group can be removed via TBAF to achieve the synthesis of the target product C9. This route starts from the readily available lactone C1 and proceeds through 7-8 steps, of which the first five steps can be performed continuously with simple post-treatment purification. The last 2-3 steps can also be carried out continuously. This route was the first to be developed. Z -io 6 The efficient synthesis of A enables large-scale production.

[0037] (2) In E-type io 6 During the synthesis and development of A, we encountered two major challenges. The first was the stereoselectivity control of allylic oxidation—existing Wittig or chlorination routes generally suffer from mixed E / Z configurations and difficulty in separation. This invention addresses this by using selenium dioxide (SeO2) / tert-butyl hydroperoxide (t... A catalytic oxidation system composed of BuOOH and salicylic acid can selectively oxidize terminal methyl groups to E under mild conditions. Allyl alcohol configuration, almost no Z-formation The isomers were identified, thus resolving the bottleneck of configurational separation at its source. However, after obtaining the allyl alcohol intermediate A3, we initially attempted to directly hydrolyze the phthalimide protecting group with hydrazine hydrate, hoping to obtain the free amine intermediate via the shortest route. However, experiments revealed that this free amine, containing both primary amino and primary hydroxyl groups, was extremely polar, exhibiting severe adsorption and significant tailing in conventional column chromatography, and could not be purified by simple extraction. This hindered subsequent purification with 6... The condensation reaction of adenosine chloride produces numerous byproducts and has a low conversion rate. To overcome this purification bottleneck, after repeated experiments and route adjustments, 4,4'- The primary hydroxyl group of A3 is selectively protected with bis(methoxytriphenylmethyl)chloro(DMTrCl) to yield A4, which is then hydrolyzed with hydrazine hydrate to obtain A5. The introduction of the bulky DMTr group significantly reduces the polarity of the intermediate, allowing for efficient purification through simple acid-base adjustments and washing. Furthermore, the DMTr group does not require additional removal in the subsequent nucleoside condensation reaction; it can ultimately be removed under high-temperature conditions during the reaction of A5 and B3, without adding any extra steps. This sequential step effectively solves the core purification problem that has limited the practical application of this route.

[0038] For Z-type io 6 The synthesis of A does not yet involve a single Z in existing technologies. configuration io 6 A highly efficient synthetic method for nucleosides A. This invention, starting from commercially available lactone raw materials, develops for the first time a complete Z-scheme synthetic route. Specifically, it efficiently reduces the carboxyl group to an alcohol without destroying the Z-scheme. The double bond configuration is the core transformation of the route, and we have studied various reducing agents (including lithium aluminum hydride, borane) A systematic screening of dimethyl sulfide and other compounds was conducted, ultimately confirming that diisobutylaluminum hydride (DIBAL-H) ​​can achieve this reduction with high selectivity under mild conditions and is well compatible with other functional groups in the molecule. Furthermore, this route, after process optimization, enables continuous operation of the first five reaction steps, requiring only simple water washing and extraction to obtain intermediates of acceptable purity. The latter three steps can also be carried out continuously, thus efficiently obtaining the target product and fundamentally solving the pain points of previous methods that were lengthy and cumbersome in purification.

[0039] (4) Furthermore, the synthetic route of this invention underwent effective chemical transformation, verifying selectivity and transformation efficiency, laying a solid foundation for further research such as activity testing and high-throughput sequencing. Taking the examples below, the synthesized modified nucleosides were tested for activity. Preliminary verification was performed in cell lines such as MCF-7 and A549. This provides a novel perspective and application scenario for subsequent drug development, and lays the foundation for subsequent systematic research. Z - formula and E -io 6 The application of A is of great research significance.

