DEUTERATED NUCLEOSIDE COMPOUNDS AND USE THEREOF

Abstract

A series of deuterated nucleoside compounds are compounds represented by formula (IV), and stereoisomers or pharmaceutically acceptable salts thereof.

##STR00001##

Claims

1-10. (canceled)

11. A compound of the following formula, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: ##STR00025##

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0084] The present disclosure is described in detail by the examples below, but it does not mean that there are any adverse restrictions on the present disclosure. The present disclosure has been described in detail herein, and its specific examples have also been disclosed; for one skilled in the art, it is obvious to make various modifications and improvements to the examples of the present disclosure without departing from the spirit and scope of the present disclosure.

Example 1

[0085] ##STR00019##

Synthetic Route:

[0086] ##STR00020##

Step 1: Synthesis of Compound 1-2

[0087] To a reaction flask were added compound 1-1 (3.0 g) and dichloromethane (20 mL), then sequentially added pyridine (29.40 g) and compound 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (3.90 g). The reaction system was stirred at room temperature for 14 hours. The reaction mixture was directly concentrated under reduced pressure to obtain a crude product. The crude product was separated by flash column chromatography (ISCO?; 40 g SepaFlash? Silica Flash Column, mobile phase: 0 to 50% EtOAc/PE, flow rate: 50 mL/min). Thus compound 1-2 (purity: 84.0%) was obtained.

[0088] MS m/z (ESI): [M+H].sup.+=534.6.

Step 2: Synthesis of Compound 1-3

[0089] To a reaction flask were added compound 1-2 (1.0 g, purity: 84%) and dichloromethane (50 mL), then added Dess-Martin periodinane (1.19 g). The reaction system was stirred at room temperature for 4 hours. The reaction mixture was filtered and directly concentrated under reduced pressure to obtain a crude product. The crude product was separated by flash column chromatography (ISCO?; 12 g SepaFlash? Silica Flash Column, mobile phase: 0 to 33% EtOAc/PE, flow rate: 35 mL/min). Thus compound 1-3 was obtained.

[0090] MS m/z (ESI): [M+H].sup.+=532.2.

Step 3: Synthesis of Compound 1-4

[0091] To a reaction flask were added compound 1-3 (200 mg) and deuterated methanol (10 mL), then added sodium borodeuteride (28.46 mg). The reaction system was stirred at room temperature for 1 hour. The reaction mixture was directly concentrated under reduced pressure to obtain a crude product. The crude product was separated by flash column chromatography (ISCO?; 12 g SepaFlash? Silica Flash Column, mobile phase: 0 to 30% EtOAc/PE, flow rate: 30 mL/min). Thus compound 1-4 was obtained.

[0092] MS m/z (ESI): [M+H].sup.+=535.2.

Step 4: Synthesis of Compound 1-5

[0093] To a reaction flask were added compound 1-4 (110 mg) and tetrahydrofuran (4 mL), then added a solution of tetrabutylammonium fluoride in tetrahydrofuran (1 M, 411.39 ?L). The reaction system was stirred at room temperature for 0.5 hours. The reaction mixture was directly concentrated under reduced pressure to obtain a crude product. Thus compound 1-5 was obtained.

[0094] MS m/z (ESI): [M+H].sup.+=293.2.

[0095] .sup.1H NMR (400 MHZ, DMSO-d6) ?=7.88 (s, 1H), 6.91-6.94 (m, 2H), 3.95 (d, J=5.3 Hz, 1H), 3.65 (br d, J=3.5 Hz, 1H), 3.62 (br d, J=3.3 Hz, 2H), 3.52 (br d, J=4.5 Hz, 2H).

Step 5: Synthesis of Compound 1-6

[0096] To a reaction flask were added compound 1-5 (100 mg) and acetone (40 mL), then sequentially added sulfuric acid (50.34 mg) and compound 2,2-dimethoxypropane (178.17 mg). The reaction system was stirred at 45? C. for 0.5 hours. Water (50 mL) was added to the reaction system, and the reaction mixture was adjusted to neutral using a 15% sodium bicarbonate solution. The mixture was then extracted with EtOAc (50 mL*3). The organic phases were combined and subsequently washed sequentially with saturated brine (50 mL) and water (50 mL). After the phases were separated, the organic phase was dried over anhydrous Na.sub.2SO.sub.4, and finally, the organic phase was directly concentrated under reduced pressure. Thus compound 1-6 was obtained.

