NUCLEOSIDE ANALOG AND USE THEREOF
20240166680 ยท 2024-05-23
Inventors
Cpc classification
A61K31/7048
HUMAN NECESSITIES
C07H19/23
CHEMISTRY; METALLURGY
A61K31/7068
HUMAN NECESSITIES
A61K31/706
HUMAN NECESSITIES
C07H19/10
CHEMISTRY; METALLURGY
A61K31/7076
HUMAN NECESSITIES
C07H19/24
CHEMISTRY; METALLURGY
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
C07H19/23
CHEMISTRY; METALLURGY
C07H19/10
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a nucleoside analog represented by the following formula and a use thereof. Specifically, the present invention relates to a nucleoside analog represented by the following formula or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition thereof, and a use thereof in preparation of (a) an inhibitor for inhibiting replication of coronaviruses, paramyxoviruses, influenza viruses, flaviviruses, filoviruses, bunya viruses and/or arenaviruses, and/or (b) a medicine for treating and/or preventing or alleviating a disease caused by infection of coronaviruses, paramyxoviruses, influenza viruses, flaviviruses, filoviruses, bunya viruses and/or arenaviruses.
Claims
1. A compound represented by formula I or a pharmaceutically acceptable salt thereof: ##STR00107## wherein B is selected from the group consisting of ##STR00108## X is selected from the group consisting of oxygen, sulfur, CH.sub.2, and NH; R.sub.1 is selected from the group consisting of hydrogen, deuterium, and cyano; R.sub.2 is selected from the group consisting of hydrogen, C.sub.1-18 alkyl, C.sub.3-8 cycloalkyl, C.sub.8-20 aryl, and 5-15 membered heteroaryl, wherein the alkyl and the cycloalkyl are unsubstituted or substituted by one to three substituents independently selected from the group consisting of halogen, hydroxyl, carboxyl and 0 1-4 alkoxy, and the aryl and the heteroaryl are unsubstituted or substituted by one to five substituents independently selected from the group consisting of R.sub.9; R 3 is selected from the group consisting of hydrogen, and C.sub.1-4 alkoxy; ##STR00109## or R.sub.2, R.sub.3 and the carbon to which they are attached form R.sub.4 is selected from the group consisting of hydrogen, deuterium, halogen, azido, cyano, C.sub.1-8 alkyl, halogenated C.sub.1-6 alkyl, azido C.sub.1-8 alkyl, cyano C.sub.1-6 alkyl, hydroxy C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-8 alkynyl, C.sub.3-8 cycloalkyl, and C.sub.1-8 alkoxy C.sub.1-6 alkyl; R.sub.5 is selected from the group consisting of hydrogen, C.sub.1-20 alkanoyl, C.sub.3-20 cycloalkanoyl, amino C.sub.1-20 alkanoyl, C.sub.1-20 alkylamino C.sub.1-8 alkanoyl, C.sub.1-6 cycloalkylamino C.sub.1-8 alkanoyl, C.sub.1-20 dialkylamino C.sub.1-8 alkanoyl, C.sub.1-20 alkoxy C.sub.1-8 alkanoyl, an amino acid group in which the carbonyl of the carboxyl group on the amino acid forms an ester bond with the connected oxygen, C.sub.6-20 arylamino C.sub.1-6 alkanoyl, 3-20 membered heterocycloalkyl C.sub.1-6 alkanoyl, ##STR00110## wherein the C.sub.1-20 alkanoyl and the C.sub.3-20 cycloalkanoyl are unsubstituted or substituted by one to three halogens, and the 3-20 membered heterocycloalkyl is unsubstituted or substituted by C.sub.1-6 alkyl; R.sub.6 is selected from the group consisting of hydroxyl, amino, hydroxylamine (NHOH), and NHOR.sub.13; R.sub.7 is selected from the group consisting of hydrogen, deuterium, and halogen; R.sub.8 is selected from the group consisting of hydrogen, deuterium, halogen, cyano, and carbamoyl; R.sub.9 is selected from the group consisting of halogen, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, C.sub.1-4 alkylthio, cyano, nitro, amino, phenyl, carboxyl, trifluoromethyl, difluoromethoxy, trifluoromethoxy, C.sub.1-4 alkylamino, di(C.sub.1-4 alkyl) amino, C.sub.1-4 alkylcarbonyl, C.sub.1-4 alkylcarbonyloxy, and C.sub.1-4 alkoxycarbonyl; R.sub.10 is selected from the group consisting of C.sub.1-6 alkyl, C.sub.3-6 cycloalkyl, C.sub.6-20 aryl, and 5-15 membered heteroaryl; R.sub.11 is selected from the group consisting of C.sub.1-18 alkyl, and methylene C.sub.6-20 aryl; R.sub.12 is selected from the group consisting of C.sub.1-6 alkyl, C.sub.3-6 cycloalkyl, C.sub.6-20 aryl, and 5-15 membered heteroaryl; R.sub.13 is selected from the group consisting of C.sub.1-20 alkanoyl, C.sub.3-29 cycloalkanoyl, amino C.sub.1-20 alkanoyl, C.sub.1-29 alkylamino C.sub.1-6 alkanoyl, C.sub.1-6 cycloalkylamino C.sub.1-6 alkanoyl, C.sub.1-20 dialkylamino C.sub.1-6 alkanoyl, C.sub.1-20 alkoxy C.sub.1-6 alkanoyl, and C.sub.1-6 alkoxycarbonyloxymethylene.
2. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is represented by formula I-I: ##STR00111## wherein, B, X, R.sub.1, R.sub.4, and R.sub.5 are defined the same as those in the claim 1.
3. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is represented by formula I-II: ##STR00112## Preferably, the formula (I-II) is selected from the group consisting of formulas I-IIA and I-IIB, ##STR00113## wherein, B, X, R.sub.1, R.sub.2, R.sub.4 and R.sub.5 are defined the same as those in the claim 1.
4. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is selected from the group consisting of the following formulas: ##STR00114## ##STR00115## wherein R.sub.1 is selected from the group consisting of hydrogen and deuterium; R.sub.2, R.sub.5, R.sub.7, R.sub.8 and R.sub.13 are defined the same as those in the claim 1; R.sub.4 is selected from the group consisting of hydrogen, deuterium and halogen.
5. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is selected from the group consisting of the following compounds: ##STR00116## ##STR00117## ##STR00118## ##STR00119## ##STR00120## ##STR00121## ##STR00122## ##STR00123## ##STR00124## ##STR00125## ##STR00126## ##STR00127## ##STR00128## ##STR00129## ##STR00130## ##STR00131## ##STR00132## ##STR00133## ##STR00134## ##STR00135## ##STR00136## ##STR00137## ##STR00138## ##STR00139## ##STR00140## ##STR00141## ##STR00142## ##STR00143## ##STR00144## ##STR00145## ##STR00146## ##STR00147## ##STR00148## ##STR00149## ##STR00150## ##STR00151## ##STR00152## ##STR00153## ##STR00154## ##STR00155## ##STR00156## ##STR00157## ##STR00158## ##STR00159##
6. A pharmaceutical composition comprising: (a) one or more selected from the group consisting of the compound and the pharmaceutically acceptable salt thereof according to claim 1, and (b) a pharmaceutically acceptable carrier.
7. A method of inhibiting virus replication or treating or preventing or alleviating a disease caused by a viral infection comprising administering to a subject the compound or the pharmaceutically acceptable salt thereof according claim 1 optionally in the presence of a pharmaceutically acceptable carrier.
8. The method according to claim 7, wherein the virus is one or more selected from the group consisting of: (1) coronaviruses, such as coronaviruses that infect humans: such as severe acute respiratory syndrome coronavirus (SARS-CoV), 2019 novel coronavirus (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), Human coronavirus OC43, Human coronavirus 229E, Human coronavirus NL63, Human coronavirus HKU1; and coronaviruses that infect animals: such as porcine epidemic diarrhea virus (PEDV), feline infectious peritonitis virus (FIFV); (2) paramyxoviruses: such as paraflu virus, measles virus, respiratory syncytial virus (RSV); (3) influenza viruses: such as influenza A virus, influenza B virus, influenza C virus, influenza D virus; (4) flaviviruses: such as hepatitis C virus (HCV), dengue virus (DENY), Zika virus (ZIKV); (5) filoviruses: such as Marburg virus (MBV), Ebola virus (EBV), Cueva virus; (6) bunyaviridae viruses: such as Bunyaviviruses, Phleboviruses, Nairoviruses, Hantaviruses; (7) arenaviruses: such as Lassa fever virus (LASV), Junin virus (JUNV), Machupo virus (MACV); in particular, the virus is SARS-CoV-2 or an influenza virus.
