阅读说明：本技术 一种核苷类似物及其制备方法和应用 (Nucleoside analogue and preparation method and application thereof ) 是由 杨庆凯 王丽娜 王凯 孙震 宋成丽 于 2021-01-21 设计创作，主要内容包括：本发明涉及一种核苷类似物及其制备方法和应用,属于抗病毒药物技术领域。本发明所述核苷类似物为苄基((((2R,3S)-3-羟基-5-(6-(甲氨基)-9H-嘌呤-9-基)四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)-L-丙氨酸酯,结构式如式I所示。本发明所述核苷类似物生物利用度高,具有抗癌、抗病毒作用,还能够提高固有免疫能力。(The invention relates to a nucleoside analogue and a preparation method and application thereof, belonging to the technical field of antiviral drugs. The nucleoside analogue is benzyl ((((2R,3S) -3-hydroxy-5- (6- (methylamino) -9H-purine-9-yl) tetrahydrofuran-2-yl) methoxyl) (phenoxy) phosphoryl) -L-alanine ester, and the structural formula is shown in a formula I. The nucleoside analogue has high bioavailability, has anticancer and antiviral effects, and can improve the inherent immunity.)

1. A nucleoside analog having a structural formula shown in formula I:

2. a process for preparing a nucleoside analog according to claim 1, comprising the steps of:

mixing the dissolved L-alanine benzyl ester hydrochloride with triethylamine, carrying out neutralization reaction to obtain free L-alanine benzyl ester, mixing the free L-alanine benzyl ester with phenyl dichlorophosphate to carry out a first substitution reaction to obtain an intermediate product A, mixing the intermediate product A with p-nitrophenol and triethylamine to carry out a second substitution reaction to obtain a compound shown in a formula 1,

dissolving N6-methyl deoxyadenosine in a mixed solution of anhydrous tetrahydrofuran and N-methylpyrrolidone, adding tert-butyl magnesium chloride for activation reaction to obtain activated N6-methyl deoxyadenosine, mixing the activated N6-methyl deoxyadenosine with a compound shown in a formula 1, and carrying out a third substitution reaction to obtain the nucleoside analogue shown in a formula I.

3. The method of claim 2, wherein the first substitution reaction is carried out under the following conditions: reacting at-75 to-80 ℃ for 25 to 35min, and then reacting at 20 to 30 ℃ for 2.5 to 3.5 h.

4. The method of claim 2, wherein the second substitution reaction is carried out under the following conditions: reacting at 0 ℃ for 25-35 min, and then reacting at 20-30 ℃ for 4.5-5.5 h.

5. The method according to claim 2, wherein the temperature of the activation reaction is 20 to 25 ℃ and the time is 20 to 60 min.

6. The preparation method according to claim 2, wherein the temperature of the third substitution reaction is 50-60 ℃ and the time is 6-8 h.

7. Use of the nucleoside analogue as claimed in claim 1 or the nucleoside analogue prepared by the preparation method as claimed in any one of claims 2 to 6 in the preparation of an antiviral medicament.

8. The use of claim 7, wherein the virus comprises herpes simplex virus, hepatitis b virus, and human papilloma virus.

9. Use of the nucleoside analogue of claim 1 or the nucleoside analogue prepared by the preparation method of any one of claims 2 to 6 for the preparation of a medicament for enhancing innate immunity.

10. Use of the nucleoside analogue of claim 1 or the nucleoside analogue prepared by the preparation method of any one of claims 2 to 6 for the preparation of an anticancer drug.

Technical Field

The invention relates to the technical field of antiviral drugs, and particularly relates to a nucleoside analogue and a preparation method and application thereof.

Background

Viral infection-like diseases are abusive worldwide, and therefore, as a global public health problem, development of novel antiviral therapeutic drugs is particularly important. Among antiviral drugs currently on the market and clinically used, nucleosides account for more than half and are of great importance in antiviral therapy, such as viral infections including cancer and Herpes Simplex Virus (HSV), adenovirus (AdV), Human Cytomegalovirus (HCMV), Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), and Hepatitis C Virus (HCV). Many nucleoside analogs are inhibitors of enzymes involved in viral replication, inhibit the activity of viral DNA polymerase and reverse transcriptase, and participate in the competitive incorporation of nucleotides into the viral DNA strand, thereby terminating or inhibiting the elongation and synthesis of the viral DNA strand, allowing the virus to function under inhibition. However, the clinical application is greatly limited due to the defects that nucleoside drugs have high toxicity, short half-life period in vivo, easy induction of drug-resistant strains during application and the like. Therefore, the research and development of novel nucleoside analogues which are efficient, low in toxicity and not easy to generate drug resistance are of great significance. However, since the nucleoside molecules themselves are mostly polar molecules, which prevent them from passing the cell boundary by the paracellular route, their intestinal permeability is low and thus oral bioavailability is also poor.

