Antiviral drug and use thereof

Abstract

The present invention discloses an antiviral drug and its use. The present invention provides a compound as shown in formula I or a pharmaceutically acceptable salt thereof. The compound as shown in formula I provided by the present invention has excellent antiviral activity, high stability in acidic, neutral and human plasma, low toxicity and high conversion efficiency.

Description

Technical Field

[0001] The invention relates to an antiviral drug and application thereof. Background Art

[0002] Nucleotide antivirals can inhibit viral polymerases by directly and competitively binding to natural deoxyribose substrates, causing termination of viral DNA chain synthesis. Among the known nucleotides for viral inhibition, tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor with a phosphonate group and can be used as a single drug or a component in combination with other drugs for the treatment of HIV or HBV infection. However, because the phosphonate group is a divalent anion at physiological pH, TFV has poor cell membrane permeability and low oral bioavailability (<10%), which limits its therapeutic effect.

[0003] Many efforts have been made to develop anti-HBV and HIV drugs for clinical application. For example, tenofovir disoproxil fumarate (TDF) is the first prodrug of tadalafil developed by Gilead Sciences Inc. However, TDF has poor stability in physiological pH and plasma. Therefore, it must be administered at high doses by oral administration in order to produce sufficient concentrations of TFV in target tissues and achieve its therapeutic effect. As a result, due to the instability of TFV, high concentrations of TFV are also formed in plasma, causing dose-dependent renal and bone toxicity. In addition, the prodrug also releases two molecules of potentially toxic formaldehyde when metabolized. Tenofovir alafenamide (TAF, also known as GS-7340) is Gilead's second prodrug of tadalafil, which has improved plasma stability, efficacy and safety over TDF. However, TAF contains phenol as a good leaving group, so the stability of TAF in physiological pH and plasma is still not good. In addition, the synthesis and manufacture of TAF are very difficult, and TAF tablets must be packaged in thick-walled bottles with a large amount of desiccant. The insufficient stability of TDF and TAF at physiological pH and in plasma makes these prodrugs unsuitable as components of long-acting intramuscular injection formulations for the treatment of HIV and HBV infections. Other prodrugs of tenofovir containing phenol or similar molecules with good leaving ability, such as HS-10234 with a methyl group added to the alanine moiety of TAF, have the same problem.

[0004] WO2022271880A1, CN103842369A and WO2012048013A2 disclose compounds with structures similar to tenofovir prodrugs, but the compounds provided in WO2022271880A1 are mainly used for the treatment of cancer and do not involve the research of antiviral drugs, and cannot solve the technical problem of poor stability of anti-HBV and HIV drugs;

[0005] The compounds provided by CN103842369A and WO2012048013A2 are mainly used to treat hepatitis C virus infection (HCV virus) and do not involve research on the effects of anti-HBV and anti-HIV drugs. The nucleosides of anti-HCV drugs are completely different from those of anti-HBV and anti-HIV drugs, and they also cannot solve the technical problem of poor stability of anti-HBV and anti-HIV drugs.

[0006] Therefore, there is still a need in the art to further develop anti-HBV and HIV drugs with further improved stability, efficacy and toxicity. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide an anti-HBV and HIV drug with good stability, efficacy and low toxicity in view of the deficiencies of the prior art. The present invention provides an antiviral drug and its use. The compound drug provided by the present invention has excellent antiviral activity, high stability in acidic, neutral and human plasma, low toxicity and high conversion efficiency.

[0008] The present invention solves the above technical problems through the following technical solutions.

[0009] The present invention provides a compound as shown in Formula I or a pharmaceutically acceptable salt thereof,

[0010] ;

[0011] The phosphorus atoms marked with "*" are of S configuration and / or R configuration.

[0012] In some embodiments, the compound as shown in Formula I is any of the following compounds:

[0013] and .

[0014] The present invention also provides a pharmaceutical composition, which comprises the compound as shown in Formula I or a pharmaceutically acceptable salt thereof according to any embodiment of the present invention, and one or more pharmaceutically acceptable excipients.