[0097] MS m/z (ESI): [M+H].sup.+=333.2.

Step 6: Synthesis of Compound 1-7

[0098] To a reaction flask were added compound 1-6 (130 mg) and EtOAc (5 mL), then sequentially added isobutyric anhydride (123.77 mg), triethylamine (118.75 mg), and N,N-lutidine (9.56 mg). The reaction system was stirred at room temperature for 1 hour. The reaction mixture was directly concentrated under reduced pressure to obtain a crude product. The crude product was separated by flash column chromatography (ISCO?; 12 g SepaFlash? Silica Flash Column, mobile phase: 0 to 33% ethyl acetate/petroleum ether, flow rate: 30 mL/min). Thus compound 1-7 was obtained.

[0099] MS m/z (ESI): [M+H].sup.+=403.2.

Step 7: Synthesis of Compound 1

[0100] To a reaction flask were added compound 1-7 (100 mg) and H.sub.2O (1 mL), then added formic acid (11.94 mg). The reaction system was stirred at room temperature for 1 hour. The reaction mixture was directly concentrated under reduced pressure to obtain a crude product. The crude product was purified by a silica gel column (eluent: petroleum ether:ethyl acetate=1:0 to 0:1) to obtain compound 1.

[0101] MS m/z (ESI): [M+H].sup.+=534.6.

[0102] .sup.1H NMR (400 MHZ, CD.sub.3OD) ?=7.88 (s, 1H), 6.91-6.94 (m, 2H), 4.63-4.44 (m, 1H), 4.30-4.38 (m, 2H), 4.15-4.16 (m, 1H), 2.55-2.57 (m, 1H), 1.88-1.91 (m, 6H).

Example 2

[0103] ##STR00021##

Synthetic Route:

[0104] ##STR00022##

Step 1: Synthesis of Compound 2-2

[0105] To a reaction flask were added compound 1-6 (300 mg), salicylic acid (311.71 mg), and dichloromethane (3 mL), then sequentially added 4-dimethylaminopyridine (275.71 mg) and N,N-dicyclohexylcarbodiimide (465.63 mg). The reaction system was stirred at 35? C. for 12 hours. The reaction system was quenched by water (30 mL), and extracted with EtOAc (10 mL*2). The organic phases were combined, washed with saturated brine (10 mL), and dried over anhydrous Na.sub.2SO.sub.4. The organic phase was filtered, and concentrated under reduced pressure. The crude product was separated by flash column chromatography (ISCO?, 4 g SepaFlash? Silica Flash Column, mobile phase: 0 to 80% ethyl acetate/petroleum ether, flow rate: 18 mL/min) to obtain compound 2-2.

[0106] MS m/z (ESI): [M+H].sup.+=453.0.

Step 2: Synthesis of Compound 2

[0107] To a reaction flask were added compound 2-2 (275 mg) and water (3 mL), then added formic acid (15 mL). The reaction system was stirred at room temperature for 4 hours. The reaction mixture was directly concentrated under reduced pressure to obtain a crude product. The crude product was purified by prep-HPLC (column: O-Welch C18 150*30 mm*5 ?m; mobile phase: [H.sub.2O (FA)-ACN]; ACN %: 18% to 58%, 10 min) to obtain compound 2.

[0108] MS m/z (ESI): [M+H].sup.+=413.3.

[0109] .sup.1H NMR: (400 MHz, DMSO-d.sub.6) ?=10.43 (s, 1H), 7.98-7.81 (m, 2H), 7.64 (d, J=6.8 Hz, 1H), 7.53 (t, J=7.7 Hz, 1H), 6.98 (d, J=8.3 Hz, 1H), 6.91 (t, J=7.7 Hz, 1H), 6.87 (d, J=4.5 Hz, 1H), 6.80 (d, J=4.5 Hz, 1H), 6.34 (s, 1H), 5.46 (br s, 1H), 4.63 (dd, J=2.6, 12.2 Hz, 1H), 4.48-4.43 (m, 1H), 4.41-4.36 (m, 1H), 4.13 (br d, J=6.3 Hz, 1H).