Disclosure of Invention

The invention aims to provide a nucleoside analogue and a preparation method and application thereof. The nucleoside analogue has high bioavailability, has anticancer and antiviral effects, and can improve the inherent immunity.

The invention provides a nucleoside analogue, which has a structural formula shown as a formula I:

the invention also provides a preparation method of the nucleoside analogue in the technical scheme, which comprises the following steps:

mixing the dissolved L-alanine benzyl ester hydrochloride with triethylamine, carrying out neutralization reaction to obtain free L-alanine benzyl ester, mixing the free L-alanine benzyl ester with phenyl dichlorophosphate to carry out a first substitution reaction to obtain an intermediate product A, mixing the intermediate product A with p-nitrophenol and triethylamine to carry out a second substitution reaction to obtain a compound shown in a formula 1,

dissolving N6-methyl deoxyadenosine in a mixed solution of anhydrous tetrahydrofuran and N-methylpyrrolidone, adding tert-butyl magnesium chloride for activation reaction to obtain activated N6-methyl deoxyadenosine, mixing the activated N6-methyl deoxyadenosine with a compound shown in a formula 1, and carrying out substitution reaction to obtain the nucleoside analogue shown in a formula I.

Preferably, the conditions of the first substitution reaction are: reacting at-75 to-80 ℃ for 25 to 35min, and then reacting at 20 to 30 ℃ for 2.5 to 3.5 h.

Preferably, the conditions of the second substitution reaction are: reacting at 0 ℃ for 25-35 min, and then reacting at 20-30 ℃ for 4.5-5.5 h.

Preferably, the temperature of the activation reaction is 20-25 ℃ and the time is 20-60 min.

Preferably, the temperature of the third substitution reaction is 50-60 ℃ and the time is 6-8 h.

The invention also provides application of the nucleoside analogue in the technical scheme or the nucleoside analogue prepared by the preparation method in the technical scheme in preparation of antiviral drugs.

Preferably, the virus includes herpes simplex virus, hepatitis b virus and human papilloma virus.

The invention also provides application of the nucleoside analogue in the technical scheme or the nucleoside analogue prepared by the preparation method in the technical scheme in preparation of a medicine for improving innate immunity.

The invention also provides application of the nucleoside analogue in the technical scheme or the nucleoside analogue prepared by the preparation method in the technical scheme in preparing anticancer drugs.

The invention provides a nucleoside analogue. The nucleoside analogue has high bioavailability, has anticancer and antiviral effects, and can improve the inherent immunity. The nucleoside analogue has an action mechanism that active triphosphate is synthesized in vivo and inserted into a virus genome through DNA polymerase. Enhancing the perception of virus DNA through cGAS, and further enhancing the DNA immunostimulation capacity; the nucleoside analogue can be applied to the process of preparing inactivated vaccines, attenuated live vaccines and DNA vaccines and can be used for immunoprophylaxis by enhancing the immune response capability. The interferon is a protein which can evoke immune response in early stage of virus infection, and is taken as a common treatment method for inhibiting disease progression in early stage of disease due to the capacity of interfering virus replication, I-type Interferon (IFNB) walks on the front line of body for resisting virus invasion, the nucleoside analogue cannot evoke immune response by itself, but enters virus DNA through virus replication when the virus is infected, so that the modified virus DNA is sensed and enhanced by cGAS, and the generation of IFNB is induced through interferon gene stimulation protein (STING), therefore, the nucleoside analogue can improve the inherent immune capacity of human body by changing virus DNA modification, and provides a new direction for anti-virus treatment.