[0040] Taking the following embodiments as examples, the present invention has also been implemented. Z -io 6 A and E -io 6Activity assay for A. Under aseptic conditions, a series of nucleoside solutions and positive control drugs of various concentrations were prepared. Target cell suspensions in the logarithmic growth phase were seeded into 96-well plates. After cell attachment, culture medium containing different drug concentrations was added. Blank and negative control wells were also included. The plates were incubated at 37°C with 5% CO2 for a specific time (usually 48 hours). The supernatant was then discarded, and MTT or CCK-8 reagents were added for further incubation to form formazan crystals or a colorimetric reaction. The absorbance of each well at a specific wavelength was measured using a microplate reader. Finally, the cell inhibition rate was calculated using a formula, and a dose-response curve was plotted to determine the half-maximal inhibitory concentration (IC50). IC 50 To assess the bioactivity of nucleosides, aseptic techniques were strictly followed throughout the process, and duplicate wells were used to ensure data reproducibility and statistical significance. Attached Figure Description

[0041] Figure 1 This is the 1H NMR spectrum of the A2 product obtained in Example 1.

[0042] Figure 2 This is the carbon NMR spectrum of the A2 product obtained in Example 1.

[0043] Figure 3 This is the 1H NMR spectrum of the A3 product obtained in Example 2.

[0044] Figure 4 This is the mass spectrum of the A3 product obtained in Example 2.

[0045] Figure 5 This is the 1H NMR spectrum of the A4 product obtained in Example 3.

[0046] Figure 6 This is the 1H NMR spectrum of the A5 product obtained in Example 4.

[0047] Figure 7 This is the 1H NMR spectrum of the A6 product obtained in Example 6.

[0048] Figure 8 This is the 1H NMR spectrum of the A7 product obtained in Example 7.

[0049] Figure 9 This is the mass spectrum of the A7 product obtained in Example 7.

[0050] Figure 10 It is the compound in Example 8 E -io 6 Results of MCF-7 cell viability assay for A.

[0051] Figure 11 It is the compound in Example 8 E -io 6Results of A549 cell viability assay.

[0052] Figure 12 This is the mass spectrum of the C4 product obtained in Example 11.

[0053] Figure 13 This is the 1H NMR spectrum of the C6 product obtained in Example 13.

[0054] Figure 14 This is the mass spectrum of the C6 product obtained in Example 13.

[0055] Figure 15 This is the mass spectrum of the C9 product obtained in Example 16.

[0056] Figure 16 This is the 1H NMR spectrum of the C9 product obtained in Example 16.

[0057] Figure 17 It is the compound in Example 17 Z -io 6 Results of MCF-7 cell viability assay for A.

[0058] Figure 18 It is the compound in Example 17 Z -io 6 Results of A549 cell viability assay.

[0059] Figure 19 The blood samples and standards from clinical stroke patients in Example 18 ( E -io 6 Comparison of LC-MS detection results for compound A. Figure 19 From left to right, the images show: the LC-MS / MS detection chromatogram of the standard, the LC-MS / MS detection chromatogram of blood samples from clinical stroke patients, and the development result of the detection peak at position 2.91 in the LC-MS / MS detection chromatogram of blood samples from clinical stroke patients. Detailed Implementation

[0060] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0061] The following examples 1-7 are described in their entirety. E -io 6 The synthesis route for A is as follows:

[0062] Compound A7, namelyE -io 6 A.

[0063] Specifically: Example 1: Isoprene bromide (A1, 5 g, 1.0 equivalent, commercially available, CAS No.: 870-63-3) and potassium phthalimide (B1, 1.05 equivalent, commercially available, CAS No.: 1074-82-4) were added to a reaction vessel, along with 300 mL of N,N-dimethylformamide (DMF) solvent. The mixture was stirred until completely dissolved. The reaction mixture was stirred at room temperature for 12 hours. After the reaction was complete, the solvent was removed under reduced pressure, and the residue was washed with saturated brine and extracted with dichloromethane (DCM). The organic phases were combined, and the solvent was removed under reduced pressure to give 9 g of a white solid product (A2, yield 78%).

[0064] The 1H NMR and 1C NMR spectra of the A2 product are as follows: Figure 1 , Figure 2 As shown.