Example 3

[0110] ##STR00023##

Synthetic Route:

[0111] ##STR00024##

Step 1: Synthesis of Compound 3

[0112] To a reaction flask were added compound 1-5 (1 g) and ethyl acetate (5 mL), then sequentially added isobutyric anhydride (1.62 g), 4-dimethylaminopyridine (83.60 mg), and triethylamine (1.04 g). The reaction system was stirred at room temperature for 4 hours. The reaction mixture was quenched with water (20 mL), and extracted with ethyl acetate (20 mL*3). The organic phases were combined and then washed with saturated brine (20 mL*2). The mixture was washed with and dried over anhydrous sodium sulfate, and directly concentrated under reduced pressure to obtain a crude product. The crude product was separated by flash column chromatography (ISCO?; 20 g SepaFlash? Silica Flash Column, mobile phase: 0 to 80% ethyl acetate/petroleum ether, flow rate@ 35 mL/min), and further purified by prep-HPLC (column: Welch Xtimate C18 150*30 mm*5 ?m; mobile phase: [H.sub.2O (FA)-ACN]; ACN %: 38% to 78%, 8 min) to obtain compound 3.

[0113] MS m/z (ESI): [M+H].sup.+=503.1.

[0114] .sup.1H NMR: (400 MHZ, DMSO-d.sub.6) ?=8.08-7.93 (m, 2H), 6.93 (d, J=4.5 Hz, 1H), 6.74 (d, J=4.5 Hz, 1H), 5.43 (d, J=3.5 Hz, 1H), 4.82-4.82 (m, 1H), 4.62 (br d, J=3.3 Hz, 1H), 4.68-4.55 (m, 1H), 2.69-2.55 (m, 2H), 2.48-2.41 (m, 1H), 1.15 (dd, J=7.0, 10.3 Hz, 6H), 1.09 (d, J=7.0 Hz, 6H), 1.02 (dd, J=7.0, 13.3 Hz, 6H).

Bioassay:

Test Example 1: In Vitro Anti-Coronavirus Activity Test

Research Purposes

[0115] To detect the in vitro anti-human coronavirus (HCOV) 229E activity of the compound using the cytopathic effect (CPE) assay, and simultaneously evaluate the cytotoxicity of the compound.

1. Experimental Materials

1.1 Compound

[0116] The test compound was prepared as a 20 mM stock solution using DMSO. The initial testing concentration of the test compound was 50 ?M, tested at 8 concentrations with 4-fold gradient dilutions, in duplicate wells. The control compound Remdesivir would be provided by WuXi AppTec. Remdesivir would be tested at 8 concentrations with 3-fold gradient dilutions, in duplicate wells.

1.2 Cells and Virus

[0117] MRC5 cells and coronavirus HCoV 229E were purchased from ATCC. MRC5 cells were cultured in EMEM (Sigma) culture medium supplemented with 10% fetal bovine serum (Excell), 1% double antibiotic (Hyclone), 1% L-glutamine (Gibco), and 1% non-essential amino acids (Gibco). The EMEM (Sigma) culture medium supplemented with 5% fetal bovine serum (Excell), 1% double antibiotic (Hyclone), 1% L-glutamine (Gibco), and 1% non-essential amino acids (Gibco) was used as the experimental culture medium.

2. Experimental Scheme:

[0118]

TABLE-US-00001 TABLE 1 Virus assay methods used in this study Compound Treatment Duration (Days)/ End-Point Control Detection Virus (Strain) Cell Method Compound Reagent HCoV 229E, 20,000 3/CPE Remdesivir CellTiter 200TCID.sub.50/ MRC5 Glo. well cells/well

[0119] Cells were seeded in a 96-well microplate at a specific density (Table 1) and cultured overnight in a 5% CO.sub.2, 37? C. incubator. The next day, serially diluted compounds (8 concentration points, in duplicate wells), 50 ?L per well, were added. Subsequently, the virus, diluted to 200 TCID.sub.50 per well, was added to the cells, 50 ?L per well. Cell control (cells without compound treatment or virus infection), virus control (cells infected with the virus without compound treatment), and culture medium control (culture medium only) were set up. The final volume of the experimental culture medium was 200 ?L, with a final DMSO concentration of 0.5%. Cells were incubated in a 5% CO.sub.2, 35? C. incubator for 3 days. Cell viability was detected using the CellTiter Glo (Promega) cell viability assay kit. The cytotoxicity experiment was conducted under the same conditions as the antiviral experiment, but without virus infection.