Drawings

FIG. 1 is a HNMR spectrum of Compound 1 provided by the present invention;

FIG. 2 is a mass spectrum peak diagram of Compound 1 provided by the present invention;

FIG. 3 is an H NMR spectrum of N6-methyldeoxyadenosine provided by the present invention;

FIG. 4 is a mass spectrum of N6-methyldeoxyadenosine according to the present invention;

FIG. 5 shows intracellular free N6-methyldeoxyadenosine triphosphate provided by the present invention;

FIG. 6 is a graph of the ratio of N6-methyldeoxyadenosine to deoxyadenosine within the genome of cells and viruses quantified as provided by the present invention;

FIG. 7 is a graph of the QRT-PCR method for detecting the changes of IFNB caused by infection of MEF cells with HSV-1 and insertion of HSV-1 modified by N6mdATP at mRNA level;

FIG. 8 is a graph of the QRT-PCR method for detecting the changes of IFNB caused by HSV-1 infection of MLF cells and HSV-1 inserted with N6mdATP modification at the mRNA level;

FIG. 9 is a graph of the QRT-PCR method for detecting the change of IFNB caused by infection of AdV by MEF cells and insertion of AdV modified by N6mdATP at mRNA level;

FIG. 10 is a graph of the QRT-PCR method for detecting the change of IFNB caused by AdV infection of MLF cells and insertion of N6mdATP modified AdV on mRNA level.

Detailed Description

The invention provides a nucleoside analogue, which has a structural formula shown as a formula I:

the name of the nucleoside analogue is as follows: benzyl ((((2R,3S) -3-hydroxy-5- (6- (methylamino) -9H-purin-9-yl) tetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) -L-alanine ester, english name: benzyl (((2R,3S) -3-hydroxy-5- (6- (methylamino) -9H-purin-9-yl) tetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) -L-alaninate (common name: QKY-613). Has a chemical formula of C₂₇H₃₁N₆O₇P, molecular weight 582.55. The nucleoside analogue is structurally different from natural nucleoside, and the aromatic group and amino acid ester are used for covering the oxygen atom of the phosphate group of the nucleoside analogue with phosphoric acid, so that the nucleoside analogue can improve the bioavailability, and the efficiency of synthesizing the active triphosphate metabolite after the nucleoside analogue enters a human body is obviously higher than that of nucleoside. After entering human body, the nucleoside analogue synthesizes active triphosphate metabolite, and can enter virus DNA through virus replication in the initial stage of virus infection to cause strong immune reaction so as to achieve the effect of resisting virus. In particular, pathogenic microorganisms are infected in vivoThe DNA of the substance stimulates the innate immune response, and cyclic GMP-AMP synthase (cGAS) activates STING by recognizing cytoplasmic DNA and signals, thereby inducing type I Interferon (IFNB) having antiviral and immunomodulatory activities. Mammalian dm6A level is very low (dm 6A/dA)<1×10^-6) Whereas most microorganisms (such as bacteria) have dm6A levels more than 10000 times higher. The fidelity of mammal DNA polymerase is very high, and the fidelity of DNA polymerase of pathogenic microorganism is very low, the nucleoside analogue of the invention synthesizes active triphosphate metabolite after entering into the body, and the nucleoside analogue is inserted into virus genome through DNA polymerase, but does not enter into human genome. The virus genome modified by the nucleoside analogue enhances the sensing capability of cGAS to the virus genome, thereby improving the inherent immunity of a human body. The nucleoside analogue can be applied to preparation of living vaccines and DNA vaccines for immunoprophylaxis.

The invention also provides a preparation method of the nucleoside analogue in the technical scheme, which comprises the following steps:

mixing the dissolved L-alanine benzyl ester hydrochloride (formula a) with triethylamine, carrying out neutralization reaction to obtain free L-alanine benzyl ester, mixing the free L-alanine benzyl ester with phenyl dichlorophosphate (b) to carry out a first substitution reaction to obtain an intermediate product A, mixing the intermediate product A with p-nitrophenol (c) and triethylamine, carrying out a second substitution reaction to obtain a compound shown in formula 1,

dissolving N6-methyl deoxyadenosine (formula 2) in a mixed solution of anhydrous tetrahydrofuran and N-methylpyrrolidone, adding tert-butyl magnesium chloride for activation reaction to obtain activated N6-methyl deoxyadenosine, mixing the activated N6-methyl deoxyadenosine with a compound shown in formula 1, and carrying out a third substitution reaction to obtain the nucleoside analogue shown in formula I.

The nucleoside analogue is prepared by reacting L-alanine benzyl ester hydrochloride, phenyl dichlorophosphate, p-nitrophenol and N6-methyl deoxyadenosine. The specific reaction formula is as follows:

the preparation process according to the invention is preferably carried out under a protective gas atmosphere, such as inert gas or nitrogen. When the protective gas is an inert gas, the inert gas is preferably argon.