[0015] In some embodiments, the pharmaceutical composition further comprises an additional agent selected from an antiviral agent, an immunotherapeutic agent, and an anti-infective agent.

[0016] The present invention also provides a use of a compound as shown in Formula I or a pharmaceutically acceptable salt thereof according to any embodiment of the present invention or a pharmaceutical composition according to any embodiment of the present invention in the preparation of a drug for preventing and / or treating viral infection; the virus may be HBV virus or HIV virus.

[0017] The compounds of the present invention may be formulated as or in the form of pharmaceutically acceptable salts. Unless specified to the contrary, the compounds provided herein include pharmaceutically acceptable salts of such compounds.

[0018] The term "pharmaceutically acceptable" means relatively non-toxic, safe, and suitable for use by patients.

[0019] The term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable acid or base. When the compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. When the compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. For details, please refer to Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P.Heinrich Stahl, Camille G. Wermuth, 2011, 2nd Revised Edition).

[0020] The term "pharmaceutically acceptable excipients" refers to all substances contained in pharmaceutical preparations other than active pharmaceutical ingredients, which are generally divided into two categories: excipients and additives. For details, please refer to the Pharmacopoeia of the People's Republic of China (2020 Edition) and Handbook of Pharmaceutical Excipients (Paul J Sheskey, Bruno C Hancock, GaryP Moss, David J Goldfarb, 2020, 9th Edition).

[0021] The term "treating" refers to removing the cause or alleviating the symptoms.

[0022] The term "prevention" refers to reducing the risk of developing a disease.

[0023] In one aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, which is useful as a TFV prodrug.

[0024] In some embodiments, the compounds of the present invention may be converted to TFV after administration. In some embodiments, the compounds of the present invention may be converted to TFV after oral administration. In some embodiments, the compounds of the present invention may be converted to TFV after parenteral administration. In some embodiments, the compounds of the present invention may be converted to TFV in target cells. In some embodiments, the compounds of the present invention may be converted to TFV in human hepatocytes and lymphocytes.

[0025] For a prodrug, it may be desirable for the prodrug to remain intact (i.e., uncleaved) while in the systemic circulation, but to be cleaved (i.e., releasing the parent drug) in the target tissue. Alternatively, it may be desirable for the prodrug to remain intact (i.e., uncleaved) while in the gastrointestinal (GI) tract, but to be cleaved (i.e., releasing the parent drug) after absorption or uptake from the GI lumen (e.g., in the enterocytes lining the GI lumen or in the blood). The useful level of stability may be determined, at least in part, by the mechanism and pharmacokinetics of the prodrug.

[0026] In some embodiments, the compounds provided herein are stable in plasma. In certain embodiments, after incubation of the compounds provided herein with plasma at 40°C for 6 days, less than about 2 mol% of the compounds are converted to TFV.

[0027] In some embodiments, the compounds of the invention exhibit stability in plasma for no less than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or 25 hours.

[0028] In some embodiments, provided herein are compounds that are stable under neutral conditions. In certain embodiments, after incubation of provided herein compounds in a phosphate buffer at pH 7.0 for 30 days at 40°C, as measured in an assay described in the following examples, more than about 90 mole % of the compound is retained.

[0029] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0030] The reagents and raw materials used in the present invention are commercially available.

[0031] The positive and progressive effects of the present invention are:

[0032] (1) The compound shown in Formula I provided by the present invention has excellent in vitro antiviral activity against HBV and HIV and has low toxicity.