[0120] The antiviral activity and cytotoxicity of the compound were expressed by the inhibition rate (%) and cell viability (%) of the compound at different concentrations on the cytopathic effect caused by virus, respectively. The calculation formula is as follows:

Inhibition rate (%)=(test well reading value?average value of virus control)/(average value of cell control?average value of virus control)?100

Cell viability (%)=(test well reading value?average value of culture medium control)/(average value of cell control?average value of culture medium control)?100

[0121] Nonlinear fitting analysis was performed using GraphPad Prism on the inhibition rate and cell viability of the compound, to calculate the median effective concentration (EC.sub.50) and median cytotoxic concentration (CC.sub.50) values of the compound.

[0122] The experimental results are shown in Table 2.

TABLE-US-00002 TABLE 2 Median effective concentration (EC.sub.50) and median cytotoxic concentration (CC.sub.50) values of the compound of the present disclosure Compound No. EC.sub.50 (?m) CC.sub.50 (?m) 1 0.22 >50

[0123] Conclusion: The compound of the present disclosure exhibits potent in vitro anti-coronavirus activity.

Test Example 2: Pharmacokinetic Evaluation of Compounds

[0124] Experimental objective: To test the pharmacokinetics of the compounds in CD-1 mice

[0125] Experimental materials: CD-1 mice (male, from Beijing Vital River Laboratory Animal Technology Co., Ltd.)

[0126] Experimental procedure: The pharmacokinetic characteristics in rodents after intragastric administration of the compound were tested using a standard protocol. In the experiment, candidate compounds are prepared into clear solutions (the solvent for intragastric administration preparations was 1% methylcellulose 4000 aqueous solution). Four male CD-1 mice were used in this project, with a drug concentration of 10 mg/ml and a dose of 100 mg/kg. Plasma samples were collected at 0 h (before administration) and post-administration at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24 h. Samples were centrifuged at 3200 g for 10 minutes at 4? C., and the supernatant was separated to obtain plasma samples. The plasma was transferred to a pre-cooled centrifuge tube, snap-frozen in dry ice, and then stored in an ultra-low temperature freezer at ?70?10? C./?60? C. or lower. Plasma drug concentration was quantitatively analyzed by LC-MS/MS analysis method, and the plasma drug concentration data of the metabolite of the compound of the present disclosure were processed by using the pharmacokinetic software of WinNonlin Version 6.3 or above (Pharsight) in a non-compartmental model. Relevant pharmacokinetic parameters such as peak concentration (C.sub.max), half-life (T.sub.1/2), and area under the curve (AUC.sub.0-inf) were calculated using the linear-log trapezoidal method. Experimental results are shown in Tables 3 and 4.

TABLE-US-00003 TABLE 3 Pharmacokinetic experimental data Pharmacokinetic Parameter Compound 2 Compound 3 C.sub.max (nmol/L) 38150 55900 T.sub.1/2 (h) 2.67 2.55 AUC.sub.0-inf (h * nmol/L) 150291 162521

[0127] Note: The compounds of the present disclosure would be rapidly metabolized in vivo, and the above data are all for the detected metabolites.

TABLE-US-00004 TABLE 4 Concentration of nucleoside Nuc in plasma and tissues at different times Concentration of Nucleoside Nuc Nucleoside PK Assay in Plasma and Tissues (nM) Time (h) Compound 2 Compound 3 1 38150 55900 2 32700 30650 4 21500 14450 8 1489 1864 24 95.8 72.5

[0128] Conclusion: After administration of the compound of the present disclosure to mice, it is rapidly metabolized in vivo into its corresponding active metabolites, exerting a pharmacological effect; the drug concentration of the compound of the present disclosure is sustained for a long duration and maintained at a high concentration, which is conducive to the continuous inhibition of the virus and the exertion of pharmacological efficacy.