9. The method according to claim 7, wherein the disease caused by viral infection is one or more selected from the group consisting of: (D1) common cold, high-risk symptom infection, respiratory tract infection, pneumonia and complications thereof caused by human coronavirus infection; (D2) porcine epidemic diarrhea caused by porcine epidemic diarrhea virus; (D3) Feline infectious peritonitis caused by feline coronavirus; (D4) common cold, high-risk symptom infection, respiratory tract infection, pneumonia and complications thereof caused by human respiratory syncytial virus infection; (D5) common cold, high-risk symptom infection, respiratory tract infection, pneumonia and complications thereof caused by influenza virus infection; (D6) chronic hepatitis C and complications thereof caused by hepatitis C virus; (D7) dengue fever and complications thereof caused by dengue virus; (D8) infection and complications thereof caused by Zika virus; (D9) hemorrhagic fever and complications thereof caused by Marburg virus or Ebola virus; (D10) infection and complications thereof caused by Bunyaviridae viruses; (D11) infection and complications thereof caused by arenaviruses.
10. The method according to claim 7, wherein the disease caused by viral infection is a disease caused by SARS-CoV-2 infection, in particular one or more selected from the group consisting of respiratory tract infection, pneumonia and complications thereof; or a disease caused by influenza virus infection; in particular, one or more selected from the group consisting of common cold, high-risk symptom infection, respiratory tract infection, pneumonia and complications thereof.
Description
EXAMPLES
Preparation Example 1: Synthesis of A1
[0120] ##STR00056##
[0121] ?-D-N4-hydroxycytidine (NHC) (0.97 g, 3.75 mmol) was added to dichloromethane (5 mL), and 4-dimethylaminopyridine (76 mg, 0.75 mmol), triethylamine (1.14 g, 11.25 mmol) and 4,4-bismethoxytrityl chloride (2.80 g, 8.25 mmol) were added in sequence, and stirred at room temperature. After 6 hours, dichloromethane (20 mL) and saturated brine were added to the reaction solution, the separated organic phase was dried over anhydrous sodium sulfate, evaporated to dryness, and separated by silica gel column chromatography (petroleum ether:ethyl acetate=1:1), to obtain A1-1 as a white solid (1.81 g, yield 56%).
[0122] Carbonyldiimidazole (134.5 mg, 0.83 mmol) and A1-1 (600 mg, 0.69 mmol) were added to dichloromethane (5 mL), and stirred at room temperature. After 2 hours, the reaction solution was evaporated to dryness and separated by silica gel column chromatography (petroleum ether: ethyl acetate=2:1) to obtain A1-2 as a white foamy solid (420 mg, yield 68%).
[0123] A1-2 (420 mg, 0.47 mmol) was added to methanol (10 mL), and trifluoroacetic acid (107 mg, 0.94 mmol) was added thereto, and stirred at room temperature. After 10 minutes, a saturated sodium bicarbonate solution was added to adjust pH to neutral. The reaction solution was concentrated and separated by silica gel column chromatography (dichloromethane:methanol=15:1) to obtain a foamy solid, which was slurried in ethyl acetate to obtain Al as a white powdery solid (30 mg, yield 22%). .sup.1H NMR (500 MHz, DMSO-d.sub.6) ? 10.10 (s, 1H), 9.85 (s, 1H), 6.96 (d, J=8.1 Hz, 1H), 5.88 (d, J=2.6 Hz, 1H), 5.60 (d, J=8.1 Hz, 1H), 5.50 (dd, J=7.8, 2.6 Hz, 1H), 5.21 (dd, J=7.8, 4.1 Hz, 1H), 5.14 (t, J=5.6 Hz, 1H), 4.20-4.15(m, 1H), 3.60 (t, J=5.8 Hz, 2H).
Preparation Example 2: Synthesis of A2
[0124] ##STR00057##
[0125] A2-0 (296 mg, 0.9 mmol) was added to dichloromethane (3 mL). Carbonyldiimidazole (225 mg, 1.4 mmol) was added under an ice bath, and after the addition was complete, it was stirred at room temperature. After the reaction of the raw materials was complete, the reaction mixture was filtered and the filter cake was washed with dichloromethane to obtain A2 as a white solid (200 mg, yield 63%). .sup.1H NMR (500 MHz, DMSO-d.sub.6) ? 10.13 (s, 1H), 9.92 (d, J=2.3 Hz, 1H), 6.96 (d, J=8.1 Hz, 1H), 5.88 (d, J=2.1 Hz, 1H), 5.60 (dd, J=8.1, 2.1 Hz, 1H), 5.57 (dd, J=7.7, 2.2 Hz, 1H), 5.29 (dd, J=7.8, 4.2 Hz, 1H), 4.44-4.40 (m, 1H), 4.32 (dd, J=11.6, 5.1 Hz, 1H), 4.21 (dd, J=11.5, 6.8 Hz, 1H), 2.62-2.54 (m, 1H), 1.12-1.08 (m, 6H).
Preparation Example 3: Synthesis of A35 and A31
[0126] ##STR00058##
[0127] A2-0 (1.00 g, 3.04 mmol) and propionaldehyde (882 mg, 15.20 mmol) were added to dichloromethane (20 mL), and p-toluenesulfonic acid monohydrate (1.16 g, 6.08 mmol) was added slowly under an ice bath, and after the addition was completed, the reaction solution was warmed to room temperature, and stirred for 2 h. A 10% sodium carbonate aqueous solution and dichloromethane were added to the reaction solution, and the organic phase was separated. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (petroleum ether: ethyl acetate=20:1 to 1:1) to obtain A35 as a white solid (897 mg, yield 80%). .sup.1H NMR (500 MHz, CD.sub.3OD) ? 6.87 (d, J=8.2 Hz, 1H), 5.69 (d, J=2.3 Hz, 1H), 5.57 (d, J=8.1 Hz, 1H), 5.07 (t, J=4.5 Hz, 1H), 4.93 (dd, J=6.7, 2.3 Hz, 1H), 4.74 (dd, J=6.7, 3.7 Hz, 1H), 4.30-4.26 (m, 2H), 4.26-4.22 (m, 1H), 2.64-2.55 (m, 1H), 1.78-1.72 (m, 2H), 1.18-1.14 (m, 6H), 0.99 (t, J=7.5 Hz, 3H).
[0128] A35 (400 mg, 1.08 mmol) was added to a 7M ammonia methanol solution (15 mL), and stirred overnight at room temperature. The reaction solution was concentrated and separated by silica gel column chromatography (dichloromethane:methanol=50:1 to 15:1) to obtain A31 as a white solid (259 mg, yield 80%). .sup.1H NMR (600 MHz, CD.sub.3OD) ? 7.03 (d, J=8.2 Hz, 1H), 5.81 (d, J=3.2 15 Hz, 1H), 5.58 (d, J=8.2 Hz, 1H), 5.07 (t, J=4.5 Hz, 1H), 4.81 (dd, J=6.7, 3.2 Hz, 1H), 4.71 (dd, J=6.7, 3.4 Hz, 1H), 4.15-4.12 (m, 1H), 3.75 (dd, J=11.9, 3.8 Hz, 1H), 3.71 (dd, J=11.9, 4.7 Hz, 1H), 1.79-1.73 (m, 2H), 1.00 (t, J=7.5 Hz, 3H).
Preparation Example 4: Synthesis of A36 and A32
[0129] ##STR00059##
[0130] A2-0 (1.00 g, 3.04 mmol) and n-heptanal (1.73 g, 15.20 mmol) were added to dichloromethane (20 mL), and p-toluenesulfonic acid monohydrate (1.16 g, 6.08 mmol) was added slowly under an ice bath, and after the addition was completed, the reaction solution was warmed to room temperature, and stirred for 2 h. A 10% sodium carbonate aqueous solution and dichloromethane were added to the reaction solution, and the organic phase was separated. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (petroleum ether:ethyl acetate=20:1 to 1:1) to obtain A36 as a white solid (1.03 g, yield 80%). .sup.1H NMR (500 MHz, CD.sub.3OD) ? 6.87 (d, J=8.2 Hz, 1H), 5.68 (d, J=2.3 Hz, 1H), 5.57 (d, J=8.2 Hz, 1H), 5.09 (t, J=4.7 Hz, 1H), 4.92 (dd, J=6.7, 2.3 Hz, 1H), 4.73 (dd, J=6.7, 3.6 Hz, 1H), 4.30-4.26 (m, 2H), 4.25-4.22 (m, 1H), 2.64-2.55 (m, 1H), 1.76-1.70 (m, 30 2H), 1.49-1.41 (m, 2H), 1.39-1.27 (m, 6H), 1.18-1.13 (m, 6H), 0.91 (t, J=6.9 Hz, 3H).
