Alkylthioarylclavudine phosphoramide compounds and their applications

Abstract

The present invention provides an alkylthioaryl clavudine phosphoramide compound, as shown in formula (I). Compared with the prior art, the alkylthioaryl clavudine phosphoramide compound provided by the present invention retains a unique anti-HBV activity while reducing the systemic exposure of clavudine, eliminating possible toxic side effects, providing a new option for combined drug use, and may provide a new functional cure solution for HBV patients in the future.

Description

Technical Field

[0001] The invention belongs to the technical field of drug synthesis, and in particular relates to an alkylthioaryl clavudine phosphoramide compound and application thereof. Background Art

[0002] Chronic hepatitis B is an infectious disease caused by hepatitis B virus (HBV) infection. Clinical manifestations include pain in the liver area, fatigue, hepatosplenomegaly, etc. As HBV continues to replicate, liver fibrosis and cirrhosis may gradually form, and even liver cancer may be induced, endangering the patient's life. At present, curing hepatitis B is a common goal in China and even the world. Currently, clinical anti-HBV treatment drugs include nucleoside (acid) analogs (NAs) and polyethylene glycol interferon α (PEG-IFNα). Long-term use of nucleotide antiviral drugs will lead to a certain degree of drug resistance, promote viral gene mutation or deletion, and easily relapse once the drug is stopped. However, polyethylene glycol interferon can significantly improve the patient's liver function. The combination of the two can enhance the therapeutic effect, maximally inhibit HBV replication, reduce liver function damage, and delay the progression of chronic hepatitis B. However, neither can directly remove the covalently closed circular DNA (cccDNA) in the nucleus of liver cells, and HBV infection is difficult to be completely "cured". Despite this, nucleoside (acid) antiviral drugs are still the most promising drugs for the treatment of chronic hepatitis B.

[0003] Claviudine is a non-natural L-nucleoside thymidine analogue, a first-generation nucleotide active site polymerase inhibitor (ASPIN), and a liver tissue targeted drug with significant viral inhibition and liver biochemical improvement effects. It still has significant persistent effects 6 months after treatment (persistent effects are a crucial advantage, as most hepatitis B patients require long-term treatment), and has a significant effect on controlling the virus. Claviudine's mechanism of action is different from other nucleoside drugs. It is not a DNA chain terminator, but through a series of intracellular phosphorylation to produce triphosphate L-FMAU-TP, which binds to the active site, induces changes in the polymerase conformation, and prevents the smooth synthesis of the viral DNA chain. It is the only nucleotide that inhibits all stages of DNA synthesis by non-competitively distorting the active site of the polymerase. Its mechanism of action may complement other programs that are seeking functional cures for HBV.

[0004] However, since clavudine does not have the advantage of liver targeting, and it was found in previous published clinical studies that clavudine can cause reversible proximal skeletal muscle myopathy in subjects, it is no longer developed for the treatment of chronic hepatitis B. This lesion may be the result of mitochondrial dysfunction in patients. Abel De La Rosa et al. reported a new liver-targeted molecule ATI2173 in WO2017 / 223421, which aims to improve the pharmacokinetic characteristics of clavudine and reduce systemic exposure levels, thereby eliminating adverse reactions such as skeletal myopathy, but the ability of its active metabolites to enter liver tissue has not been significantly improved.

[0005] Therefore, developing a new generation of ASPIN with the same mechanism of action, higher activity and fewer toxic side effects is an urgent clinical need. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide an alkylthioaryl clavudine phosphoramide compound and its application, wherein the alkylthioaryl clavudine phosphoramide compound reduces the systemic exposure of clavudine while retaining the unique anti-HVB activity, thereby eliminating possible toxic and side effects; and compared with ATI-2173, the compound involved in the present invention has an unexpectedly higher bioconversion efficiency, and therefore has the advantages of low dosage and less toxic and side effects in clinical practice.