[0065] Example 2: Compound A2 (720 mg, 1.0 equivalent) was dissolved in dichloromethane (DCM), and salicylic acid (0.1 equivalent), tert-butyl hydroperoxide (t-BuOOH, 3 equivalent), and selenium dioxide (SeO2, 0.05 equivalent) were added sequentially at 0 °C. After stirring at room temperature for 12 hours, the solvent was removed by vacuum distillation, and the residue was washed with saturated brine and extracted with dichloromethane (DCM). The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography to give the target alcohol product A3 (60% yield).

[0066] The 1H NMR spectrum of the A3 product is as follows: Figure 3 As shown, the mass spectrometry is as follows Figure 4 As shown.

[0067] Example 3: Compound A3 (300 mg, 1.0 equivalent) was dissolved in pyridine, and 4,4'-bismethoxytriphenylmethyl chloride (DMTrCl, 1.5 equivalent, commercially available, CAS No.: 40615-36-9) was added. After stirring at room temperature for 12 hours, the solvent was removed under reduced pressure. The residue was washed with saturated brine and extracted with dichloromethane (DCM). The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography to give the target alcohol product A4 (95% yield).

[0068] The 1H NMR spectrum of the A4 product is as follows: Figure 5 As shown.

[0069] Example 4: Compound A4 (35 mg, 1.0 equivalent) was dissolved in ethanol (EtOH), and 85% hydrazine hydrate (N2H4·H2O, 2 equivalent) was added. The mixture was stirred at 55 °C for 3 hours. After filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by rapid column chromatography to obtain the target product A5 (100% yield).

[0070] The 1H NMR spectrum of the A5 product is as follows: Figure 6 As shown.

[0071] Example 5: Compound B2 (1 g, commercially available, CAS No.: 5399-87-1) was dissolved in tetrahydrofuran (40 mL), and imidazole (1.5 equivalents) and tert-butyldimethylchlorosilane (TBSCl, 1.5 equivalents, commercially available, CAS No.: 18162-48-6) were added. The mixture was stirred at room temperature for 12 hours. After the reaction was completed, the crude product was purified by silica gel column chromatography to give a white solid TBS-protected product B3 (2 g, 91% yield).

[0072] Example 6: Compound A5 (206 mg, 1.0 equivalent) and compound B3 (1.05 equivalent) were dissolved in n-butanol and refluxed at 110 °C for 12 hours with stirring. The reaction solution was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the crude product was purified by rapid column chromatography to give compound A6 (90% yield).

[0073] The 1H NMR spectrum of the A6 product is as follows: Figure 7 As shown.

[0074] Example 7: Compound A6 (200 mg, 1.0 equivalent) was dissolved in 1 mL of tetrahydrofuran (THF) solvent, and 1 M tetrabutylammonium fluoride (TBAF, 5.0 equivalent; commercially available, CAS No.: 429-41-4) reagent was added. The mixture was stirred at room temperature for 12 hours. After the reaction was completed, the solvent was removed by vacuum distillation, and the crude product was purified by rapid column chromatography to obtain compound A7. The 1H NMR spectrum of A7 was analyzed using databases and literature (e.g., Tranová L, Bu...). ek J, Zatloukal M, Canka P, Stýskala J. Synthesis of [15N4]purine labeled cytokinin glycosides derived from zeatins and topolins with 9-β-d, 7-β-d-glucopyranosyl, or 9-β-d-ribofuranosyl group. JLabel Compd Radiopharm. 2019;62:118–125.) The target E-type was determined by comparison. 6 Product A (A7) (81 mg, 80% yield).

[0075] The 1H NMR spectrum of the A7 product is as follows: Figure 8 As shown, the mass spectrometry is as follows Figure 9 As shown.

[0076] The synthetic routes formed by the systematic optimization and screening of reaction conditions in Examples 1-7 above possess comprehensive advantages such as simple steps, easy operation, readily available raw materials, high stereoselectivity of key steps, and recyclable materials, providing a basis for... E io 6 This provides a feasible new approach for the large-scale, high-purity preparation of A and its analogues.