[0131] A36 (600 mg, 1.41 mmol) was added to a 7M ammonia methanol solution (15 mL), and stirred overnight at room temperature. The reaction solution was concentrated and separated by silica gel column chromatography (dichloromethane:methanol=50:1 to 15:1) to obtain A32 as a white solid (375 mg, yield 75%). 1H NMR (500 MHz, CD.sub.3OD) ? 7.04 (d, J=8.2 Hz, 1H), 5.80 (d, J=3.1 Hz, 1H), 5.58 (d, J=8.2 Hz, 1H), 5.10 (t, J=4.7 Hz, 1H), 4.80 (dd, J=6.6, 3.1 Hz, 1H), 4.70 (dd, J=6.6, 3.4 Hz, 1H), 4.15-4.11 (m, 1H), 3.75 (dd, J=11.9, 3.8 Hz, 1H), 3.70 (dd, J=11.9, 4.7 Hz, 1H), 1.77-1.70 (m, 2H), 1.51-1.42 (m, 2H), 1.40-1.28 (m, 6H), 0.91 (t, J=6.9 Hz, 3H).
Preparation Example 5: Synthesis of A37 and A33
[0132] ##STR00060##
[0133] A2-0 (0.33 g, 1 mmol), anhydrous zinc chloride (0.68 g, 5 mol) and 4-chlorobenzaldehyde (1.40 g, 10 mmol) were sequentially added to anhydrous tetrahydrofuran (10 mL), and stirred at 70? C. overnight. The reaction solution was poured into a sodium carbonate aqueous solution and extracted with ethyl acetate, and the organic phase was separated. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography (dichloromethane:methanol=50:1) to obtain A37 as a white solid (0.15 g, yield 33%). .sup.1H NMR (500 MHz, CD.sub.3OD) ? 7.57-7.54 (m, 2H), 7.49-7.44 (m, 2H), 6.93 (d, J=8.2 Hz, 1H), 6.01 (s, 1H), 5.81 (d, J=2.3 Hz, 1H), 5.61 (d, J=8.2 Hz, 1H), 5.18 (dd, J=6.8, 2.2 Hz, 1H), 4.44-4.30 (m, 3H), 2.67-2.58 (m, 1H), 1.20-1.17 (m, 6H). ESI-MS m/z=450.0 [M?1].sup.?.
[0134] A37 (0.15 g, 0.33 mmol) and potassium carbonate (0.04 g, 0.33 mmol) were added into anhydrous methanol (5 mL), and stirred at room temperature for 4 hours. The reaction solution was evaporated to dryness and added with ethyl acetate and water, and the organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (dichloromethane:methanol=30:1) to obtain A33 as a white solid (0.1 g, yield 78.8%). .sup.1H NMR (500 MHz, CD.sub.3OD) ? 7.59-7.56 (m, 2H), 7.47-7.44 (m, 2H), 7.10 (d, J=8.2 Hz, 1H), 6.02 (s, 1H), 5.95 (d, J=3.1 Hz, 1H), 5.62 (d, J=8.2 Hz, 1H), 5.05 (dd, J=6.7, 3.1 Hz, 1H), 4.94 (dd, J=6.7, 3.3 Hz, 1H), 4.31-4.27 (m, 1H), 3.84-3.75 (m, 2H).
[0135] Preparation Example 6: Synthesis of A38 and A34
##STR00061##
[0136] To N,N-dimethylformamide (20 mL) were sequencely added p-toluenesulfonic acid monohydrate (1.16 g, 6.08 mmol), p-methoxybenzaldehyde (2.07 g, 15.20 mmol) and 2,2-dimethoxypropane (1.58 g, 15.20 mmol) under an ice bath. After the addition, the reaction solution was warmed to room temperature and stirred for 2 hours. A2-0 (1.00 g, 3.04 mmol) was added and the stirring continued for 4 hours. The reaction solution was added to water and extracted with ethyl acetate, and the organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness. The residue was sluried in a mixture of ethyl acetate and methyl tert-butyl ether to obtain A38, which is a pair of diastereomers (6:4), as a white solid (1.08 g, yield 80%). The .sup.1H NMR data of the main isomer is as follows: .sup.1H NMR (600 MHz, DMSO-d.sub.6) ? 10.07 (s, 1H), 9.73 (d, J=2.2 Hz, 1H), 7.47-7.43 (m, 2H), 6.99-6.96 (m, 2H), 6.95 (d, J=8.1 Hz, 1H), 5.93 (s, 1H), 5.82 (d, J=2.3 Hz, 1H), 5.58 (dd, J=8.2, 1.9 Hz, 1H), 5.08 (dd, J=6.8, 2.4 Hz, 1H), 4.85 (dd, J=6.9, 3.8 Hz, 1H), 4.33-4.29 (m, 1H), 4.26 (dd, J=11.5, 4.7 Hz, 1H), 4.20 (dd, J=11.5, 6.5 Hz, 1H), 3.78 (s, 3H), 2.60-2.52 (m, 1H), 1.10-1.07 (m, 6H).
[0137] Anhydrous potassium carbonate (62 mg, 0.46 mmol) and A38 (335 mg, 0.75 mmol) were added to methanol (8 mL), stirred at 35? C. for 3 h, and adjusted the pH to neutral with a 2M dilute hydrochloric acid. The reaction solution was evaporated to dryness; saturated brine and ethyl acetate were added, and the organic phase was separated, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography (dichloromethane:methanol=20:1) to obtain A34 as a white solid (143 mg, yield 50.6%).
Preparation Example 7: Synthesis of A39
[0138] ##STR00062##
[0139] Cytidine A39-0 (1.2 g, 4.94 mmol) was added to anhydrous pyridine, and imidazole (1.34 g, 19.69 mmol) and tert-butyldimethylsilyl chloride (1.12 g, 7.41 mmol) were added sequentially under an ice bath, and stirred under the ice bath. After 2 hours, methanol (5 mL) was added to the reaction solution, and the reaction solution was evaporated to dryness, and separated by silica gel column chromatography (dichloromethane:methanol=25:1) to obtain A39-1 as an oil (1.62 g, yield 92%).
[0140] A39-1 (2.72 g, 7.61 mmol) was added into waterhydroxylamine sulfate (1.51 g, 9.14 mmol) was added therein at room temperature, and the mixture was stirred overnight at 70? C. After staying overnight, ethyl acetate was added to the reaction solution, and the organic phase was separated, washed with saturated sodium bicarbonate and saturated sodium chloride aqueous solution successively, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain the crude A39-2 as a foamy solid (1.32 g, yield 47%).
[0141] A39-2 (54.6 mg, 0.147 mmol) was added to dichloromethane, and triethylamine (30 mg, 0.294 mmol) and isobutyric anhydride (24 mg, 0.147 mmol) were added successively under an ice bath, and stirred under the ice bath. After 4 hours, methanol (1 mL) was added to the reaction solution; the reaction solution was concentrated, then water was added therein, and the mixture was extracted with ethyl acetate. The organic phase was separated, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (dichloromethane:methanol=75:1) to obtain A39-3 as a white solid (40 mg, yield 62%).
[0142] A39-3 (125 mg, 0.282 mmol) was added into dichloromethane, then carbonyldiimidazole (46 mg, 0.282 mmol) was slowly added therein, and the mixture was stirred at room temperature. After 20 15 minutes, the reaction solution was evaporated to dryness; water was added, and the mixture was extracted with ethyl acetate. The organic phase was separated, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (ethyl acetate:petroleum ether=2:1) to obtain A39-4 as a white foamy solid (127 mg, yield 97%).
[0143] A39-4 (71 mg, 0.152 mmol) was added into tetrahydrofuran; acetic acid (4.6 mg, 0.076 mmol) and tetrabutylammonium fluoride (0.15 mL, 0.152 mmol) were added in sequence, and the mixture was stirred at room temperature. After 2 hours, water and ethyl acetate were added to the reaction solution. The organic phase was separated, washed successively with saturated sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (ethyl acetate: petroleum ether:methanol=10:10:1) to obtain A39 as a white solid (45 mg, yield 83%). .sup.1H NMR shows that A39 has tautomers in deuterated methanol, and the ratio of the two is about 6:1. .sup.1H NMR (500 MHz, CD.sub.3OD) ? 7.36-7.26 (m, 1H), 5.85 (d, J=2.2 Hz, 1H), 5.74 (d, J=8.1 Hz, 1H), 5.57 (dd, J=7.7, 2.2 Hz, 1H), 5.33 (dd, J=7.6, 3.9 Hz, 1H), 4.33 (q, J=4.8 Hz, 1H), 3.85-3.79 (m, 2H), 2.91-2.71 (m, 1H), 1.24 (d, J=6.9 Hz, 6H).
Preparation Example 8: Synthesis of A41
[0144] ##STR00063##
[0145] A39-2 (208 mg, 0.557 mmol) was added to dichloromethane, and triethylamine (113 mg, 1.114 mmol) and isopropyl chloroformate (76 mg, 0.613 mmol) were added successively under an ice bath, and stirred under the ice bath. After 4 hours, methanol (1 mL) was added to the reaction solution. The reaction solution was concentrated, then water was added therein, and the mixture was extracted with ethyl acetate. The organic phase was separated, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (dichloromethane:methanol=75:1) to obtain A41-1 as a white foamy solid (148 mg, yield 58%).