[0007] The present invention provides an alkylthioaryl clavudine phosphoramide compound, as shown in formula (I):

[0008]

[0009] wherein R1 to R3 are each independently selected from hydrogen, deuterium, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic group;

[0010] R4 is selected from substituted or unsubstituted alkyl;

[0011] A is selected from substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclic.

[0012] Preferably, the substituents in the substituted alkyl, substituted aryl, substituted cycloalkyl and substituted heterocyclic are independently selected from one or more of deuterium, halogen, C1-C10 alkoxy, C1-C10 deuterated alkoxy, C6-C20 aryl, C6-C20 deuterated aryl, cyano, carboxyl, ester, amide, amino and hydroxyl.

[0013] Preferably, the R1 to R3 are each independently selected from hydrogen, deuterium, substituted or unsubstituted C1 to C10 alkyl, substituted or unsubstituted C6 to C20 aryl, substituted or unsubstituted C3 to C20 cycloalkyl, substituted or unsubstituted C2 to C20 heterocyclic group;

[0014] R4 is selected from substituted or unsubstituted C1-C10 alkyl;

[0015] A is selected from a substituted or unsubstituted C6-C20 aryl group, a substituted or unsubstituted C3-C20 cycloalkyl group, and a substituted or unsubstituted C2-C20 heterocyclic group.

[0016] Preferably, the alkylthioarylclavudine phosphoramide compound has a structure shown in formula (II) and / or formula (III):

[0017]

[0018] Preferably, the alkylthioarylclavudine phosphoramide compound has a structure shown in formula (IV):

[0019]

[0020] Among them, the carbon atom with an * is a chiral carbon atom, and its configuration is R-configuration or S-configuration.

[0021] Preferably, the alkylthioarylclavudine phosphoramide compound has a structure shown in formula (V):

[0022]

[0023] Preferably, the alkylthioarylclavudine phosphoramide compound has a structure shown in formula (VI) or formula (VII):

[0024]

[0025] Preferably, the alkylthioarylclavudine phosphoramide compound has a structure shown in any one of formula (VI-1) to formula (VI-4) or formula (VII-1) to formula (VII-4):

[0026]

[0027] The present invention also provides a pharmaceutical composition, comprising one or more of the above-mentioned alkylthioarylclavudine phosphoramide compounds, their pharmaceutically acceptable salts, their hydrates, their solvates and their crystals.

[0028] The present invention also provides a use of one or more of the above-mentioned alkylthioaryl clavudine phosphoramide compounds, their pharmaceutically acceptable salts, their hydrates, their solvates and their crystals in the preparation of drugs for preventing and / or treating diseases caused by hepatitis viruses.

[0029] The present invention provides an alkylthioaryl clavudine phosphoramide compound, as shown in formula (I). Compared with the prior art, the alkylthioaryl clavudine phosphoramide compound provided by the present invention retains a unique anti-HBV activity while reducing the systemic exposure of clavudine, eliminating possible toxic side effects, providing a new option for combined drug use, and may provide a new functional cure solution for HBV patients in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a graph showing the change of Clevudine-TP content in Huh 7 cells over time after treatment with the alkylthioaryl clevudine phosphoramide compounds obtained in Examples 1 and 2 of the present invention. DETAILED DESCRIPTION

[0031] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0032] The present invention provides an alkylthioaryl clavudine phosphoramide compound, as shown in formula (I):

[0033]