[0077] Example 8: The A7 product obtained in Example 7 was used in cell viability experiments, and the above-mentioned cell viability was detected using CCK-8 assay. E -io 6 The toxicity of nucleoside A to MCF-7 and A549 cells was detected, specifically: Cells were spaced at 9 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in 96-well plates and incubated at 37°C for 24 hours. Then, different concentrations of [missing information] were used. E -io 6 Cells were treated with nucleosides A (10 μM, 20 μM, 50 μM, 100 μM, 200 μM, 400 μM) for 24 hours. Cells were incubated for 1.5 hours per well with 10 μl of CCK-8 at 37 °C. Absorbance was measured at 450 nm using a microplate reader. Cells treated with the A7 product (10-400 μM) showed the lowest cell death rate, indicating that the use of... E -io 6 This labeling method (up to 100 μM) is fully compatible. Three independent experiments were conducted for each group.

[0078] compound E -io 6Results of MCF-7 cell viability assay for A, such as Figure 10 As shown (where "CTL" stands for the control group, meaning no nucleosides are used), it can be seen that... E -io 6 A exhibited a concentration-dependent, mild inhibitory effect on the survival rate of MCF-7 cells within the concentration range of 10–400 µM.

[0079] compound E -io 6 The results of the A549 cell viability assay for A, such as Figure 11 As shown (where "CTL" stands for the control group, meaning no nucleosides are used), it can be seen that... E -io 6 The survival rate of A549 cells showed a similar concentration-dependent decreasing trend.

[0080] The following examples 9-16 are described in their entirety. Z -io 6 The synthesis of A is shown below:

[0081] C9 compounds, i.e. Z -io 6 A.

[0082] Specifically: Example 9: Compound C1 (500 mg, commercially available, CAS No.: 6124-79-4) was dissolved in ethanol (10 mL), and 3 M sodium hydroxide aqueous solution was slowly added at room temperature to adjust the final concentration to 0.3 M (equivalent ratio of compound C1 to sodium hydroxide 1:5). After reacting for 12 hours, the solvent was removed by rotary evaporation under reduced pressure to give crude product C2 (430 mg, yield 73%).

[0083] Example 10: Compound C2 (400 mg, 1.0 equivalent) was dissolved in tetrahydrofuran (20 mL), and imidazole (2 equivalent) and tert-butyldimethylchlorosilane (TBSCl, 2 equivalent) were added. The mixture was stirred at room temperature for 12 hours. After the reaction was completed, the crude product was purified by silica gel column chromatography to give a colorless oily TBS protected product C3 (610 mg, 72% yield).

[0084] Example 11: Compound C3 (100 mg, 1.0 equivalent) was dissolved in methanol (MeOH), and sulfuric acid (2 equivalents; the sulfuric acid solution used was 98%) was slowly added. The mixture was heated and stirred at 50°C for 5 hours. After the reaction was complete, the solvent was removed by rotary evaporation under reduced pressure. After washing and extraction with saturated brine, an ester intermediate was obtained. The ester intermediate was dissolved in dichloromethane, and diisobutylaluminum hydride (DIBAL-H, 3 equivalents, commercially available, CAS No.: 1191-15-7) was slowly added at -20°C. The mixture was reacted at -20°C for 3 hours. After the reaction was complete, the crude product was purified by silica gel column chromatography to give a colorless oily alcohol product C4 (85 mg, yield 91%). [M+Na] + = 239.1443. The mass spectrum of the C4 product is as follows: Figure 12 As shown.

[0085] Example 12: Compound C4 (85 mg, 1.0 equivalent) was dissolved in dichloromethane solution (5 mL), and triphenylphosphine (1.5 eq) and carbon tetrabromide (1.2 eq) were added in a single reaction at room temperature. The reaction was carried out at room temperature for 12 hours. After the solvent was evaporated, the crude product was separated by silica gel column chromatography to obtain C5 (53 mg, yield 48%).