[0146] A41-1 (275 mg, 0.599 mmol) was added into dichloromethane, then carbonyldiimidazole (146 mg, 0.899 mmol) was slowly added therein, and the mixture was stirred at room temperature. After minutes, the reaction solution was evaporated to dryness, water was added, and the mixture was extracted with ethyl acetate. The organic phase was separated, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (ethyl acetate:petroleum ether=2:1) to obtain A41-2 as a white powdery solid (233 mg, yield 80%).
[0147] A41-2 (233 mg, 0.481 mmol) was added into tetrahydrofuran, acetic acid (15 mg, 0.241 mmol) and tetrabutylammonium fluoride (0.48 mL, 0.152 mmol) were added in sequence, and the mixture was stirred at room temperature. After 2 hours, water and ethyl acetate were added to the reaction solution, the organic phase was separated, washed successively with saturated sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (ethyl acetate: petroleum ether:methanol=10:10:1) to obtain A41 as a white solid (112 mg, yield 63%). .sup.1H NMR shows that A41 has tautomers in deuterated methanol, and the ratio of the two is about 7:1. .sup.1H NMR (500 MHz, CD.sub.3OD) ? 7.34-7.24 (m, 1H), 5.83 (d, J=2.1 Hz, 1H), 5.72 (d, J=8.1 Hz, 1H), 5.56 (dd, J=7.6, 2.2 Hz, 1H), 5.32 (dd, J=7.6, 3.9 Hz, 1H), 5.02-4.94 (m, 1H), 4.32 (q, J=4.9 Hz, 1H), 3.84-3.78 (m, 2H), 1.36 (d, J=6.2 Hz, 6H).
Preparation Example 9: Synthesis of A52
[0148] ##STR00064##
[0149] 30
[0150] A39-2 (373 mg, 1.0 mmol) was added into pyridine (10 mL), then dimethylcarbamoyl chloride (113 mg, 1.114 mmol) was added under an ice bath, and the mixture was reacted overnight at room temperature. The reaction solution was evaporated to dryness, water was added, and the mixture was extracted with ethyl acetate. The organic phase was separated, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (dichloromethane:methanol=60:1) to obtain A52-1 as a white foamy solid (186 mg, yield 42%).
[0151] Referring to the reaction conditions of the second and third steps in Example 8, using A52-1 (186 mg, 0.42 mmol) as a raw material, A52 was prepared as a foamy solid (60 mg, yield 40%).
[0152] Preparation Example 10: Synthesis of B1
##STR00065##
[0153] Triphenylphosphine (11.79 g, 45 mmol) was added to pyridine (50 mL), and iodine (11.42 g, 45 mmol) was added under an ice bath, stirred for 10 minutes, and then warmed to room temperature. Uridine B1-0 (7.32 g, 20 mmol) was added, and stirred overnight at 25? C. Saturated sodium thiosulfate and saturated sodium bicarbonate were added in sequence, and the reaction solution was rotary evaporated to dryness. Tetrahydrofuran and saturated saline were added to the concentrate, and the organic phase and the aqueous phase were separated. The aqueous phase was extracted twice with tetrahydrofuran, and the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography (dichloromethane:methanol=15:1) to obtain B1-1 as a yellow solid (5.30 g, yield 75%).
[0154] Sodium (1.04 g, 45 mmol) was added to methanol (40 mL) under an ice bath to prepare a solution of sodium methoxide in methanol. B1-1 (5.30 g, 15 mmol) was added into methanol (50 mL), the solution of sodium methoxide in methanol prepared above was added, and the mixture was stirred under reflux at 67? C. After 2 h, a dilute hydrochloric acid was added to adjust pH to 8-9, and the reaction solution was rotary evaporated to dryness, and separated by silica gel column chromatography (dichloromethane:methanol=15:1) to obtain B1-2 as a white solid (2.1 g, yield 62%).
[0155] B1-2 (2.10 g, 9.3 mmol) and triethylamine trihydrofluoride (1.80 g, 11.2 mmol) were added to acetonitrile (30 mL), and N-iodosuccinimide (2.51 g, 11.2 mmol) was added in batches under an ice bath, stirred for 30 minutes, naturally settled for 1 hour, filtered, rinsed with dichloromethane to obtain B1-3 (1.5 g, yield 43%).
[0156] B1-3 (950 mg, 2.55 mmol) was added to anhydrous tetrahydrofuran (5 mL), then carbonyldiimidazole (616 mg, 3.8 mmol) was added therein, and the mixture was stirred at room temperature for 30 min. The reaction solution was evaporated to dryness, and separated by silica gel column chromatography (petroleum ether:ethyl acetate=1:1 to 1:2) to obtain B1-4 as a white foamy solid (520 mg, yield 51%).
[0157] Trifluoroacetic acid was added to an aqueous solution of tetrabutylammonium hydroxide (1.45 g, 5.5 mmol) to adjust pH to about 4, and the above solution was added to a solution of B1-4 (440 mg, 1.1 mmol) in dichloromethane (5 mL); m-chloroperoxybenzoic acid (952 mg, 5.5 mmol) was added therein, and the mixture was stirred at room temperature. After 7 h, saturated sodium thiosulfate solution was added, and then saturated brine and ethyl acetate were added. The ethyl acetate layer and the water layer were separated. Then the aqueous layer was extracted with tetrahydrofuran, and the tetrahydrofuran layer was separated. The organic phase was combined, dried with anhydrous sodium sulfate, concentrated, separated by silica gel column chromatography (petroleum ether:ethyl acetate=1:3), and slurried in ethyl acetate to obtain B1 as a white solid (220 mg, yield 69%). 1 H NMR (500 MHz, DMSO-d.sub.6) ? 11.62 (s, 1H), 7.80 (d, J=8.1 Hz, 1H), 6.29 (d, J=1.3 Hz, 1H), 5.75 (dd, J=7.3, 1.3 Hz, 1H), 5.71 (d, J=8.1 Hz, 1H), 5.61 (t, J=6.6 Hz, 1H), 5.55 (dd, J=12.4, 7.3 Hz, 1H), 3.78-3.69 (m, 1H), 3.67-3.58 (m, 1H).
Preparation Example 11: Synthesis of B2
[0158] ##STR00066##
[0159] B1 (57 mg, 0.2 mmol), triethylamine (81 mg, 0.8 mmol) and 4-dimethylaminopyridine (12 mg, 0.1 mmol) were sequentially added to dichloromethane (3 mL), isobutyryl chloride (32 mg, 0.3 mmol) was added therein, and the mixture was stirred at room temperature. After 2 hours, dichloromethane and saturated saline were added, and the organic phase was separated, dried over anhydrous sodium sulfate, concentrated, and subjected to silica gel column chromatography (dichloromethane:methanol=30:1) to obtain B2 as a white solid (54 mg, yield 75%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 11.63 (s, 1H), 7.79 (d, J=8.0 Hz, 1H), 6.35 (s, 1H), 5.77 (d, J=7.3 Hz, 1H), 5.71 (d, J=8.0 Hz, 1H), 5.62 (dd, J=11.7, 7.3 Hz, 1H), 4.47-4.35 (m, 2H), 2.66-2.57 (m, 1H), 1.11 (d, J=7.0 Hz, 6H).
Preparation Example 12: Synthesis of C1
[0160] ##STR00067##
[0161] GS-441524 (85 mg, 0.292 mmol) was added to N,N-dimethylformamide, then carbonyldiimidazole (48 mg, 0.292 mmol) was added therein, and the mixture was stirred at room temperature. After 15 minutes, water and ethyl acetate were added to the reaction solution, and the organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (dichloromethane:methanol=20:1) to obtain Cl as a white solid (19 mg, yield 21%). .sup.1H NMR (500 MHz, DMSO-d.sub.6) ? 8.16 (s, 1H), 8.02 (s, 1H), 8.00 (s, 1H), 7.02-6.98 (m, 2H), 5.96 (d, J=7.9 Hz, 1H), 5.40 (dd, J=7.8, 4.0 Hz, 1H), 5.28 (t, J=5.7 Hz, 1H), 4.50 (q, J=4.7 Hz, 1H), 3.72-3.65 (m, 1H), 3.64-3.58 (m, 1H).