The invention mixes the dissolved L-alanine benzyl ester hydrochloride (formula a) with triethylamine for neutralization reaction to obtain free L-alanine benzyl ester. The invention preferably uses dichloromethane as solvent to dissolve L-alanine benzyl ester hydrochloride, and the volume ratio of the L-alanine benzyl ester hydrochloride to the dichloromethane is preferably 0.1 mol: 150 to 220ml, more preferably 0.1 mol: 200 ml. In the present invention, the molar ratio of the mixture of the L-alanine benzyl ester hydrochloride and the triethylamine is preferably 100: (200-220), preferably 100:210, and reacting triethylamine with hydrochloric acid of L-alanine benzyl ester hydrochloride to obtain free L-alanine benzyl ester. The neutralization reaction condition is preferably normal temperature, more preferably 20-25 ℃, and the reaction time is preferably 10-20 min, more preferably 15 min. In the present embodiment, 21.568g L-alanine benzyl ester hydrochloride (0.1mol) is preferably dissolved in 200ml dichloromethane and 21.25g triethylamine (0.21mol) is added for neutralization reaction.

After obtaining free L-alanine benzyl ester, the invention mixes the free L-alanine benzyl ester with phenyl dichlorophosphate (b) to carry out a first substitution reaction, and obtains an intermediate product A. The free benzyl L-alanine is exchanged for one chlorine of phenyl dichloride phosphate to obtain an intermediate product A (benzyl (chloro (phenoxy) phosphoryl) -L-alanine, and the reaction is exothermic at the beginning, so the temperature is preferably reduced to-75 to-80 ℃ before the mixing, more preferably to 78 ℃, the invention preferably dropwise adds the phenyl dichloride phosphate into the free benzyl L-alanine, the molar ratio of free benzyl L-alanine to phenyl dichlorophosphate is preferably 1: (10-12), more preferably 1: 11. the conditions of the first substitution reaction of the invention are preferably-75 to-80 ℃ for 25-35 min, then 20-30 ℃ for 2.5-3.5 h, more preferably-78 ℃ for 30min, then 20-30 ℃ for 3 h.

After the intermediate product A is obtained, the intermediate product A is mixed with p-nitrophenol (c) and triethylamine to carry out a second substitution reaction, and the compound shown in the formula 1 is obtained. In the present invention, the amounts of the p-nitrophenol and triethylamine added are preferably 0.9 to 1.1 times the amount of the L-alanine benzyl ester hydrochloride, and more preferably the same as the amount of the L-alanine benzyl ester hydrochloride. The reaction is exothermic after the p-nitrophenol is added, so after mixing, the conditions of the second substitution reaction are preferably 0 ℃ for 25-35 min, then 20-30 ℃ for 4.5-5.5 h, more preferably 0 ℃ for 30min, then 20-30 ℃ for 5 h. The reaction conditions are set to ensure that the second reaction is carried out more completely, and the second substitution reaction can exchange p-nitrophenol for phenyl dichlorophosphate and the other chlorine to obtain the compound (colorless oily substance) shown in the formula 1. The invention preferably adopts a dot plate method to detect whether the second substitution reaction is finished. The dot plate of the invention is preferably petroleum ether: ethyl acetate system, petroleum ether: the volume ratio of ethyl acetate is preferably 3: 1. After the reaction is finished, the invention preferably carries out spin drying and column passing for separating products. The preferable condition of the spin-drying is 45-55 ℃, and the vacuum spin-drying is carried out in a water bath at 50 ℃. The conditions of the column passing of the invention are preferably SiO₂(silica gel column), the eluent is a mixed solvent of petroleum ether and ethyl acetate, and the ratio of petroleum ether: the volume ratio of ethyl acetate is 3:1, and other ratios can result in poor product separation.