[0086] Example 13: The brominated derivative C5 (53 mg, 1.0 equivalent) and potassium phthalimide (B1, 1.05 equivalent) were added to a reaction vessel, followed by 3 mL of N,N-dimethylformamide (DMF) solvent. The mixture was stirred until completely dissolved. The reaction mixture was stirred at room temperature for 12 hours. After the reaction was complete, the solvent was removed under reduced pressure, and the residue was washed with saturated brine and extracted with dichloromethane (DCM). The organic phases were combined, and the solvent was removed under reduced pressure to give solid product C6 (55 mg, 84% yield).

[0087] The 1H NMR spectrum of the C6 product is as follows: Figure 13 As shown; the mass spectrum of the C6 product, as shown. Figure 14 As shown.

[0088] Example 14: Compound C6 (55 mg, 1.0 equivalent) was dissolved in ethanol (EtOH), and 85% hydrazine hydrate (N2H4·H2O, 2 equivalent) was added. The mixture was stirred at 55 °C for 3 hours. After filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by rapid column chromatography to obtain the target alkanolamine product C7 (24 mg, 70% yield).

[0089] Example 15: Compound C7 (22 mg, 1.0 equivalent) and compound B3 (1.05 equivalent) were dissolved in n-butanol and refluxed at 110 °C for 12 hours with stirring. The reaction solution was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the crude product was purified by rapid column chromatography to obtain the target C8 product (69 mg, 84% yield).

[0090] Example 16: Compound C8 (69 mg, 1.0 equivalent) was dissolved in 1 mL of tetrahydrofuran (THF) solvent, and 150 μL of 1M tetrabutylammonium fluoride (TBAF) solvent was added. The mixture was stirred at room temperature for 12 hours. After the reaction was completed, the solvent was removed by vacuum distillation, and the crude product was purified by rapid column chromatography to obtain the target product. Z -io 6 Product A (C9) (24 mg, 80% yield).

[0091] The mass spectrum of the C9 product is as follows: Figure 15 As shown, the 1H NMR spectrum is as follows Figure 16 As shown.

[0092] Example 17: Referring to the operation of Example 8, only [the following is taken] E -io 6 Nucleoside A was replaced with the one prepared in Example 16. Z -io 6 A nucleoside.

[0093] compound Z -io 6 Results of MCF-7 cell viability assay for A, such as Figure 17 As shown, it can be seen from this that Z -io 6 The effect of A on the survival rate of MCF-7 cells showed a concentration-dependent inhibitory effect.

[0094] compound Z -io 6 The results of the A549 cell viability assay for A, such as Figure 18 As shown, it can be seen from this that Z -io 6 A also showed a concentration-dependent inhibitory effect on the survival rate of A549 cells.

[0095] Example 18: Blood sample testing: RNA extracted from 1 μg of blood sample from a stroke patient was added to a 20 μl system of 20 mM sodium acetate, along with 1 U of nuclease P1 enzyme. The reaction was carried out at 42°C for 2 h. After the reaction, 1 μL of LFast Ap and 3 μL of 10xFast Ap Buffer were added, and the reaction was carried out at 37°C for 4 h. The reaction solution was then filtered through a 0.22 μm filter membrane, and the filtered sample solution was freeze-dried under vacuum until completely dry. Finally, the dried sample was redissolved in 500 μL of an acetonitrile-water mixture (acetonitrile to water volume ratio of 1:1) and sent to a liquid chromatography-mass spectrometry (LC-MS) instrument.

[0096] Standard sample testing: The A7 compound obtained in Example 7 was dissolved in 500 μL of acetonitrile-water mixed solution (the volume ratio of acetonitrile to water was 1:1), wherein the concentration of the A7 compound was 100 ppm; the resulting solution was sent to a liquid chromatography-mass spectrometry (LC-MS) instrument.

[0097] The results of the liquid chromatography-mass spectrometry (LC-MS) analysis of the above blood samples and the above standards are as follows: Figure 19 As shown, both have a detection peak at position 2.91 (corresponding to the same elution time); further, by expanding the detection peak at position 2.91 in the blood sample, its mass spectrometry data ([(C 15 H 21 N5O5)+H] + :352.1551) and Figure 9 The mass spectrometry data of product A7 shown ([(C 15 H 21 N5O5)+H] + The values ​​of 352.1619 are almost identical, which indicates that... E -io 6 A may be related to diseases such as cerebral infarction.