[0034] wherein R1 to R3 are each independently hydrogen, deuterium, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocyclic group; preferably hydrogen, deuterium, substituted or unsubstituted C1 to C10 alkyl, substituted or unsubstituted C6 to C20 aryl, substituted or unsubstituted C3 to C20 cycloalkyl, or substituted or unsubstituted C2 to C20 heterocyclic group; more preferably hydrogen, deuterium, substituted or unsubstituted C1 to C8 alkyl, substituted or unsubstituted C6 to C15 aryl, substituted or Unsubstituted C3~C15 cycloalkyl, substituted or unsubstituted C2~C15 heterocyclic group; more preferably, it is hydrogen, deuterium, substituted or unsubstituted C1~C5 alkyl, substituted or unsubstituted C6~C10 aryl, substituted or unsubstituted C3~C10 cycloalkyl, substituted or unsubstituted C2~C10 heterocyclic group; most preferably, it is hydrogen, deuterium, substituted or unsubstituted C1~C3 alkyl, substituted or unsubstituted C6~C8 aryl, substituted or unsubstituted C3~C8 cycloalkyl, substituted or unsubstituted C2~C8 heterocyclic group.

[0035] R4 is a substituted or unsubstituted alkyl group, preferably a substituted or unsubstituted C1-C10 alkyl group, more preferably a substituted or unsubstituted C1-C8 alkyl group, further preferably a substituted or unsubstituted C1-C5 alkyl group, further preferably a substituted or unsubstituted C1-C3 alkyl group, and most preferably a substituted or unsubstituted C1-C2 alkyl group.

[0036] A is a substituted or unsubstituted aryl, a substituted or unsubstituted cycloalkyl, or a substituted or unsubstituted heterocyclic group; preferably, it is a substituted or unsubstituted C6-C20 aryl, a substituted or unsubstituted C3-C20 cycloalkyl, or a substituted or unsubstituted C2-C20 heterocyclic group; more preferably, it is a substituted or unsubstituted C6-C15 aryl, a substituted or unsubstituted C3-C15 cycloalkyl, or a substituted or unsubstituted C2-C15 heterocyclic group; further preferably, it is a substituted or unsubstituted C6-C10 aryl, a substituted or unsubstituted C3-C10 cycloalkyl, or a substituted or unsubstituted C2-C10 heterocyclic group; most preferably, it is a substituted or unsubstituted C6-C8 aryl, a substituted or unsubstituted C3-C8 cycloalkyl, or a substituted or unsubstituted C2-C8 heterocyclic group.

[0037] In the present invention, the heteroatom in the heterocyclic group may be any heteroatom known to those skilled in the art without any particular limitation. In the present invention, the heteroatom is preferably one or more of an oxygen atom, a sulfur atom and a nitrogen atom.

[0038] In the present invention, the substituents in the substituted alkyl, substituted aryl, substituted cycloalkyl and substituted heterocyclic group are independently one or more of deuterium, halogen, C1-C10 alkoxy, C1-C10 deuterated alkoxy, C6-C20 aryl, C6-C20 deuterated aryl, cyano, carboxyl, ester, amide, amino and hydroxyl; more preferably, one or more of deuterium, halogen, C1-C8 alkoxy, C1-C8 deuterated alkoxy, C6-C15 aryl, C6-C15 deuterated aryl, cyano, carboxyl, ester, amide, amino and hydroxyl; more preferably, one or more of deuterium, halogen, C1-C6 alkoxy , one or more of C1-C6 deuterated alkoxy, C6-C10 aryl, C6-C10 deuterated aryl, cyano, carboxyl, ester, amide, amino and hydroxyl; more preferably, it is one or more of deuterium, halogen, C1-C4 alkoxy, C1-C4 deuterated alkoxy, C6-C10 aryl, C6-C10 deuterated aryl, cyano, carboxyl, ester, amide, amino and hydroxyl; most preferably, it is one or more of deuterium, halogen, C1-C2 alkoxy, C1-C2 deuterated alkoxy, C6-C10 aryl, C6-C10 deuterated aryl, cyano, carboxyl, ester, amide, amino and hydroxyl.

[0039] The alkylthioarylclavudine phosphoramide compounds provided by the present invention may be chiral, wherein the chiral atom may be a phosphorus atom or a carbon atom.