[0033] (2) The compound of formula I provided by the present invention has good stability in acidic, human plasma and neutral conditions;

[0034] (3) The compound of formula I provided by the present invention has excellent conversion efficiency in PBMC or Hep G2 cells. DETAILED DESCRIPTION

[0035] For illustrative purposes, the following examples are included. However, it should be understood that these examples do not limit the present invention and are intended only to illustrate methods of practicing the present invention. Those skilled in the art will recognize that the chemical reactions described may be easily applicable to the preparation of a variety of other compounds of the present invention, and alternative methods for preparing the compounds of the present invention are considered to be within the scope of the present invention. For example, non-exemplary compounds according to the present invention may be successfully synthesized by modifications apparent to those skilled in the art, such as by appropriately protecting interfering groups, by utilizing other suitable reagents and building blocks known in the art in addition to the reagents and building blocks, and / or by routinely changing reaction conditions. Alternatively, other reactions disclosed herein or known in the art will be considered to be applicable to the preparation of other compounds of the present invention.

[0036] It should be understood that the chiral center in the compound can exist in the "S" or "R" stereoconfiguration, or as a mixture of the two. In the molecule, each bond drawn straight from the chiral center includes both (R) and (S) stereoisomers and mixtures thereof. The compounds of the examples provided herein contain a phosphorus chiral center. The isomer mixture in each of the examples in the following examples section was separated by HPLC to obtain Isomer #A and Isomer #B. The structural formulas of these two are represented by the corresponding phosphorus atoms marked with * in the corresponding mixture structural formula. The absolute stereochemistry (R or S) of the phosphorus chiral center in each of the "A" and "B" isomers in the following examples section has not been determined.

[0037] LCMS conditions:

[0038] Instrument: Shimadzu equipped with LC20AD pump and PDA detector; Column: Restek Ultra C18 (50 mm × 2.1 mm × 5.0 μm); Mobile phase: 0.1% HCOOH in H2O / ACN; Gradient: ACN from 5% to 95% in 0.1 min to 2.5 min, ACN from 95% to 98% in 2.5 min to 3.0 min, ACN from 95% to 98% in 3.0 min to 4.0 min, ACN from 98% to 5% in 4.0 min to 4.15 min, ACN from 5% to 4.55 min; Column temperature: 25 o C; detection wavelength: 214 / 254 nm; flow rate: 1.0 mL / min; injection volume: 5 μL.

[0039] HPLC analysis conditions:

[0040] Instrument: Agilent 1200 system with DAD detector; Column: Kinetex 5u EVO C18 100Å (150 mm × 4.6 mm); Mobile phase: 0.1% TFA in H2O / ACN; Gradient: ACN from 5% to 95% in 0.1 min to 7.5 min, ACN from 95% to 5% in 7.5 min to 9.5 min, ACN from 95% to 5% in 9.5 min to 10 min; Column temperature: 25 o C; detection wavelength: 214 / 254 nm; flow rate: 1.0 mL / min; injection volume: 5 μL.

[0041] Chiral HPLC analysis conditions:

[0042] Instrument: Agilent 1200 system with DAD; Column: Daicel AD-H (250 mm × 4.6 mm × 5.0 μm); Mobile phase: Hexane: IPA: MeOH = 80:15:5, 0.1% DEA; Column temperature: 35 o C; detection wavelength: 214 / 254 nm; flow rate: 1.0 mL / min; running time: 30 min.

[0043] Preparative HPLC (normal phase) conditions:

[0044] Instrument: Varian PREP-HPLC system; Column: Uni Silica (250 mm × 21.2 × 10 μm, 120 Å); Mobile phase and gradient: DCM:MeOH = 97:3 to 90:10 in 20 min; Column temperature: 25 o C; detection wavelength: 214 / 254 nm; flow rate: 20 mL / min; injection volume: 0.5 mL (sample concentration 50 mg / mL).

[0045] Preparative HPLC (reverse phase) conditions:

[0046] Method A: Instrument: Shimadzu LC8AP PREP-HPLC system; Column: Boston ODS (250 mm × 21.2 mm × 10 μm, 120 Å); Mobile phase: 0.1% TFA in H2O / ACN; Gradient: ACN 5% from 1 min to 5 min, ACN from 5% to 25% from 5 min to 20 min, ACN 25% from 20 min to 25 min; Column temperature: 25 o C; detection wavelength: 214 / 254 nm; flow rate: 20 mL / min; injection volume: 1 mL (sample concentration 50 mg / mL).