Preparation Example 13: Synthesis of C22
[0162] ##STR00068##
[0163] C22-0 (48.7 mg, 0.17 mmol) was added to N,N-dimethylformamide (1 mL), then carbonyldiimidazole (28 mg, 0.17 mmol) was added therein, and the mixture was stirred at room temperature. After 5 minutes, water and ethyl acetate were added, and the aqueous layer and the ethyl acetate layer were separated. The aqueous layer was extracted three times with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (dichloromethane:methanol=20:1) to obtain 18 mg of C22 as a white solid. .sup.1H NMR (500 MHz, DMSO-d.sub.6) ? 8.12 (s, 1H), 8.02 (s, 1H), 8.00 (s, 1H), 6.99 (s, 1H), 5.95 (d, J=7.8 Hz, 1H), 5.39 (dd, J=7.8, 4.0 Hz, 1H), 5.25 (t, J=5.7 Hz, 1H), 4.52-4.48 (m, 1H), 3.71-3.66 (m, 1H), 3.64-3.58 (m, 1H).
Preparation Example 14: Synthesis of C38
[0164] ##STR00069##
[0165] Propionaldehyde (290 mg, 5.0 mmol) and C22-0 (292 mg, 1.0 mmol) were added to dichloromethane (10 mL), then p-toluenesulfonic acid monohydrate (380 mg, 2.0 mmol) was added therein under an ice bath, the mixture was stirred for 10 minutes, warmed to room temperature, and continued to stir for 2 hours. The reaction solution was poured into saturated sodium bicarbonate solution and extracted with dichloromethane. An ammonia ethanol solution was added to the organic layer and concentrated to obtain an oil. Ethyl acetate and saturated brine were added to the concentrate, and the organic phase was seperated, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (dichloromethane:methanol=50:1 to 15:1) to obtain C38 as a white solid (96 mg, yield 30%). .sup.1H NMR (500 MHz, DMSO-d.sub.6) ? 8.05-7.88 (m, 3H), 6.91 (s, 1H), 5.35 (d, J=6.7 Hz, 1H), 5.17 (t, J=5.0 Hz, 1H), 5.03 (t, J=5.6 Hz, 1H), 4.80 (dd, J=6.7, 2.9 Hz, 1H), 4.38-4.35 (m, 1H), 3.59-3.48 (m, 2H), 1.90-1.83 (m, 2H), 0.99 (t, J=7.5 Hz, 3H).
Preparation Example 15: Synthesis of A54
[0166] ##STR00070##
[0167] NHC (15.0 g, 61.67 mmol), imidazole (12.6 g, 185.29 mmol) were added to N,N-dimethylformamide (50 mL), and tert-butyldiphenylchlorosilane (25.4 g, 92.51 mmol) was added dropwise under an ice bath. After the addition was completed, the reaction solution was warmed to room temperature and stirred. After 4 hours, distilled water (100 mL) was added dropwise to the reaction solution to precipitate a solid, which was filtered and the filter cake was air-dried at 50? C. to obtain A54-1 as a white solid (25 g, yield 84%).
[0168] A54-1 (25.0 g, 51.90 mmol), hydroxylamine sulfate (25.0 g, 152.44 mmol) were added into acetonitrile/water (120 mL/120 mL). After the addition was complete, the reaction solution was heated to 60? C. and stirred overnight. After the reaction was complete, the reaction solution was cooled to room temperature, and the organic layer was separated. The aqueous layer was extracted with ethyl acetate, and the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain A54-2 as a white solid, which was directly used in the next reaction.
[0169] The product obtained in the previous step, 4-methoxytriphenylchloromethane (19.2 g, 62.30 mmol), triethylamine (10.5 g, 103.80 mmol) were added into dichloromethane (200 mL), and stirred at room temperature. After 2-3 hours, methanol (1 mL) was added to the reaction solution and concentrated to obtain A54-3 as a yellow foamy solid, which was directly used for the next reaction. The product obtained in the previous step was added into dichloromethane (200 mL), then carbonyldiimidazole (10.1 g, 62.28 mmol) was added under an ice bath, and the mixture was stirred at room temperature after the addition was complete. After 2 hours, the reaction solution was poured into water, extracted with dichloromethane, the organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography (dichloromethane: methanol=30:1) to obtain A54-4 as an off-white solid (31.5 g, yield 76% over three steps).
[0170] A54-4 (31.5 g, 39.57 mmol), acetic acid (1.2 g, 19.79 mmol) were added to tetrahydrofuran (200 mL), then a 1M tetrabutylammonium fluoride solution (43.5 mL, 43.5 mmol) in tetrahydrofuran was added therein at room temperature, and the mixture was stirred at room temperature. After 2 hours, the reaction solution was concentrated, water and ethyl acetate were added, and the organic phase was separated, washed successively with saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography (dichloromethane: methanol=30:1) to obtain A54-5 as an off-white solid (15 g, yield 68%).
[0171] A54-5 (800 mg, 1.43 mmol), triethylamine (289 mg, 2.86 mmol), 4-dimethylaminopyridine (35 mg, 0.29 mmol), acetic anhydride (220 mg, 2.15 mmol) were sequentially added to dichloromethane (10 mL), and stirred at room temperature. After 2 hours, dichloromethane and water were added to the reaction solution, and the organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain A54-6, which was directly used in the next reaction.
[0172] The product obtained in the previous step was added to methanol (5 mL), then trifluoroacetic acid (326 mg, 2.86 mmol) was added therein, and the mixture was stirred at room temperature. After 1 hour, the reaction solution was concentrated and separated by silica gel column chromatography (dichloromethane:methanol=30:1) to obtain A54 as a white solid (40 mg, yield 9%). .sup.1H NMR (500 MHz, DMSO-d.sub.6) ? 10.10 (s, 1H), 9.88 (s, 1H), 6.95 (d, J=8.1 Hz, 1H), 5.86 (d, J=2.1 Hz, 1H), 5.59 (d, J=8.1 Hz, 1H), 5.56 (dd, J=7.7, 2.2 Hz, 1H), 5.27 (dd, J=7.7, 4.2 Hz, 1H), 4.43-4.37 (m, 1H), 4.31 (dd, J=11.6, 4.7 Hz, 1H), 4.18 (dd, J=11.6, 7.0 Hz, 1H), 2.04 (s, 3H). MS m/z=328.2 [M+1].sup.+.
Preparation Example 16: Synthesis of A55
[0173] ##STR00071##
[0174] A54-5 (800 mg, 1.43 mmol), palmitic acid (554 mg, 2.16 mmol), 1-hydroxybenzotriazole (350 mg, 2.59 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (663 mg, 3.46 mmol), 4-dimethylaminopyridine (176 mg, 1.44 mmol) were added to dichloromethane (10 mL), and stirred at room temperature. After 2 hours, dichloromethane and water were added to the reaction solution, and the organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain A55-0, which was directly used in the next reaction.
[0175] A55-0 and trifluoroacetic acid (328 mg, 2.88 mmol) were added into methanol (5 mL), and stirred at room temperature. After 1 hour, the reaction solution was concentrated and separated by silica gel column chromatography (dichloromethane:methanol=30:1) to obtain A55 as a white solid (70 mg, yield 9%). .sup.1H NMR (500 MHz, DMSO-d.sub.6) ? 10.11 (s, 1H), 9.88 (s, 1H), 6.95 (d, J=8.1 Hz, 1H), 5.86 (d, J=2.2 Hz, 1H), 5.58 (d, J=8.1 Hz, 1H), 5.55 (dd, J=7.7, 2.2 Hz, 1H), 5.26 (dd, J=7.8, 4.1 Hz, 1H), 4.42-4.36 (m, 1H),4.31 (dd, J=11.5, 4.9 Hz, 1H), 4.19 (dd, J=11.6, 7.0 Hz, 1H), 2.31 (t, J=7.4 Hz, 2H), 1.31-1.14 (m, 27H), 0.85 (t, J=6.8 Hz, 3H). MS m/z=524.5 [M+1].sup.+.
Preparation Example 17: Synthesis of A56
[0176] ##STR00072##
[0177] A54-5 (600 mg, 1.08 mmol), triethylamine (216 mg, 2.16 mmol), 4-dimethylaminopyridine (27 mg, 0.22 mmol), pivaloyl chloride (195 mg, 1.62 mmol) were added to dichloromethane (10 mL), and stirred at room temperature. After 2 hours, water and dichloromethane were added, and the organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain A56-0, which was directly used in the next reaction.
[0178] A56-0 and trifluoroacetic acid (246 mg, 2.16 mmol) were added into methanol (5 mL), and stirred at room temperature. After 1 hour, the reaction solution was concentrated and separated by silica gel column chromatography (dichloromethane:methanol=30:1) to obtain A56 as a white solid (140 mg, yield 35%). 1 H NMR (500 MHz, DMSO-d.sub.6) ? 10.10 (s, 1H), 9.88 (d, J=2.0 Hz, 1H), 6.94 (d, J=8.1 Hz, 1H), 5.86 (d, J=2.0 Hz, 1H), 5.61-5.54 (m, 2H), 5.27 (dd, J=7.7, 4.2 Hz, 1H), 4.40 (q, J=5.6 Hz, 1H), 4.31-4.18 (m, 2H), 1.15 (s, 9H). MS m/z=370.0 [M+1].sup.+.