[0040] When the chiral atom is a phosphorus atom, the alkylthioarylclavudine phosphoramide compound has a structure shown in formula (II) and / or formula (III); that is, the phosphorus atom is a chiral atom, and its configuration can be R-configuration, S-configuration or a mixture of the two.

[0041]

[0042] Wherein, R1 to R3, R4 and A are the same as described above and will not be described in detail here.

[0043] When the chiral atom is a carbon atom, the alkylthioarylclavudine phosphoramide compound has a structure shown in formula (IV):

[0044]

[0045] Among them, the carbon atom with an * is a chiral carbon atom, and its configuration is R-configuration or S-configuration, or a mixture of the two.

[0046] According to the present invention, it is further preferred that the alkylthioarylclavudine phosphoramide compound has a structure shown in formula (V):

[0047]

[0048] According to the present invention, it is further preferred that the alkylthioarylclavudine phosphoramide compound has a structure shown in formula (VI) or formula (VII):

[0049]

[0050] According to the present invention, it is further preferred that the alkylthioarylclavudine phosphoramide compound has a structure shown in any one of formula (VI-1) to formula (VI-4) or formula (VII-1) to formula (VII-4):

[0051]

[0052] The alkylthioarylclavudine phosphamide compounds provided by the present invention are typical ProTide (McGuigan) prodrugs, and the design principle thereof is to connect nucleoside phosphonic acid / phosphate drugs with polar groups through phosphoester bonds / phosphoamide bonds (aryl modules / amino acid ester modules) to form phosphoester / phosphoamide prodrugs, respectively, and to reduce the polarity of the molecule and increase the membrane permeability by masking the polar groups. After the prodrug is absorbed into the body, it is hydrolyzed by a specific enzyme to release the prototype drug.

[0053] The alkylthioaryl clavudine phosphoramide compounds provided by the present invention retain unique anti-HBV activity while reducing the systemic exposure of clavudine and eliminating possible toxic side effects, thereby providing a new option for combined drug use and potentially providing a new functional cure solution for HBV patients in the future.

[0054] The present invention also provides a method for preparing the alkylthioaryl clavudine phosphoramide compound, comprising: reacting the compound represented by formula (A) with clavudine to obtain the alkylthioaryl clavudine phosphoramide compound.

[0055]

[0056] wherein R1 to R3 are each independently selected from hydrogen, deuterium, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclic group;

[0057] R4 is selected from substituted or unsubstituted alkyl;

[0058] A is selected from substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclic.

[0059] The reaction is preferably carried out in the presence of a catalyst; the catalyst is preferably tert-butyl magnesium chloride; the reaction is preferably carried out in an organic solvent; the organic solvent is preferably a mixed solvent of tetrahydrofuran and N-methylpyrrolidone; the volume ratio of tetrahydrofuran to N-methylpyrrolidone is preferably 10:(1-5), more preferably 10:(2-4), and more preferably 10:3; in the present invention, it is preferred that clavudine, an organic solvent and a catalyst are first mixed, heated and stirred, and then the compound represented by formula (A) is added to react to obtain an alkylthioaryl. Cyclavudine phosphoramide compounds; the compound represented by the formula (A) is added in the form of a solution thereof; the solvent of the solution is preferably tetrahydrofuran; the temperature of the heating and stirring is preferably 40°C to 60°C, more preferably 45°C to 55°C, and more preferably 50°C; the time of the heating and stirring is preferably 20 to 50 min, and more preferably 30 to 40 min; the temperature of the reaction is preferably 20°C to 30°C, and more preferably 25°C; the time of the reaction is preferably 10 to 15 h, more preferably 10 to 14 h, and more preferably 12 to 13 h.