The method comprises the steps of dissolving N6-methyl deoxyadenosine (formula 2) in a mixed solution of anhydrous tetrahydrofuran and N-methylpyrrolidone, and adding tert-butyl magnesium chloride for activation reaction to obtain activated N6-methyl deoxyadenosine. The source of the N6-methyldeoxyadenosine (formula 2) is not particularly limited in the invention, and the N6-methyldeoxyadenosine (formula 2) can be synthesized by using a conventional commercial product or a conventional synthetic method of N6-methyldeoxyadenosine (formula 2) (the N6-methyldeoxyadenosine is preferably obtained by self-synthesis in the invention, and the detailed synthetic method is specifically described below). In the present invention, the ratio of the amount of the substance of N6-methyldeoxyadenosine to the volume of anhydrous tetrahydrofuran and the volume of N-methylpyrrolidone is preferably 18.85mmol (90-150) ml: (30-50) ml, more preferably 18.85mmol:120 ml: 40 ml. In the present invention, the tert-butyl magnesium chloride is preferably dissolved in anhydrous tetrahydrofuran before mixing, and the concentration of the tert-butyl magnesium chloride in the anhydrous tetrahydrofuran is preferably 1M. In the invention, the temperature of the activation reaction is preferably 20-25 ℃, and the time is preferably 20-60 min, and more preferably 20 min. In the present invention, the activation reaction is preferably carried out under stirring.

After the compound shown in the formula 1 and the activated N6-methyl deoxyadenosine are obtained, the activated N6-methyl deoxyadenosine is mixed with the compound shown in the formula 1 to carry out a third substitution reaction, and the nucleoside analogue shown in the formula I is obtained. In the present invention, the compound represented by the formula 1 is preferably dissolved in anhydrous tetrahydrofuran before being mixed with N6-methyldeoxyadenosine. In the invention, the molar ratio of the N6-methyl deoxyadenosine to the compound shown in the formula 1 is preferably (18-19): (37-38), and more preferably 18.85: 37.7. In the invention, the temperature of the third substitution reaction is preferably 50-60 ℃, and the time is preferably 6-8 h; more preferably at 55 ℃ for 7h to complete the reaction. After the third substitution reaction is finished, the temperature is preferably reduced to room temperature (20-30 ℃), the reaction liquid is poured into a 10% ammonia chloride aqueous solution, and alkaline substances in the reaction liquid are neutralized. After the alkaline substances are neutralized, the extraction is preferably performed, and the number of times of extraction is preferably 2-4, more preferably 3. The extraction of the invention preferably uses dichloromethane or chloroform, after the extraction, the dichloromethane layer or the chloroform layer is combined, preferably dried by anhydrous sodium sulfate, and is dried on a column by spinning, so that the compound shown in the formula I (white solid) is obtained. The preferable condition of the spin-drying is 45-55 ℃, and the vacuum spin-drying is carried out in a water bath at 50 ℃. The conditions of the upper column of the present invention are preferably SiO₂(silica gel column), the eluent is preferably a mixed solvent of Dichloromethane (DCM) and methanol (MeOH), and the volume ratio of DCM to MeOH is preferably (98:2) - (95:5), and more preferably 95: 5.

In the present invention, the method for synthesizing N6-methyldeoxyadenosine (formula 2) preferably comprises the steps of:

dissolving methyl tosylate in a solvent DMF, mixing with 2' -deoxyadenosine, and stirring at room temperature; adding diatomaceous earth, filtering, mixing the filtrate with acetone, stirring at room temperature, filtering to obtain solid, washing with acetone, and vacuum drying; dissolving the obtained solid in an aqueous solution of NaOH or KOH, stirring at room temperature, neutralizing with an aqueous solution of 8-12% by mass of p-toluenesulfonic acid, and spin-drying to obtain a (N6-methyl deoxyadenosine) white solid.

The invention dissolves methyl tosylate in a solvent DMF, mixes with 2 '-deoxyadenosine, stirs at room temperature, methyl tosylate can provide methyl, and the nitrogen at the 1 position of the 2' -deoxyadenosine is changed into methyl quaternary ammonium salt. In the present invention, the mixing molar ratio of the methyl tosylate and the 2' -deoxyadenosine is preferably (3-5): 1, and more preferably 4: 1. In the invention, the room-temperature stirring condition is preferably 20-30 ℃ for 8-12 h.

After stirring overnight, the invention adds diatomaceous earth, filters, mixes the filtrate with acetone, stirs at room temperature, filters to get solid, washes with acetone, dries in vacuum. In the present invention, the mass ratio of the amount of the substance of methyl p-toluenesulfonate to diatomaceous earth is preferably 0.1 mol: (18-22) g, more preferably 0.1 mol: 20 g. Diatomaceous earth helps to filter out solid impurities in the reaction solution, and acetone can precipitate the product out of solution. In the present invention, when the amount of methyl p-toluenesulfonate is 0.1mol, the amount of acetone added to the filtrate is preferably 800 to 950ml, and more preferably 900 ml. In the invention, the room-temperature stirring condition is preferably 20-30 ℃ for 1 h.