Preparation Example 18: Synthesis of A4
[0179] ##STR00073##
[0180] A54-5 (700 mg, 1.26 mmol), triethylamine (255 mg, 2.52 mmol), 4-dimethylaminopyridine (31 mg, 0.25 mmol), cyclopropylformyl chloride (198 mg, 1.89 mmol) were added to dichloromethane (10 mL), and stirred at room temperature. After 2 hours, water and dichloromethane were added, and the organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain A4-0, which was directly used in the next reaction.
[0181] A4-0 and trifluoroacetic acid (287 mg, 2.52 mmol) were added into methanol (5 mL), and stirred at room temperature. After 1 hour, the reaction solution was concentrated and separated by silica gel column chromatography (dichloromethane:methanol=30:1) to obtain A4 as a white solid (150 mg, yield 34%). .sup.1H NMR (500 MHz, DMSO-d.sub.6) ? 10.10 (s, 1H), 9.87 (d, J=2.1 Hz, 1H), 6.95 (d, J=8.1 Hz, 1H), 5.87 (d, J=2.1 Hz, 1H), 5.59 (dd, J=8.1, 2.0 Hz, 1H), 5.56 (dd, J=7.7, 2.2 Hz, 1H), 5.27 (dd, J=7.7, 4.2 Hz, 1H), 4.44-4.38 (m, 1H), 4.32 (dd, J=11.6, 4.9 Hz, 1H), 4.20 (dd, J=11.6, 7.0 Hz, 1H), 1.69-1.61 (m, 1H), 0.93-0.88 (m, 2H), 0.87-0.82 (m, 2H). MS m/z=354.2 [M+1].sup.+.
Preparation Example 19: Synthesis of A6
[0182] ##STR00074##
[0183] A54-5 (15.27 g, 27.39 mmol) was added to dichloromethane (200 mL), and triethylamine (11.09 g, 109.56 mmol), Boc-L-valine (8.34 g, 38.35 mmol), 1-hydroxybenzotriazole (5.56 g, 41.09 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (11.56 g, 60.26 mmol), 4-dimethylaminopyridine (3.35 g, 27.39 mmol) were sequencely added under an ice bath. After the addition, the reaction solution was warmed to room temperature and stirred. After 3 hours, the reaction solution was concentrated, and water and ethyl acetate were added. The organic phase was separated, washed with saturated sodium bicarbonate and saturated brine successively, dried over anhydrous sodium sulfate, and concentrated to obtain A6-0.
[0184] A6-0 (20.73 g, 27.39 mmol) was added to dichloromethane/methanol (10:1), then trifluoroacetic acid (6.37 g, 55.82 mmol) was added therein, and the mixture was stirred at room temperature for 4 hours. The reaction solution was concentrated, and separated by silica gel column chromatography (dichloromethane:methanol=40:1) to obtain A6-1.
[0185] A6-1 (5.81 g, 12.00 mmol) was added to tetrahydrofuran (50 mL), then concentrated hydrochloric acid (12 mL, 144.00 mmol) was added therein, and the mixture was stirred overnight at room temperature. The reaction solution was concentrated, slurried with isopropanol, and filtered to obtain A6 hydrochloride as a white solid (3.4 g, yield 29% over three steps). .sup.1H NMR (500 MHz, CD.sub.3OD) ? 7.87 (d, J=7.9 Hz, 1H), 6.08 (d, J=7.9 Hz, 1H), 5.96 (d, J=1.2 Hz, 1H), 5.81 (dd, J=7.4, 1.3 Hz, 1H), 5.49 (dd, J=7.5, 3.8 Hz, 1H), 4.75 (dd, J=11.5, 7.6 Hz, 1H), 4.64 (dt, J=7.8, 4.2 Hz, 1H), 4.54 (dd, J=11.6, 4.5 Hz, 1H), 4.04 (d, J=4.4 Hz, 1H), 2.41-2.26 (m, 1H), 1.10 (dd, J=6.9, 4.5 Hz, 6H).
Preparation Example 20: Synthesis of B4
[0186] ##STR00075##
[0187] B1 (100.0 mg, 0.35 mmol) was added to dichloromethane (6 mL), and cyclopropanecarboxylic acid (42.0 mg, 0.49 mmol), 1-hydroxybenzotriazole (112.4 mg, 0.53 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (147.8 mg, 0.77 mmol), 4-dimethylaminopyridine (170.8 mg, 1.40 mmol) were sequencely added under an ice bath. After the addition, the reaction solution was warmed to room temperature and stirred. After 3 hours, the reaction solution was concentrated; water and ethyl acetate were added, and the organic phase was separated, washed successively with dilute hydrochloric acid, saturated sodium bicarbonate, and saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (petroleum ether:acetone=1:1) to obtain B4 as a white solid (95 mg, yield 80%). .sup.1H NMR (500 MHz, DMSO-d.sub.6) ? 11.64 (s, 1H), 7.79 (d, J=8.0 Hz, 1H), 6.35 (d, J=1.2 Hz, 1H), 30 5.78 (dd, J=7.2, 1.2 Hz, 1H), 5.72 (dd, J=8.0, 2.2 Hz, 1H), 5.64 (dd, J=11.7, 7.3 Hz, 1H), 4.47 -4.35 (m, 2H), 1.73-1.67 (m, 1H), 0.99-0.88 (m, 4H).
Preparation Example 21: Synthesis of B41
[0188] ##STR00076##
[0189] B1 (60.0 mg, 0.21 mmol) was added to dichloromethane (4 mL), and palmitic acid (74.5 mg, 0.29 mmol), 1-hydroxybenzotriazole (67.8 mg, 0.32 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (88.3 mg, 0.46 mmol), 4-dimethylaminopyridine (102.5 mg, 0.84 mmol) were sequencely added under an ice bath. After the addition, the reaction solution was warmed to room temperature and stirred. After 3 hours, the reaction solution was concentrated;water and ethyl acetate were added, and the organic phase was separated, sequencely washed with dilute hydrochloric acid, saturated sodium bicarbonate, and saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (dichloromethane:methanol=30:1) to obtain B41 as a white solid (90 mg, yield 82%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 11.63 (s, 1H), 7.78 (d, J=8.1 Hz, 1H), 6.34 (s, 1H), 5.77 (d, J=7.5 Hz, 1H), 5.70 (d, J=7.9 Hz, 1H), 5.62 (t, J=9.7 Hz, 1H), 4.48-4.31 (m, 2H), 2.38-2.32 (m, 2H), 1.56-1.48 (m, 2H), 1.31-1.21 (m, 24H), 0.85 (t, J=6.6 Hz, 3H).
Preparation Example 22: Synthesis of B6
[0190] ##STR00077##
[0191] B1 (200.0 mg, 0.695 mmol) was added to dichloromethane (10 mL), and Boc-L-valine (211.0 mg, 0.973 mmol), 1-hydroxybenzotriazole (221.1 mg, 1.043 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (293.6 mg, 1.529 mmol), 4-dimethylaminopyridine (339.2 mg, 2.780 mmol) were sequencely added under an ice bath. After the addition, the reaction solution was warmed to room temperature and stirred. After 3 hours, the reaction solution was concentrated; water and ethyl acetate were added, and the organic phase was separated, washed successively with dilute hydrochloric acid, saturated sodium bicarbonate, saturated brine, and dried over anhydrous sodium sulfate, and concentrated to obtain B1-1.
[0192] B1-1 was added to dichloromethane (2 mL), then trifluoroacetic acid (1 mL) was added therein, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was concentrated, and separated by silica gel column chromatography (dichloromethane:methanol=20:1) to obtain B6 as a pale yellow solid (245 mg, yield 90%). 1 H NMR (500 MHz, DMSO-d.sub.6) ? 11.66 (s, 1H), 8.44 (s, 2H), 7.80 (d, J=8.0 Hz, 1H), 6.38 (s, 1H), 5.81 (d, J=7.2 Hz, 1H), 5.74-5.66 (m, 2H), 4.72 (dd, J=15.7, 12.3 Hz, 1H), 4.48 (dd, J=16.2, 12.4 Hz, 1H), 4.02 (d, J=4.4 Hz, 1H), 2.22-2.15 (m, 1H), 0.98 (d, J=6.9 Hz, 3H), 0.94 (d, J=6.9 Hz, 3H).