[0060] After the reaction is completed, the reaction solution is preferably poured into a saturated ammonium chloride solution, extracted with ethyl acetate, and the organic phase is washed with a saturated sodium chloride solution, dried, and concentrated to obtain an alkylthioaryl clavudine phosphoramide compound; the concentrated substance can also be separated by high performance liquid chromatography to obtain an alkylthioaryl clavudine phosphoramide compound; the stationary phase of the high performance liquid chromatography is preferably a C18 chromatographic column, more preferably a Welch Xtimate C18; the mobile phase of the HPLC comprises a mobile phase A and a mobile phase B; the mobile phase A is preferably ammonia water; the mobile phase B is preferably acetonitrile; in terms of volume percentage, the elution procedure of the HPLC is preferably that the mobile phase B is increased from 20% to 30% to 50% to 60% within 15 to 25 minutes, more preferably that the mobile phase B is increased from 20% to 30% to 50% to 60% within 20 minutes, and more preferably that the mobile phase B is increased from 25% to 30% to 55% to 60% within 20 minutes; furthermore, chiral separation is preferably performed after HPLC separation; the stationary phase preferably used for chiral separation is DAICEL CHIRALPAK AD; the mobile phase used for the chiral separation includes mobile phase A and mobile phase B; the mobile phase A during the chiral separation is preferably 0.1% ammonia water; the mobile phase B is preferably ethanol and / or isopropylamine; the elution procedure for the chiral separation is preferably isocratic elution; in terms of volume percentage, the concentration of the mobile phase B is preferably 40% to 60%, more preferably 45% to 55%, and even more preferably 50%.

[0061] In the present invention, the compound represented by formula (A) is preferably prepared according to the following steps: reacting the compound represented by formula (B) with the compound represented by formula (C) and / or its salt compound in the presence of an organic base to obtain the compound represented by formula (A).

[0062]

[0063] The salt compound of the compound represented by formula (C) is preferably its hydrochloride; the organic base is preferably triethylamine; the reaction is preferably carried out in an organic solvent; the organic solvent is preferably dichloromethane; the reaction temperature is preferably -5°C to 5°C, more preferably 0°C; the reaction time is preferably 1 to 2 hours; after the reaction is completed, the mixture is concentrated, ethyl acetate solvent is added, filtered, concentrated, and purified by column chromatography to obtain the compound represented by formula (A); the eluent for column chromatography purification is preferably petroleum ether and ethyl acetate; the volume ratio of petroleum ether to ethyl acetate is preferably 50:1 to 1:1, more preferably 20:1 to 1:1, more preferably 10:1 to 1:1, more preferably 6:1 to 2:1, and most preferably 4:1; the stationary phase for column chromatography purification is preferably silica gel; the particle size of the stationary phase is preferably 200 to 300 meshes.

[0064] The compound represented by formula (B) is preferably prepared according to the following steps: 4-nitrobenzene phosphorus dichloride is reacted with a compound represented by formula (D) in the presence of an organic base to obtain the compound represented by formula (B).

[0065]

[0066] Among them, the organic base is preferably triethylamine; the reaction is preferably carried out in an organic solvent; the organic solvent is preferably dichloromethane; in the present invention, it is preferred to first dissolve 4-nitrobenzene phosphorus dichloride in an organic solvent, cool to -30°C to -25°C, then dropwise add an organic solution containing the compound represented by formula (D) and the organic base, and after the dropwise addition is completed, heat to 20°C to 30°C, more preferably 25°C, and react to obtain the compound represented by formula (B); the reaction time is preferably 30 to 90 min, more preferably 50 to 80 min, and more preferably 60 min.

[0067] The present invention also provides a pharmaceutical composition, comprising one or more of the above-mentioned alkylthioarylclavudine phosphoramide compounds, their pharmaceutically acceptable salts, their hydrates, their solvates and their crystals.

[0068] The present invention also provides the use of one or more of the above-mentioned alkylthioaryl clavudine phosphoramide compounds, their pharmaceutically acceptable salts, their hydrates, their solvates and their crystals in the preparation of drugs for preventing and / or treating diseases caused by hepatitis viruses.

[0069] In the present invention, the hepatitis virus is preferably hepatitis B virus.