After vacuum drying, dissolving the solid obtained by the method in an aqueous solution of NaOH or KOH, stirring at room temperature, then neutralizing with an 8-12% by mass aqueous solution of p-toluenesulfonic acid, and spin-drying to obtain N6-methyl deoxyadenosine (white solid). In the present invention, the concentration of the aqueous NaOH or KOH solution is preferably 2M. In the present invention, the molar ratio of the solid to NaOH or KOH is preferably 1: (15-20). In the present invention, the conditions for stirring at room temperature are preferablyStirring for 1h at 20-30 ℃. In the present invention, the mass percentage of the p-toluenesulfonic acid in the aqueous solution of the p-toluenesulfonic acid is preferably 10%. The preferable condition of the spin-drying is 45-55 ℃, and the vacuum spin-drying is carried out in a water bath at 50 ℃. In the present invention, the condition of the upper column is preferably SiO₂，DCM:MeOH＝(10～20):1。

The invention also provides application of the nucleoside analogue in the technical scheme or the nucleoside analogue prepared by the preparation method in the technical scheme in preparation of antiviral drugs.

In the present invention, the virus preferably includes herpes simplex virus, adenovirus, hepatitis b virus and human papilloma virus.

The invention also provides application of the nucleoside analogue in the technical scheme or the nucleoside analogue prepared by the preparation method in the technical scheme in preparation of a medicine for improving innate immunity. The nucleoside analogue enters into the body and synthesizes active triphosphate metabolites (N6-Methyl-dATP, N6mdATP), the fidelity of the virus nuclease is low, and the fidelity of mammalian DNA polymerase is higher, so that when a human body is infected with virus, N6mdATP can be incorporated into the virus genome by the virus DNA polymerase and hardly enters into the human genome. Since the presence of DNA in the cytoplasm is often a hallmark of infection by pathogenic microorganisms, it can be rapidly detected by cyclic GMP-AMP synthase (cGAS), thereby eliciting an anti-infectious immune response. Therefore, the virus modified by the N6mdATP can cause stronger immune response. The nucleoside analogue can enhance the perception of DNA through cGAS, thereby enhancing the immunostimulatory capacity of the DNA.

The invention also provides application of the nucleoside analogue in the technical scheme or the nucleoside analogue prepared by the preparation method in the technical scheme in preparing anticancer drugs.

The nucleoside analog, the preparation method and the application thereof according to the present invention will be described in further detail with reference to the following specific examples, and the technical solutions of the present invention include, but are not limited to, the following examples.

Example 1

The synthesis of the nucleoside analogues of the present invention is carried out according to the following reaction scheme:

1) a first step of synthesizing a compound 1 (i.e., a compound represented by formula 1):

21.568g L-alanine benzyl ester hydrochloride (0.1mol, 1eq) was dissolved in 200ml of dichloromethane under argon protection, 21.25g of triethylamine (0.21mol, 2.1eq) was added, the temperature was reduced to-78 ℃, 23.2g of phenyl dichlorophosphate (0.11mol, 1.1eq) was added dropwise, and the mixture was kept at-78 ℃ for 30min after the addition. The temperature is raised to room temperature for reaction for 3 h. Then cooled to 0 ℃ and 13.91g of p-nitrophenol (0.1mol, 1eq) and 10.12g of triethylamine (0.1mol, 1eq) were added. After the addition, the temperature is kept for 30min, and then the reaction is carried out for 5h after the temperature is raised to room temperature, and plates (petroleum ether: ethyl acetate) are spotted. After the reaction is finished, the solution is directly spin-dried and passes through a column (SiO)₂Petroleum ether: ethyl acetate 3:1) to give 23g of a colorless oil in 50.4% yield.

The H NMR spectrum of Compound 1 is shown in FIG. 1. As can be seen from FIG. 1, the 1H NMR spectrum of Compound 1 gives 5 sets of peaks with an integration ratio (from low field to high field) of 2:12:2:2:3, giving a total of 21 hydrogen protons, corresponding to the number of hydrogen protons in the structure shown in formula 1.

The mass spectrum results of compound 1 are shown in fig. 2. The conclusion that can be drawn from the mass spectrometry results of fig. 2 is that: compound 1 has a mass to charge ratio of 457.1 and a mass spectrum adopting a positive mode, so that the precise molecular weight is 456.11.