[0047] Method B: Instrument: Shimadzu LC8AP PREP-HPLC system; Column: Boston ODS (250 mm × 21.2 mm × 10 μm, 120 Å); Mobile phase: 0.1% TFA in H2O / ACN; Gradient: 5% ACN from 1 min to 5 min, 10% to 30% ACN from 5 min to 20 min, 25% ACN from 20 min to 25 min; Column temperature: 25 o C; detection wavelength: 214 / 254 nm; flow rate: 20 mL / min; injection volume: 1 mL (sample concentration 50 mg / mL).

[0048] Method C: Instrument: Shimadzu LC8AP PREP-HPLC system; Column: Boston ODS (250 mm × 21.2 mm × 10 μm, 120 Å); Mobile phase: 0.1% TFA in H2O / ACN; Gradient: 5% ACN from 1 min to 5 min, 20% to 50% ACN from 5 min to 20 min, 25% ACN from 20 min to 25 min; Column temperature: 25 o C; detection wavelength: 214 / 254 nm; flow rate: 20 mL / min; injection volume: 1 mL (sample concentration 50 mg / mL).

[0049] Chiral preparative HPLC conditions:

[0050] Instrument: Shimadzu LC8AP PREP-HPLC system; Column: Daicel AD-H Semi-Prep (250 mm × 10.0 mm × 5.0 μm); Mobile phase: Hexane: IPA: MeOH = 80:15:5, 0.1% DEA; Column temperature: 35 o C; detection wavelength: 214 / 254 nm; flow rate: 5.0 mL / min; running time: 15 min.

[0051] Example 1: Synthesis of intermediates

[0052] Synthesis of intermediate s1:

[0053]

[0054] In N2 and 0 oTo a mixture of (R)-(((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphonic acid (200 mg, 0.7 mmol, 1.0 equiv) in ACN (10 mL) was added SOCl2 (828.7 mg, 7.0 mmol, 10 equiv) at 4 °C. The resulting mixture was heated to reflux for 4 h. The mixture was then cooled to room temperature and concentrated under reduced pressure to give crude s1 (226 mg, yellow solid) as a yellow solid, which was used in the next step without further purification.

[0055] Example 2 Synthesis of Compounds

[0056] Comparison of compounds 1A and 1B:

[0057]

[0058] Cool down to 0 under N2 o To a solution of dipropylamine (1.39 g, 13.75 mmol, 5.0 equiv.) of C, (S)-isopropyl 2-aminopropanoate hydrochloride (462 mg, 2.75 mmol, 1.0 equiv.) and DIPEA (1.77 g, 13.75 mmol, 5.0 equiv.) in DCM (10 mL) was slowly added a solution of crude s1 in anhydrous DCM (10 mL), and then the mixture was stirred at room temperature for 30 minutes. The reaction mixture was quenched with H2O (50 mL) and extracted with DCM (60 mL×3), and the combined organic layers were washed with brine (60 mL×3), dried over Na2SO4, filtered and concentrated to dryness. The residue was purified by flash column chromatography on silica gel (DCM / MeOH=93:7) to give 1 (225 mg, 13% yield) as a light yellow solid, which was separated by chiral HPLC to give 1A (first eluting, 118.8 mg, 52% yield) as a white solid and 1B (latter eluting, 67.4 mg, 30% yield) as a white solid.

[0059] 1A:

[0060] LCMS: Rt = 2.6 min; m / z= 484 [M+1] + ;

[0061] 1H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 7.95 (s, 1H), 5.86 (brs, 2H), 5.08 – 4.97 (m, 1H), 4.30 (dd, J = 14.3, 3.0 Hz, 1H), 4.18 – 4.08 (m, 1H),3.98 – 3.83 (m, 2H), 3.80 – 3.66 (m, 1H), 3.47 – 3.36 (m, 1H), 3.36 – 3.26(m, 1H), 2.84 – 2.65 (m, 4H), 1.42 – 1.31 (m, 7H), 1.25 – 1.20 (m, 9H), 0.78– 0.70 (m, 6H).