[0098] Comparative Example 1: This comparative experiment investigated whether compound A5, which does not contain the DMTr group, could be directly synthesized from compound A3. Specifically: Compound A3 (20 mg, 1.0 equivalent) was dissolved in ethanol (EtOH), and 85% hydrazine hydrate (N2H4·H2O, 2 equivalent) was added. The mixture was stirred at 55 °C for 3 hours. After filtration, the filtrate was concentrated under reduced pressure. Thin-layer chromatography showed that the product spots were mixed, and the target product was not obtained after purification by column chromatography.

[0099] For Z-type io 6 The synthesis of A involved various reducing agents (including lithium aluminum hydride, borane, etc.). A systematic screening was conducted on substances such as dimethyl sulfide, but the results were not ideal. Specifically: Comparative Example 2 Compound C3 (20 mg, 1.0 equivalent) was dissolved in tetrahydrofuran, and lithium aluminum hydride (LiAlH4, 2 equivalent, commercially available, CAS No.: 16853-85-3) was slowly added at 0°C. The reaction was carried out at room temperature for 8 hours. Thin-layer chromatography was used to detect the target product during the reaction.

[0100] Comparative Example 3 Compound C3 (20 mg) was dissolved in tetrahydrofuran, and borane-dimethyl sulfide (BH3-Me2S, 2 equivalents, commercially available, CAS No.: 13292-87-0) was slowly added at -20°C. The reaction was carried out at room temperature for 4 hours. During the reaction, thin-layer chromatography showed that the starting material gradually transformed and disappeared, but potassium permanganate analysis did not show any new compound spots, suggesting that the double bond of the starting compound C3 was reduced.

[0101] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A nucleoside E -Mode N 6 The method for synthesizing 1-hydroxyisopentene adenosine is characterized by, The synthesis route is as follows: 。 2. The synthesis method as described in claim 1, characterized in that, in, The synthetic route for compound B3 is as follows: 。 3. The synthesis method as described in claim 1, characterized in that, The synthesis method includes the following steps: (1) Compound A1 and compound B1 are dissolved together in N,N-dimethylformamide (DMF) solvent and stirred at 20°C to 50°C to generate compound A2; preferably, the stirring reaction is carried out at room temperature; (2) Dissolve compound A2 in dichloromethane (DCM), add salicylic acid, tert-butyl hydroperoxide (t-BuOOH) and selenium dioxide, and stir at 20℃~40℃ to generate compound A3; preferably, the stirring reaction is carried out at room temperature; (3) Dissolve compound A3 in pyridine, add 4,4'-bismethoxytriphenylmethyl chloride (DMTrCl), and stir the reaction at 10℃~40℃ to generate compound A4; preferably, the stirring reaction is carried out at room temperature; (4) Dissolve compound A4 in ethanol (EtOH), add hydrazine hydrate, and stir at 50℃~70℃ to generate compound A5; preferably, the stirring reaction is carried out at 55℃; (5) Dissolve compound A5 and compound B3 in n-butanol solvent and heat them under reflux at 90°C to 120°C with stirring to generate compound A6; preferably, the reaction is carried out at 110°C. (6) Dissolve compound A6 in tetrahydrofuran (THF) solvent, add tetrabutylammonium fluoride (TBAF) reagent, and stir the reaction at 0℃~50℃ to generate compound A7; preferably, the stirring reaction is carried out at room temperature.

4. The synthesis method as described in claim 3, characterized in that, In step (1), the equivalent ratio of compound A1 to compound B1 is 1:(1~1.2). In step (2), the equivalent ratio of compound A2, salicylic acid, tert-butyl hydroperoxide, and selenium dioxide is 1:(0.1~0.2):(2~4):(0.05~0.1). In step (3), the equivalent ratio of compound A3 to 4,4'-bismethoxytriphenylmethyl chloride is 1:(1~2). In step (4), the equivalent ratio of compound A4 to hydrazine hydrate is 1:(1~2). In step (5), the equivalent ratio of compound A5 to compound B3 is 1:(1~1.2). In step (6), the equivalent ratio of compound A6 to tetrabutylammonium fluoride is 1:(3~5).