2) The second step of synthesis of N6-methyldeoxyadenosine (dm6A) (shown in formula 2)

Under argon, 18.62g of methyl p-toluenesulfonate (0.1mol, 4eq) were dissolved in 60ml of DMF, and 6.28g of 2' -deoxyadenosine (0.025mol, 1eq) were added. Inverse directionThe reaction was stirred at room temperature overnight. 20g of diatomaceous earth was added and filtered. 900ml of acetone is added to the filtrate, stirred at room temperature for 1 hour until a solid is produced, and filtered. The solid was washed with acetone and dried in vacuo. The resulting solid was dissolved in 150ml of 2M NaOH and stirred at room temperature for 1 h. The reaction solution was neutralized with 10% aqueous solution of p-toluenesulfonic acid, and the resulting mixture was applied to a column (SiO)₂DCM: MeOH ═ 10:1) gave 0.8g of white solid in 12.1% yield. 3) The third step obtains N6-methyl deoxyadenosine.

The H NMR spectrum of N6-methyldeoxyadenosine is shown in FIG. 3. The 1H NMR spectrum of N6-methyldeoxyadenosine gave 13 sets of peaks with an integration ratio (low to high field) of 1:1:1:1:1:1:1:1:1:1:3:1:1, for a total of 15 hydrogen protons, consistent with the number of hydrogen protons for the structure shown in formula 2. The mass spectrum result of N6-methyldeoxyadenosine is shown in FIG. 4. According to FIG. 4, the mass-to-charge ratio of N6-methyldeoxyadenosine mass spectrum is 266.1, the mass spectrum adopts a positive mode, and therefore the accurate molecular weight is 265.12.

3) The third step is to obtain the final product

5g of N6-methyldeoxyadenosine (18.85mmol, 1eq) were dissolved in a mixture of 120ml of anhydrous tetrahydrofuran and 40ml of N-methylpyrrolidone under argon protection, and 37.7ml of tert-butylmagnesium chloride (1M inTHF, 37.7mmol, 2eq) were added at room temperature. After stirring for 20min, a solution of 17.2g of Compound 1(37.7mmol, 2eq) dissolved in 50ml of anhydrous tetrahydrofuran was added, followed by warming to 55 ℃ for 7 h. After the reaction is finished, the temperature is reduced to room temperature, the reaction solution is poured into 10% ammonium chloride aqueous solution, dichloromethane is added for extraction for three times, dichloromethane layers are combined, anhydrous sodium sulfate is used for drying, and the mixture is applied to a column in a spin-drying mode (SiO)₂DCM: MeOH: 95:5) gave 0.67g of white solid in 6.1% yield.

Example 2

Examples of enhancing bioavailability

0.1mM of N6-methyl deoxyadenosine (dm6A) and the nucleoside analogues QKY-613 of the invention were added to Hela cells, DMSO was added to the control (Ctrl), PBS buffer was added after 12 hours to wash three times, 100. mu.l of an extraction reagent for cell metabolites (methanol, acetonitrile and water in a volume ratio of 40: 40: 20) was added to every 1X 10^7 cells, freeze-thawing was repeated with liquid nitrogen at 37 ℃, and then centrifugation was carried out at 1,3000rpm/min for 15min, and the supernatant was taken to obtain cell metabolites. And (3) quantitatively detecting N6-Methyl-dATP in the extracted metabolite by using liquid phase tandem mass spectrometry. The results show that the cell availability of the newly synthesized drug QKY-613 is significantly higher than dm6A, as shown in table 1 and fig. 5, and table 1 and fig. 5 show the content of the provided intracellular free N6-methyldeoxyadenosine triphosphate (N6-Methyl-dATP).

TABLE 1 intracellular free N6-methyldeoxyadenosine triphosphate (N6-Methyl-dATP) content under different conditions

Number of repetitions	Ctrl	dm6A	QKY-613
				1 st time	0	101	1187
2 nd time	0	81	1002
				3 rd time	0	44	852