[0062] 1B:

[0063] LCMS: Rt = 2.6 min; m / z= 484 [M+1] + ;

[0064] 1 H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 8.01 (s, 1H), 5.80 (brs, 2H), 5.00 – 4.91 (m, 1H), 4.39 (dd, J = 14.3, 3.2 Hz, 1H), 4.23 – 4.16 (m, 1H), 4.15-3.96 (m, 2H), 3.85 – 3.76 (m, 1H), 3.52 – 3.42 (m, 1H), 3.14 (t, J =10.0 Hz, 1H), 2.89-2.85 (m, 4H), 1.51 – 1.40 (m, 4H), 1.33 – 1.29 (d, J = 7.1Hz, 3H), 1.22 – 1.15 (m, 9H), 0.84 – 0.79 (m, 6H).

[0065] The remaining compounds were prepared by methods similar to the above methods via the corresponding intermediates.

[0066] Compounds 2A and 2B: The dipropylamine in the synthesis method of comparative compounds 1A and 1B was replaced with diisoamylamine (5.0 equivalents) and prepared with s1

[0067] 2A or 2B

[0068] 2B or 2A

[0069] 2A:

[0070] LCMS: Rt = 3.2 min; m / z = 540 [M+1] + ;

[0071] 1 H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 7.96 (s, 1H), 5.78 (s, 2H), 5.07 – 4.98 (m, 1H), 4.31 (dd, J = 14.5, 2.5 Hz, 1H), 4.13 (dd, J = 15.0, 7.1 Hz, 1H), 3.97 – 3.83 (m, 2H), 3.73 (dd, J = 12.6, 9.7 Hz, 1H), 3.43 (dd, J = 12.9, 8.5 Hz, 1H), 3.25 (t, J = 11.3 Hz, 1H), 2.87 – 2.73 (m, 3H), 1.41 (dd, J = 13.9, 7.3 Hz, 4H), 1.29 – 1.19 (m, 14H), 0.82 (dd, J = 6.4, 3.3 Hz, 12H).

[0072] 2B:

[0073] LCMS: Rt = 3.3 min; m / z = 540 [M+1] + ;

[0074] 1H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 8.02 (s, 1H), 5.74 (s, 2H), 4.95 (s, 1H), 4.41 (d, J = 14.7 Hz, 1H), 4.18 (dd, J = 14.9, 6.7 Hz, 1H),4.00 – 3.87 (m, 2H), 3.81 (dd, J = 12.9, 7.1 Hz, 1H), 3.45 (t, J = 11.6 Hz,1H), 3.12 (t, J = 8.1 Hz, 1H), 3.01-2.84 (m, 10.5 Hz, 4H), 1.54-1.42 (m, 2H), 1.36-1.27 (m, 7H), 1.22 – 1.15 (m, 9H), 0.88-0.84 (m, 12H).

[0075] Example 3: Antiviral Experiment

[0076] In vitro antiviral activity against HBV

[0077] In vitro anti-HBV activity and cytotoxicity were evaluated using HepG2.2.15 cells (source: ATCC) as described below.

[0078] Antiviral activity assay: HepG2.2.15 cells were cultured at 1×10 5 Cells were seeded at a density of 10 cells / well in 96-well plates and maintained at 37°C / 5% CO2. After 24 hours of incubation, different concentrations of test compounds and positive controls (including GS-7340 (TAF) and HS-10234) and control samples without inhibitors (DMSO only) were added. Test compounds were evaluated by an 8-point serial dilution method, with 3-fold dilutions at each point. After 72 hours of incubation, the original culture medium was replaced with fresh culture medium containing the same test compounds / positive controls at the same concentrations and cultured for another 3 days. After 6 days, the supernatant of the cell culture medium was collected and the HBV DNA content was determined by quantitative polymerase chain reaction (qPCR) assay. The inhibition % was calculated by the following equation: Inhibition (%) = 100-(DNA replication of test sample) / (DNA replication of DMSO) × 100. EC 50 The results were determined by inhibition curve (Graphpad Prism was used to fit the data using Log(inhibitor) vs. response-Variable slope (four parameters) curve fitting), and are shown in Table 1.