[0070] To further illustrate the present invention, an alkylthioaryl clavudine phosphoramide compound and its application provided by the present invention are described in detail below in conjunction with examples.

[0071] The reagents used in the following examples are all commercially available.

[0072] Example 1

[0073]

[0074] Synthesis of intermediate 3

[0075] Dissolve 4-nitrobenzene phosphorus dichloride (5.00 g) in dichloromethane (10.0 mL), cool to -30°C to -25°C, control the temperature and dropwise add a solution of compound 1A (3.01 g) and triethylamine (2.17 g, 2.99 mL) in dichloromethane (10.0 mL), raise the temperature to 25°C and stir for 1 hour, dropwise add a solution of L-alanine isopropyl ester hydrochloride (3.93 g) in dichloromethane (10.0 mL) and triethylamine (4.15 g, 5.71 mL) at 0°C, and then keep the temperature at 0°C for 1 hour. LCMS (RT = 1.06 min) shows that the raw material is completely consumed. The solution is concentrated and ethyl acetate (20.0 mL) is added to dissolve and filter, the filter cake is rinsed with ethyl acetate (20.0 mL) and concentrated. The residue was purified by column chromatography (200-300 mesh silica gel, petroleum ether / ethyl acetate = 4 / 1) to give white solid compound 3 (4.80 g, yield 54.0%).

[0076] LCMS (ESI) m / z: 455.1 [M+H]+

[0077] Synthesis of 4A and 4B

[0078] Weigh clavudine (570 mg) and dissolve it in a mixed solvent of tetrahydrofuran (10.0 mL) and N-methylpyrrolidone (3.00 mL). Add tert-butylmagnesium chloride (1.7 M, 2.58 mL) dropwise to the above system, stir at 50 ° C for 30 minutes, add compound 3A (1.99 g) in tetrahydrofuran (10.0 mL), stir at 25 ° C for 12 hours. LCMS (RT = 1.656 min) shows that the raw material is completely consumed. The reaction mixture is poured into a saturated ammonium chloride solution (15.0 mL) and extracted with ethyl acetate (15.0 mL, 10.0 mL). The organic phase is washed with a saturated sodium chloride solution (10.0 mL), dried over sodium sulfate, and concentrated under reduced pressure. The crude product was purified by HPLC (chromatographic column: Welch Xtimate C18 250*70mm#10um; mobile phase: [0.1% ammonia-acetonitrile]; B%: 25%-55%, 20min) and chiral separation (chromatographic column: DAICEL CHIRALPAK AD (250mm*30mm, 10μm); mobile phase: [0.1% ammonia ethanol]; B%: 50%-50%, 11min) to obtain white solid compounds 4A (59.4mg, purity 100%) and 4B (127mg, purity 100%).

[0079] LCMS (ESI) m / z: 576.2 [M+H]+;

[0080] The obtained compound 4A was analyzed by nuclear magnetic resonance, and its nuclear magnetic resonance hydrogen spectrum result was obtained as follows: 1 H NMR(400MHz,MeOD)δppm 1.23-1.27(m,6H),1.36-1.42(m,3H),1.84(d,J=1.20Hz,3H),2.47(s,3H),3.88-3.98(m,1H),4.10-4.15(m,1H),4.28-4. 42(m,3H),4.95(br,dd,J=3.60,2.12Hz,1H),4.98-5.02(m,1H),6.18-6.29(m,1H),7.18-7.31(m,4H),7.49-7.54(m,1H).

[0081] The obtained compound 4B was analyzed by nuclear magnetic resonance, and its nuclear magnetic resonance hydrogen spectrum result was obtained as follows: 1H NMR(400MHz,MeOD)δppm 1.22-1.27(m,6H)1.32-1.38(m,3H)1.82-1.87(m,3H)2.46-2.51(m,3H)3.88-3.98(m,1H)4.09-4.16(m,1H)4.34-4.4 7(m,3H)4.94-4.97(m,1H)4.97-5.02(m,1H)6.19-6.29(m,1H)7.16-7.23(m,2H)7.26-7.34(m,2H)7.54-7.61(m,1H).