5. The synthesis method as described in claim 2, characterized in that, The synthesis method of compound B3 is as follows: Compound B2 was dissolved in tetrahydrofuran, and imidazole and tert-butyldimethylchlorosilane (TBSCl) were added. The mixture was stirred at 0°C to 50°C to generate compound B3. Preferably, the stirring reaction was carried out at room temperature. More preferably, the equivalent ratio of compound B2, imidazole, and tert-butyldimethylchlorosilane is 1:(1~3):(1~3).

6. A nucleoside Z -Mode N 6 The method for synthesizing 1-hydroxyisopentene adenosine is characterized by, The synthesis route is as follows: 。 7. The synthesis method as described in claim 6, characterized in that, The synthesis method includes the following steps: S1: Dissolve compound C1 in ethanol, and slowly add an aqueous sodium hydroxide solution at 10℃~40℃ to react and generate compound C2; preferably, the reaction is carried out at room temperature; S2: Dissolve compound C2 in tetrahydrofuran, add imidazole and tert-butyldimethylchlorosilane (TBSCl), and stir the reaction at 10°C to 40°C to generate compound C3; preferably, the stirring reaction is carried out at room temperature. S3: Dissolve compound C3 in methanol, slowly add sulfuric acid, react at 40~70℃, wash the product with saturated brine after rotary drying, dissolve it in dichloromethane, slowly add diisopropylaluminum hydride (DIBAL-H), and react at -40℃~0℃ to generate compound C4. S4: Dissolve compound C4 in a dichloromethane solution, add triphenylphosphine and carbon tetrabromide, and react at 10°C to 40°C to generate compound C5; preferably, the reaction is carried out at room temperature; S5: Compound C5 and compound B1 are dissolved together in N,N-dimethylformamide (DMF) solvent and reacted by stirring at 10°C to 50°C to generate compound C6; preferably, the stirring reaction is carried out at room temperature; S6: Dissolve compound C6 in ethanol, add hydrazine hydrate, and stir at 50°C to 70°C to generate compound C7; preferably, the stirring is carried out at 55°C; S7: Compound C7 and compound B3 are dissolved in n-butanol solvent and heated under reflux at 90°C~120°C with stirring to generate compound C8; preferably, the reaction is carried out at 110°C. S8: Dissolve compound C8 in tetrahydrofuran (THF) solvent, add tetrabutylammonium fluoride (TBAF) solvent, and stir the reaction at 0℃~40℃ to generate compound C9; preferably, the stirring reaction is carried out at room temperature.

8. The synthesis method as described in claim 7, characterized in that, In step S1, the equivalent ratio of compound C1 to NaOH is 1:(2~10). In step S2, the equivalent ratio of compound C2: imidazole and tert-butyldimethylchlorosilane is 1:(1~3):(1~3); In step S3, the equivalence ratio of compound C3 to diisopropylaluminum hydride is 1:(2.5~4). In step S4, the equivalent ratio of compound C4, triphenylphosphine, and carbon tetrabromide is 1:(1~4):(1~2). In step S5, the equivalent ratio of compound C5 to compound B1 is 1:(1~1.2). In step S6, the equivalence ratio of compound C6 to hydrazine hydrate is 1:(1~2). In step S7, the equivalent ratio of compound C7 to compound B3 is 1:(1~1.2). In step S8, the equivalent ratio of compound C8 to tetrabutylammonium fluoride is 1:(3~6).

9. The synthesis method as described in claim 6, characterized in that, The synthetic route for compound B3 is as follows: 。 10. Nucleosides E -Mode N 6 -Hydroxyisopentene adenosine or nucleoside Z -Mode N 6 Application of 1,5-hydroxyisoprene adenosine in screening potential biomarkers for the treatment of cerebral infarction.

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