Example 3

Because of the fidelity difference between viral DNA polymerase and mammalian DNA polymerase, supplementation with nucleoside QKY-613 drugs provided by the present invention results in 10000 times more N6mdATP entering viral DNA than mammalian host DNA. The verification process is as follows: as shown in Table 2 and FIG. 6, FIG. 6 is a graph showing the results of the ratios of N6-methyldeoxyadenosine (dm6A) and deoxyadenosine (dA) in the genomes of cells and viruses, wherein the ordinate represents the ratio of dm6A to dA in the respective genomes after the drug enters human cells, Vero cells and viruses from QKY-613, and the abscissa represents the ratio of human cells (including human embryonic kidney cell HEK293, cervical cancer cell Hela, monocyte macrophage THP-1), Vero cells and viruses (AdV and HSV-1, specifically, HEK293 cells infected with AdV virus and Vero cells infected with HSV-1). In the specific process, 0.1mM QKY-613 is added into HEK293, Hela, THP-1 and Vero cells, and the contrast is DMSO; the cells replicated with AdV and HSV-1 were HEK293 and Vero cells respectively, so the appropriate AdV virus was added to HEK293 cells and HSV-1 virus was added to Vero cells, while 0.1mM QKY-613 was added to HEK293 and Vero cells containing the virus, respectively, in DMSO control. After 48h, the genome was extracted using a commercial kit, QPCR standardizes viral and host cell genomes. Taking 1 microgram genome respectively, adding 2U Nuclease (Nuclease P) and 0.2U dephosphorizing enzyme (CIAP) for enzymolysis for 12h at 37 ℃. The ratio of the adenovirus AdV to the herpes simplex virus (HSV-1) genome dm6A/dA is found to be remarkably higher than that of human cells and vero cells by 10000 times by virtue of liquid phase tandem mass spectrometry, namely, QKY-613 supplementation is shown to cause that N6mdATP enters viral DNA to be remarkably higher than that of the viral DNA entering mammalian host DNA.

Table 2 QKY-613 supplements the ratio of N6-methyldeoxyadenosine and deoxyadenosine that results in quantification of DNA following entry of N6mdATP into viral and mammalian DNA

Example 4

The antiviral ability of the nucleoside analogues of the present invention is further illustrated by herpes simplex virus (HSV-1) and adenovirus (AdV) experiments.

The experimental method comprises the following steps: the test group nucleoside analogues (QKY-613) are respectively added into HEK293 cells replicated by HSV-1 virus replication cells Vero and AdV at a final concentration of 0.1 mM; deoxyadenosine (dA) is used as a control, and viruses are harvested after 48h, wherein N6mdATP in a test group enters the genome of the test group through virus replication to obtain HSV-1 modified by inserting N6mdATP and AdV modified by inserting N6 mdATP; HSV-1 and AdV were obtained for the control group. The virus titer was determined by dilution by fold and Mouse Embryonic Fibroblasts (MEF) and Mouse Lung Fibroblasts (MLF) were infected at different MOI (Mock represents 0 multiplicity of infection, MOI: 1 represents 1 multiplicity of infection, MOI: 10 represents 10 multiplicity of infection). The immunostimulatory capacity (IFNB) caused by viral infection was determined by fluorescent quantitative PCR. The results are shown in fig. 7-fig. 10 (corresponding to tables 3-6, respectively), and fig. 7 (table 3) is a graph of changes of IFNB at mRNA level caused by QRT-PCR detection of MEF cell infection HSV-1 and insertion of N6mdATP modified HSV-1 provided by the present invention; FIG. 8 (Table 4) is a graph of the changes of IFNB on mRNA level caused by QRT-PCR detection of MLF cell infection with HSV-1 and insertion of N6mdATP modified HSV-1 provided by the present invention; FIG. 9 (Table 5) is a graph of changes at mRNA level of IFNB by QRT-PCR detection of MEF cell infection with AdV and insertion of N6mdATP modified AdV; FIG. 10 (Table 6) is a graph of QRT-PCR detection of the changes at mRNA level of IFNB by AdV infected with MLF cells and AdV modified with inserted N6 mdATP. From FIGS. 7 to 10, it is clear that N6mdATP entering the viral genome can cause a strong immune response and IFNb at the mRNA level can be increased by at least 10 to 100 times as compared to the normal virus.

TABLE 3 QRT-PCR assay for IFNB-induced changes at mRNA level caused by MEF cell infection with HSV-1 and insertion of N6 mdATP-modified HSV-1

TABLE 4 QRT-PCR detection of IFNB-induced changes at mRNA level by MLF cell infection with HSV-1 and HSV-1 modified by insertion of N6mdATP

TABLE 5 QRT-PCR assay for IFNB-induced changes at mRNA level caused by infection of MEF cells with AdV and insertion of N6 mdATP-modified AdV

TABLE 6QRT-PCR detection of IFNB-induced changes at mRNA level by AdV infected MLF cells and AdV modified by insertion of N6mdATP

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.