Preparation Example 23: Synthesis of B13
[0193] ##STR00078##
[0194] B1 (50.0 mg, 0.17 mmol), N-[(S)-(2,3,4,5,6-pentafluorophenoxy)phenoxyphosphoryl]-L-alanine isopropyl ester (86.7 mg, 0.19 mmol), anhydrous magnesium chloride (24.8 mg, 0.26 mmol) were added to anhydrous tetrahydrofuran (4 mL), then N,N-diisopropylethylamine (44.9 mg, 0.35 mmol) was added therein under an ice bath, the mixture was stirred for 10 minutes and then warmed to room temperature. After 4 hours, the reaction solution was concentrated; and water and ethyl acetate were added, and the organic phase was separated, sequencely washed with dilute hydrochloric acid, saturated sodium bicarbonate, and saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (dichloromethane:methanol=30:1) to obtain B13 as a white solid (56 mg, yield 58%). .sup.1H NMR (500 MHz, CD.sub.3OD) ? 7.67 (d, J=8.0 Hz, 1H), 7.37 (t, J=7.8 Hz, 2H), 7.26 (d, J=8.1 Hz, 2H), 7.20 (t, J=7.5 Hz, 1H), 6.15 (s, 1H), 5.70 (d, J=8.0 Hz, 1H), 5.68-5.64 (m, 2H), 5.02-4.94 (m, 1H), 4.43-4.36 (m, 1H), 4.35-4.27 (m, 1H), 3.96-3.89 (m, 1H), 1.36 (d, J=7.1 Hz, 3H), 1.25-1.21 (m, 6H).
Preparation Example 24: Synthesis of B14
[0195] ##STR00079##
[0196] B1 (50.0 mg, 0.17 mmol), N-[(S)-(2,3,4,5,6-pentafluorophenoxy)phenoxyphosphoryl]-L-alanine ethyl-n-butyl ester (95.1 mg, 0.19 mmol), anhydrous magnesium chloride (24.8 mg, 0.26 mmol) were added to anhydrous tetrahydrofuran (3 mL), then N,N-diisopropylethylamine (44.9 mg, 0.35 mmol) was added under an ice bath, the mixture was stirred for 10 minutes and then warmed to room temperature. After 12 hours, the reaction solution was concentrated; and water and ethyl acetate were added, and the organic phase was separated, sequencely washed with dilute hydrochloric acid, saturated sodium bicarbonate, and saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (dichloromethane:methanol=30:1) to obtain B14 as a white solid (51 mg, yield 50%). .sup.1H NMR (500 MHz, CD.sub.3OD) ? 7.67 (d, J=7.9 Hz, 1H), 7.37 (t, J=7.8 Hz, 2H), 7.26 (d, J=8.1 Hz, 2H), 7.20 (t, J=7.5 Hz, 1H), 6.15 (s, 1H), 5.70 (d, J=8.0 Hz, 1H), 5.68-5.63 (m, 2H), 4.42-4.27 (m, 2H), 4.10-4.02 (m, 2H), 4.01-3.95 (m, 1H), 1.55-1.49 (m, 1H), 1.40-1.34 (m, 4H), 1.22 (d, J=6.2 Hz, 3H), 0.90 (t, J=7.4 Hz, 6H).
Preparation Example 25: Synthesis of B44
[0197] ##STR00080##
[0198] B1 (30.0 mg, 0.10 mmol), 4-dimethylaminopyridine (15.3 mg, 0.125 mmol), pyridine (0.1 mL) were added to dichloromethane (2 mL), and ethyl chloroformate (13.6 mg, 0.125 mmol) was added under an ice bath. After the addition was completed, the reaction solution was warmed to room temperature and stirred. After 4 hours, the reaction solution was concentrated; water and ethyl acetate were added, and the organic phase was separated, sequencely washed with dilute hydrochloric acid, saturated sodium bicarbonate, and saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (dichloromethane:methanol=20:1) to obtain B44 as a white solid (32 mg, yield 89%). .sup.1H NMR (500 MHz, DMSO-d.sub.6) ? 11.64 (s, 1H), 7.79 (d, J=8.0 Hz, 1H), 6.35 (s, 1H), 5.78 (d, J=8.0 Hz, 1H), 5.71 (dd, J=8.0, 2.2 Hz, 1H), 5.67 (dd, J=11.4, 7.3 Hz, 1H), 4.49-4.39 (m, 2H), 4.16 (q, J=7.1 Hz, 2H), 1.23 (t, J=7.1 Hz, 3H).
Preparation Example 26: Synthesis of B38
[0199] ##STR00081##
[0200] B1 (73.0 mg, 0.25 mmol) was added into dichloromethane (3 mL), and triethylamine (38.4 mg, 25 0.38 mmol), 4-dimethylaminopyridine (6.1 mg, 0.05 mmol), acetic anhydride (31.0 mg, 0.30 mmol) were sequencely added under an ice bath. After the addition, the reaction solution was warmed to room temperature and stirred. After 1 hour, dichloromethane (20 mL) was added to the reaction solution, and washed with 1 M hydrochloric acid aqueous solution, saturated sodium bicarbonate solution, and saturated brine successively. The organic phase was separated, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography (petroleum ether : ethyl acetate=1:1) to obtain B38 as a white solid (63 mg, yield 76%). .sup.1H NMR (500 MHz, DMSO-d.sub.6) ? 11.63 (s, 1H), 7.79 (d, J=8.0 Hz, 1H), 6.34 (s, 1H), 5.77 (d, J=7.3 Hz, 1H), 5.71 (d, J=8.0 Hz, 1H), 5.64 (dd, J=11.7, 7.3 Hz, 1H), 4.46-4.31 (m, 2H), 2.08 (s, 3H).
Preparation Example 27: Synthesis of C2
[0201] ##STR00082##
[0202] Referring to the reaction conditions of Preparation Example 11, C1 (95 mg, 0.3 mmol) was reacted with isobutyryl chloride (38 mg, 0.36 mmol) to obtain C2 as a white solid (27 mg, yield 23%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.10 (s, 1H), 8.02 (s, 1H), 7.99 (s, 1H), 6.96 (d, J=4.6 Hz, 1H), 6.91 (d, J=4.6 Hz, 1H), 6.00 (d, J=7.7 Hz, 1H), 5.50 (dd, J=7.7, 3.7 Hz, 1H), 4.86-4.79 (m, 1H), 4.34 (dd, J=12.3, 4.0 Hz, 1H), 4.24 (dd, J=12.2, 5.2 Hz, 1H), 2.49-2.39 (m, 1H), 1.03 (d, J =7.0 Hz, 3H), 0.99 (d, J=7.0 Hz, 3H).
Preparation Example 28: Synthesis of C23
[0203] ##STR00083##
[0204] Referring to the reaction conditions of Preparation Example 11, C22 (477 mg, 1.5 mmol) was reacted with isobutyryl chloride (192 mg, 0.18 mmol) to obtain C23 as a white solid (105 mg, yield 18%). 1 H NMR (500 MHz, DMSO-d.sub.6) ? 8.11 (s, 1H), 8.03 (s, 1H), 7.98 (s, 1H), 6.90 (s, 1H), 5.99 (d, J=7.7 Hz, 1H), 5.49 (dd, J=7.6, 3.7 Hz, 1H), 4.82 (dt, J=5.2, 3.8 Hz, 1H), 4.33 (dd, J=12.3, 3.9 Hz, 1H), 4.22 (dd, J=12.3, 5.2 Hz, 1H), 2.47 -2.39 (m, 1H), 1.01 (d, J=7.0 Hz, 3H), 0.98 (d, J=7.0 Hz, 3H). 13 C NMR (126 MHz, DMSO-d.sub.6) ? 175.5, 155.5, 152.9, 148.5, 120.0, 117.1, 114.1, 110.5, 82.7, 81.5, 80.1, 79.5, 62.1, 33.0, 18.6, 18.5.
Preparation Example 29: Synthesis of C54
[0205] ##STR00084##
[0206] GS-441524 (611 mg, 2.1 mmol) was added to pyridine (5 mL), then N,N-dimethylformamide dimethyl acetal (1 g, 8.4 mmol) was added therein, and the mixture was stirred overnight at room temperature. The reaction solution was concentrated to dryness to obtain the crude C54-1, which was directly used for the next step.
[0207] C54-1 was added to dichloromethane (5 mL), and triethylamine (142 mg, 1.4 mmol), cyclobutylformyl chloride (125 mg, 1.05 mmol) and 4-dimethylaminopyridine (86 mg, 0.7 mmol) were sequencely added under an ice bath, and stirred at room temperature. After 1 hour, methanol was added to the reaction solution, the reaction solution was concentrated, ethyl acetate and water were added, the mixture was stirred and layered. The organic phase was separated, washed with dilute hydrochloric acid aqueous solution, saturated sodium bicarbonate and saturated sodium chloride successively, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain C54-2.