[0079] Cytotoxicity assay: Cytotoxicity was evaluated using a luminescent cell viability assay. HepG2.2.15 cells were seeded using the same method as the antiviral activity assay and incubated in the presence of test compounds / positive controls. After 6 days of incubation, Cell-Titer Glo (Promega) was added to each well and luminescence was recorded for cell viability assay. Cell viability % was calculated by the following equation:

[0080] Cell viability (%) = (luminescence of test sample - average luminescence of culture medium) / (average luminescence of control sample - average luminescence of culture medium) × 100. 50 The results were determined from the viability curve (Graphpad Prism was used to fit the data using Log(inhibitor) vs. response-Variable slope (four parameters) curve fitting), and are shown in Table 1.

[0081] Table 1 In vitro anti-HBV activity and cytotoxicity

[0082]

[0083] EC 50 :A:<100 nM; B:100-1000nM; C:>1000nM.

[0084] In vitro antiviral activity against HIV

[0085] In vitro anti-HIV activity and cytotoxicity were evaluated using MT-4 cells (source: ATCC) as described below.

[0086] Antiviral activity assay: MT4 cells were incubated with HIV-1 IIIB at 37°C / 5% CO2 for 1 hour. 4 Cells were seeded at a density of 10 cells / well in a 96-well plate. Test compounds and positive controls (GS-7340) were added in an 8-point serial dilution format (with 3-fold dilutions at each point), as well as virus control samples without inhibitors (DMSO only) and uninfected cell control samples. After incubation for 5 to 6 days at 37°C / 5% CO2, virus-induced cytopathic effect was determined by luminescent cell viability assay using Cell-TiterGlo (Promega). Inhibition % was calculated by the following equation: Inhibition (%) = (luminescence of test sample - average luminescence of virus control) / (average luminescence of uninfected cell control - average luminescence of virus control) × 100. EC 50The inhibition curve was determined (the data were fitted with Log(inhibitor) vs. response-Variable slope (four parameters) curve using Graphpad Prism), and the results are shown in Table 2.

[0087] Cytotoxicity assay: Cytotoxicity was evaluated using a luminescent cell viability assay. Uninfected MT-4 cells were seeded using the same method as the antiviral activity assay and incubated in the presence of test compounds / positive controls. After 5 to 6 days of incubation, Cell-Titer Glo (Promega) was added to each well and luminescence was recorded for cell viability assay. Cell viability % was calculated by the following equation:

[0088] Cell viability (%) = (luminescence of test sample - average luminescence of culture medium) / (average luminescence of uninfected cell control - average luminescence of culture medium). 50 The results were determined from the viability curve (Graphpad Prism was used to fit the data using Log(inhibitor) vs. response-Variable slope (four parameters) curve fitting), and are shown in Table 2.

[0089] Table 2 In vitro anti-HIV activity and cytotoxicity

[0090]

[0091] EC 50 :A:<1 nM; B:1-100nM; C:>100nM

[0092] It can be seen from Table 1 and Table 2 that the compounds of the present invention have strong inhibitory effects on HBV and HIV viruses, and their anti-HBV activity is comparable to that of the control compound, while their anti-HIV activity is superior to that of the control compound.