[0082] Example 2

[0083]

[0084] Synthesis of intermediate 6

[0085] 4-Mercaptophenol (12.6 g) and iodoethane (15.6 g.) were added to acetone (100 ml), potassium carbonate (11.7 g.) was added. After stirring at room temperature for 17 hours, the mixture was filtered through diatomaceous earth and the filtrate was concentrated under reduced pressure. The obtained residue was dissolved in Et2O (100 ml), then washed with 2M NaOH (2x60 ml). The combined aqueous layer was acidified with 1M HCl and extracted with Et2O (2x100 ml). The combined organic layer was dried over anhydrous Na2SO4, concentrated, and the desired product was separated by column chromatography (CyH / EtOAc 5:1) as a white crystalline solid (11.55 g, yield 75%).

[0086] Synthesis of intermediate 8

[0087] Dissolve 4-nitrobenzene phosphorus dichloride (5.00 g) in dichloromethane (10.0 mL), cool to -30 ℃ ~ -25 ℃, control the temperature and drop compound 6 (3.31 g) and triethylamine (2.96 g, 4.08 mL) in dichloromethane (10.0 mL), stir for 1 hour after adding, drop L-alanine isopropyl ester hydrochloride (3.93 g) in dichloromethane (10.0 mL) and triethylamine (4.15 g, 5.71 mL) at 0 ℃, and keep at 0 ℃ for 1 hour. LCMS (RT = 1.103 min) shows that the raw material is completely consumed. The solution is concentrated and ethyl acetate (20.0 mL) is added to dissolve and filter, the filter cake is rinsed with ethyl acetate (20.0 mL) and concentrated. The residue was purified by column chromatography (silica, petroleum ether / ethyl acetate = 50 / 1-1 / 1) to give white solid compound 8 (4.80 g, yield 52.4%).

[0088] LCMS (ESI) m / z: 469.2 [M+H]+ .

[0089] Synthesis of 9A & 9B

[0090] Weigh clavudine (555 mg) and dissolve it in a mixed solvent of tetrahydrofuran (10.0 mL) and N-methylpyrrolidone (3.00 mL). Add tert-butylmagnesium chloride (1.7 M, 2.51 mL) dropwise to the above system, stir at 50 ° C for 30 minutes, add compound 8 (2.00 g) in tetrahydrofuran (10.0 mL), stir at 25 ° C for 12 hours. LCMS (RT = 1.77 min) shows that the raw material is completely consumed. The reaction mixture is poured into a saturated ammonium chloride solution (15.0 mL) and extracted with ethyl acetate (15.0 mL, 10.0 mL). The organic phase is washed with a saturated sodium chloride solution (10.0 mL), dried over sodium sulfate, and concentrated under reduced pressure. The crude product was purified by HPLC (chromatographic column: Welch Xtimate C18 250*70mm#10μm; mobile phase: [0.1% ammonia-acetonitrile]; B%: 30%-60%, 20min) and chiral separation (chromatographic column: DAICEL CHIRALPAK AD (250mm*30mm, 10μm); mobile phase: [0.1% ammonia isopropylamine]; B%: 50%-50%, 11min) to give white solid compounds 9A (60.2mg, purity 98.9%) and 9B (70.6mg, purity 100%).

[0091] LCMS (ESI) m / z: 590.2 [M+H]+;

[0092] The obtained compound 9A was analyzed by nuclear magnetic resonance, and its nuclear magnetic resonance hydrogen spectrum result was obtained as follows: 1 H NMR(400MHz,MeOD)δppm 1.16-1.25(m,6H),1.25-1.29(m,3H),1.36-1.42(m,3H),1.79-1.87(m,3H),2.89-2.97(m,2H),3.90-3.96(m,1H),4.10-4.15(m,1H), 4.28-4.43(m,3H),4.94-4.97(m,1H),4.98-5.03(m,1H),6.19-6.27(m,1H),7.20-7.25(m,2H),7.31-7.36(m,2H),7.50-7.54(m,1H).