[0093] Example 4: Stability test

[0094] Aqueous solution stability

[0095] Phosphate buffer at a concentration of 100 mM at pH 1.0 and pH 7.0 was prepared by using KH2PO4 and K2HPO4 and phosphoric acid. Solution stability samples prepared from the test compound and the positive control (GS-7340 or HS-10234) were placed in 20 mL transparent glass bottles (CNW Tech Co.) with tightly closed screw caps. The sample bottles were placed in a water bath maintained at 40 ° C. At certain time points, the samples were removed from the water bath, immediately diluted 5 times with a solvent mixture of water: CH3CN = 70:30 (v / v) and stored at 8 ° C before HPLC analysis. HPLC analysis was performed under the following conditions.

[0096] Human plasma stability

[0097] Phosphate buffer at pH 7.4 was prepared from NaCl (137 mM), KCl (2.7 mM), Na2HPO4 (10 mM), and KH2PO4 (1.8 mM). Diluted plasma samples were prepared by diluting human plasma 1:1 with PBS. 10 μM plasma samples were prepared by diluting 40-fold the 400 μM working solution of the test compound using diluted plasma. Plasma stability samples prepared from the test compound were aliquoted into incubation plates (2 mL 96-well plates (AXYGEN Tech Co.)). The sample vials were placed in an incubator maintained at 37°C. At certain time points, the reaction was quenched with CH3CN 4-fold. The resulting mixture was further gently mixed for 5 min, centrifuged at 3700 rpm for 15 min, and then 100 µL of the supernatant was transferred to a clean deep-well plate, diluted with 100 µL of water, and mixed thoroughly. The samples were stored at 2°C-8°C before HPLC analysis. HPLC analysis was performed under the same conditions as in the aqueous solution stability section.

[0098] HPLC method for indicating stability

[0099] The stability-indicating HPLC method employed the following column and chromatographic conditions.

[0100] Column: Agilent Zorbax SB-C18, 4.6×150nm, 3.5μm

[0101] Detection: UV at 280nm

[0102] Flow rate: 1.0mL / min

[0103] Run time: 15 minutes

[0104] Injection volume: 20 μL

[0105] Column temperature: 30°C

[0106] Mobile phase A: 0.1% CF3COOH in water

[0107] Mobile phase B: CH3CN

[0108] The HPLC gradient run started with mobile phase A 70% and mobile phase B 30% at time 0. Then, mobile phase B was increased to 40% at 2 minutes, increased to 75% at 12 minutes, decreased to 30% at 12.01 minutes, and then maintained at 30% until 15 minutes to complete the gradient run cycle. The stability results are shown in Tables 3 to 5.

[0109] As can be seen from Tables 3 and 5, under acidic conditions and in human plasma, the exemplary compounds provided herein have improved stability compared to GS-7340 and similar stability compared to HS-10234. However, under neutral conditions as shown in Table 4, the exemplary compounds provided herein exhibit significantly better stability than both GS-7340 and HS-10234. This improved stability under neutral conditions allows the exemplary compounds provided herein to be formulated into tablets suitable for low-cost packaging (e.g., blister packaging, etc.), as well as into long-acting intramuscular injection preparations.

[0110] Table 3. Stability of prodrugs in aqueous solution at pH 1.0 and 40°C

[0111]

[0112] Table 4. Stability of prodrugs in aqueous solution at pH 7 and 40°C

[0113]

[0114] Table 5. Stability of prodrugs in human plasma at 37°C

[0115]

[0116] Example 5: Prodrug conversion evaluation

[0117] Evaluation of prodrug conversion in human peripheral blood mononuclear cells (PBMCs)