[0093] The obtained compound 9B was analyzed by nuclear magnetic resonance, and its nuclear magnetic resonance hydrogen spectrum result was obtained as follows: 1H NMR(400MHz,MeOD)δppm 1.18-1.25(m,6H),1.27(t,J=7.20Hz,3H),1.33-1.37(m,3H),1.79-1.89 (m,3H),2.86-2.97(m,2H),3.86-3.98(m,1H),4.11-4.20(m,1H),4.34-4 .51(m,3H),4.95-4.96(m,1H),4.97-5.02(m,1H),5.08-5.10(m,1H),6.1 7-6.28(m,1H),7.17-7.24(m,2H),7.33-7.38(m,2H),7.54-7.62(m,1H).

[0094]

[0095] As can be seen from the above reaction process, clavudine produces active clavudine 5'-triphosphate molecules through a series of intracellular phosphorylation to exert its efficacy. The prodrug design can bypass the first phosphorylation step and deliver clavudine monophosphate to the liver, where it is further converted into active clavudine triphosphate. Quantitative analysis of the concentration of clavudine triphosphate converted into active product in cells after the compound is incubated in Huh7 cells can indirectly reflect the biological activity of the compound.

[0096] On the first day, Huh7 cells were plated into 12-well plates at a density of 0.5*10^6 cells / well and cultured overnight in a cell culture incubator at 5% CO2 and 37°C. On the second day, the compound was converted into a 20mM stock solution based on the reference molecular weight, and further diluted to a 20μM working concentration and added to Huh7 cells. Three replicate wells were set for each compound, and a total of 8 cell plates were plated with 7 compounds. Cells were collected at 4 different time points (1h, 3h, 6h, 24h) for determination of the intracellular concentration of clavudine triphosphate. Specifically, the cells were rinsed with DPBS and the residual liquid in the wells was aspirated. 800μL of internal standard solution (6in 1internal standard in 1) was added to each tube of cell samples, calibration standards and quality control samples. MeOH / Water (v / v, 70:30). After the mixture was thoroughly vortexed, it was centrifuged at 12000xg at 4°C for 15 minutes. 70 μL of the supernatant was placed in a 96-well plate and centrifuged at 4000 rpm at 4°C for 5 minutes. 10 μL was taken and the sample was analyzed by LC-MS / MS using an ACQUITY UPLC System with a BEH C18 column. The retention times of the compounds and internal standards, the acquisition of the chromatograms, and the integration of the chromatograms were processed by the software MultiQuant 3.0.2 Software (SCIEX, MA, USA).

[0097] The Huh 7 cells were treated with the test compound at a specified concentration, and then the cells were collected at different time points. The Clevudine-TP content in the Huh 7 cells after the compound treatment was quantitatively analyzed by ultra-high performance liquid chromatography triple quadrupole mass spectrometry. The results are shown in the figure below. Figure 1 As shown. Figure 1 The results showed that all the tested compounds were converted into clavudine triphosphate, and over time, the content of clavudine triphosphate in Huh 7 cells gradually increased, and the level was significantly better than that of ATI-2173.

[0098] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An alkylthioaryl clavudine phosphoramide compound, wherein the alkylthioaryl clavudine phosphoramide compound has a structure shown in any one of 4A, 4B, and 9A: 。 2. A pharmaceutical composition comprising one or more of the alkylthioaryl clavudine phosphoramide compounds of claim 1 and pharmaceutically acceptable salts thereof.

3. Use of the alkylthioaryl clavudine phosphamide compound and one or more of its pharmaceutically acceptable salts according to claim 1 in the preparation of a medicament for preventing and / or treating diseases caused by hepatitis B virus.

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