[0118] Cryopreserved human PBMC (source: SAILYBIO) were used to evaluate the conversion of the prodrug to the active triphosphate form. 6 / mL of cells were resuspended in culture medium and incubated at 37°C / 5% CO2 for 1 hour. After cell recovery, 5mL of cell suspension was cut into a 15mL tube and mixed with 5mL of 5μM test compound (prodrug) or control (tenofovir alafenamide, TAF) in culture medium. After incubation for another 3 hours at 37°C / 5% CO2, the samples were centrifuged at 300g for 10 minutes and the supernatant was discarded. The cell pellet was washed twice with 10mL of ice-cold PBS and lysed with 700μL of 70% methanol. The lysate was placed at -20°C overnight and centrifuged at 10000rpm at 4°C for 10 minutes. Finally, 650μL of supernatant was transferred to a clean 96-well plate and evaporated to dryness with nitrogen. The dried residue was then dissolved with 200μL of the initial LC mobile phase and loaded into the LC-MS / MS instrument. The response of triphosphate metabolites was measured for each sample. To evaluate the conversion efficiency, the response ratio of triphosphate metabolites determined from the test compounds relative to the control was calculated and the corresponding results are shown in Table 6.

[0119] Evaluation of prodrug conversion in Hep G2 cells

[0120] Hep G2 cells (source: ATCC) were also used to evaluate the conversion of the prodrug to the active triphosphate form. Hep G2 cells were digested with 0.25% trypsin-0.53 mM EDTA and plated at 2 × 10 6 / mL density suspension. To seed the cells, add 1.5mL of culture medium and 1mL of cell suspension to each well of a 6-well plate. Gently shake and place the plate in an incubator at 37°C / 5% CO2 overnight to allow cell attachment. After cell attachment, replace the medium with new medium containing 5μM test compound (prodrug) or control (tenofovir alafenamide, TAF) and incubate at 37°C / 5% CO2. At each specific time point (1 hour, 4 hours, 7 hours, 24 hours), remove the medium and wash the cells twice with 3mL of ice-cold PBS. After washing, dissociate the cells with 100μL of 0.25% trypsin-0.53mM EDTA at 20°C for 5 minutes and lyse with 500μL of ice-cold 70% methanol. The lysate was placed at -20°C for lysis and centrifuged at 10000rmp at 4°C for 10 minutes. Finally, 600 μL of supernatant was transferred to a clean 96-well plate and evaporated to dryness with nitrogen. The dried residue was then dissolved with 200 μL of initial LC mobile phase and loaded into the LC-MS / MS instrument. The response of the triphosphate metabolites was determined for each sample. In order to evaluate the conversion efficiency, the response ratio of the triphosphate metabolites determined from the test compound at each time point relative to the control was calculated, and the corresponding results are shown in Table 7.

[0121] Table 6 Prodrug conversion in human peripheral blood mononuclear cells (PBMCs)

[0122]

[0123] Table 7 Prodrug conversion in Hep G2 cells

[0124]

[0125] Tables 6 and 7 show the ratio of the conversion rate of the prodrug to active triphosphate of the Example compounds of the present invention compared with TAF in Hep G2 cells. As can be seen from the table, the conversion efficiency of the compound of Example 2 of the present invention has been greatly increased compared with TAF, whether in PBMC or in Hep G2 cells, indicating that the compound of the present invention has a better ability to be converted into the active substance triphosphate in vivo than the comparative example, so that it can better exert its drug effect.

[0126] The above description is only regarded as an illustration of the principle of the present invention. In addition, since many modifications and variations will be apparent to those skilled in the art, it is not desirable to limit the present invention to the exact configuration and process as shown above. Therefore, all suitable modifications and equivalents may be regarded as belonging to the scope of the present invention as defined by the appended claims.

Claims

1. A compound as shown in formula I or a pharmaceutically acceptable salt thereof, The phosphorus atoms marked with "*" are of S configuration and / or R configuration.

2. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The compound shown in formula I is any of the following compounds: and .

3. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the compound of formula I or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 2, and one or more pharmaceutically acceptable excipients.

4. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical composition may further comprise an additional agent which is an anti-infective agent.

5. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical composition may further comprise an additional agent which is an antiviral agent.

6. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical composition may further comprise an additional immunotherapeutic agent.

7. Use of a compound of formula I or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 2, or a pharmaceutical composition as described in any one of claims 3 to 6 in the preparation of a medicament for preventing and / or treating viral infection; the virus is HBV virus or HIV virus.

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