[0208] C54-2 (0.35 mmol) was added to ethanol (3 mL), then acetic acid (0.6 mL, 10.5 mmol) was added therein, the mixture was heated and stirred at 50? C. overnight. The reaction solution was concentrated, saturated brine was added, and the mixture was extracted with ethyl acetate. The organic phase was separated, washed with saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, concentrated, and slurried in isopropyl acetate to obtain C54-3 as a white powdery solid (93 mg, yield 66.0% over three steps). .sup.1H NMR (500 MHz, DMSO-d.sub.6) ? 8.06-7.79 (m, 3H), 6.93 (d, J=4.5 Hz, 1H), 6.82 (d, J=4.5 Hz, 1H), 6.34 (d, J=6.0 Hz, 1H), 5.38 (d, J=5.9 Hz, 1H), 4.74-4.68 (m, 1H), 4.31 (dd, J=12.2, 2.9 Hz, 1H), 4.26-4.21 (m, 1H), 4.15 (dd, J=12.2, 5.0 Hz, 1H), 4.00-3.94 (m, 1H), 2.30-2.22 (m, 1H), 1.81-1.54 (m, 5H), 1.34-1.11 (m, 5H).
[0209] C54-3 (100 mg, 0.25 mmol, 1 eq) was added to tetrahydrofuran (4 mL) under an ice bath, and carbonyldiimidazole (83 mg, 0.51 mmol, 2 eq) was added thereto. The ice bath was removed, and the reaction solution was stirred at room temperature for 3 h. Methanol was added, then 1 N dilute hydrochloric acid solution was added therein, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, evaporated to dryness, and separated by column chromatography to obtain C54 as a white solid (72 mg, yield 67%). 1 H NMR (500 MHz, DMSO-d.sub.6) ? 8.10 (s, 1H), 8.02 (s, 1H), 7.98 (s, 1H), 6.95 (d, J=4.6 Hz, 1H), 6.89 (d, J=4.6 Hz, 1H), 5.99 (d, J=7.5 Hz, 1H), 5.49 (dd, J=7.5, 3.4 Hz, 1H), 4.85 (q, J=3.8 Hz, 1H), 4.31 (dd, J=12.3, 3.7 Hz, 1H), 4.21 (dd, J=12.3, 5.0 Hz, 1H), 1.71-1.50 (m, 5H), 1.28-1.02 (m, 5H). MS m/z=428.4 [M+1].sup.+.
Preparation Example 30: Synthesis of B50
[0210] ##STR00085##
[0211] B0 was synthesized by the method in reference (WO2014100505). B0 (700.0 mg, 2.67 mmol, 1 eq) was added to trimethyl orthoformate (7 mL), then p-toluenesulfonic acid monohydrate (507.4 mg, 2.67 mmol, 1 eq) was added therein, and the mixture was reacted at room temperature. The reaction liquid gradually becomes clear. After TLC showed that the reaction was complete, the reaction solution was adjusted pH to 6-7 with a 7 N ammonia methanol solution. The reaction was filtered, and the filtrate was concentrated and separated by silica gel column chromatography (dichloromethane:methanol=60:1 to 40:1) to obtain B50 as a white foamy solid (yield 84%). .sup.1H NMR (500 MHz, DMSO-d.sub.6) ? 11.52 (d, J=2.2 Hz, 1H), 7.73 (d, J=8.1 Hz, 1H), 6.09-6.05 (m, 2H), 5.67 (dd, J=8.0, 2.2 Hz, 1H), 5.47 (t, J=6.4 Hz, 1H), 5.29 (dd, J=14.3, 6.4 Hz, 1H), 5.11 (dd, J=6.4, 1.3 Hz, 1H), 3.65-3.53 (m, 2H), 3.22 (s, 3H).
Test Example 1: In Vivo Pharmacokinetic Evaluation in Rats
Experimental Method:
[0212] 6 Male SD rats for each compound were divided into 2 groups (gastric administration group and intravenous injection group), 3 rats in each group. The rats were fasted for 12 h before the experiment (the intravenous experiment group was not fasted), had free access to water, and ate uniformly 4 hours after the administration. The dosage of A1 and A2 was 20 mg/kg for intragastric administration and 5 mg/kg for intravenous injection, and the administration vehicle was 5% DMSO +5% solutol+90% saline. The dosage of C38 was 10 mg/kg for intragastric administration, 2 mg/kg for intravenous injection, and the administration vehicle was DMSO/EtOH/PEG300/0.9% NaCl (5/5/40/50, v/v/v/v). 0.2 mL of blood was collected from the jugular vein at 5 min (intravenous group only), 0.25 h, 0.5 h, 1.0 h, 2.0 h, 4.0 h, 6.0 h, 8.0 h and 24 h after the administration, placed in an EDTA-K2 test tube and centrifuged at 11,000 rpm for 5 minutes, and the plasma was separated, and stored in a ?70? C. refrigerator for testing. The operation was under an ice water bath. The concentration of nucleoside metabolites in plasma was determined by LC-MS-MS, and the pharmacokinetic parameters were calculated.
TABLE-US-00001 TABLE 1 In vivo pharmacokinetic parameters of nucleoside metabolites of A1, A2 and C38 in rats Compound A1 A1 A2 A2 C38 C38 PK parameters (po-20mpk) (iv-5mpk) (po-20mpk) (iv-5mpk) (po-10mpk) (iv-2mpk) T.sub.max(h) 1.0 0.25 0.75 C.sub.max(ng/ml) 678 531 937 871 586 217 T.sub.1/2(h) 11.4 8.4 13.5 3.1 1.9 AUC(0-t) 2192 448 1559 418 2388 551 (h*ng/mL) AUC(0-?) 2374 495 1704 424 2454 586 (h*ng/mL) F % 122% 93% 87% mpk: mg/kg body weight
[0213] The PK test in rats showed that when A1, A2 and C38 were administered orally, the exposure of nucleoside metabolites was higher, and the bioavailability could reach 122.3%, 93% and 87% respectively.
Test Example 2: Inhibition of Compounds on Viral Replication
[0214] Determination of the inhibitory activity of compounds on the replication of 2019 novel coronavirus (SARS-CoV-2): anti-novel coronavirus activity test methods of NHC and GS-441524 are as reported in the literature. To Vero cells infected with the 2019 novel coronavirus, different concentrations of test compounds were added. After 48 hours of incubation, the virus copy number in the cell supernatant was quantified by quantitative real-time RT-PCR (qRT-PCR) to evaluate the inhibitory activity of the compound on the virus (Sci Transl Med, 2020, 12:eabb5883; Cell Rep, 2020, 32:107940; Chinese Patent application No. 202010313870.X).
[0215] Determination of the inhibitory activity of compounds on the replication of respiratory syncytial virus (RSV), human coronavirus OC43, influenza A virus, and Zika virus by Cytopathogenic effect (CPE): the experimental cells were inoculated at a certain cell density to 96 well cell culture plates, and cultured overnight in a 5% CO.sub.2, 37? C. incubator. The compounds and viruses were added the next day. Depending on the tested virus, the cells were cultured in an incubator for 3-7 days under 5% CO.sub.2, 33? C. or 37? C., until 80-95% of the cells in the virus-infected control wells without compound had pathological changes. Cell viability in each well was then detected with CellTiter-Glo or CCK-8. If the cell viability of the compound-containing well is higher than that of the virus-infected control well, that is, the CPE is weakened, it indicates that the compound has an inhibitory effect on the tested virus. The cytotoxicity test method is the same as the corresponding antiviral test method, but without viral infection.
[0216] The antiviral activity and cytotoxicity of the compound are represented by the inhibition rate (%) of the compound on the virus-induced cellular viral effect and the cell viability (%), respectively. Calculation equations are as follows:
Inhibition rate (%)=(reading value of test well?average value of virus control)/(average value of cell control?average value of virus control)?100;
Cell viability (%)=(reading value of test well?average value of medium control)/(average value of cell control?average value of medium control)?100;
[0217] EC50 and CC50 values were calculated by Prism software, and the inhibition curve fitting method was log (inhibitor) vs. responseVariable slope.
[0218] Determination of the inhibitory activity of compounds against dengue virus by plaque reduction assay: Vero cells were inoculated into 6-well cell culture plates at a density of 600,000 cells per well, and cultured overnight in a 5% CO.sub.2, 37? C. incubator. The compounds and virus (40-50 PFU/well) were added the next day. The cells were cultured in an incubator at 5% CO.sub.2 and 37? C. for 2 hours, then the supernatant was aspirated, and the low-melting point agarose culture medium containing the corresponding concentration of the compound was added. The cells were cultured in an incubator at 5% CO.sub.2, 33? C. or 37? C. for 6-7 days, until obvious virus plaques could be observed in the virus-infected control wells without compound under the microscope. Cells were fixed with 4% paraformaldehyde and stained with crystal violet. The number of plaques in each well was calculated. Cytotoxicity experiments were performed in parallel with antiviral experiments. Vero cells were inoculated into 96-well cell culture plates at a density of 20,000 cells per well, and cultured overnight in a 5% CO.sub.2, 37? C. incubator. The compounds were added the next day (1-5 concentration points, single point). The cells were cultured in an incubator at 5% CO.sub.2, 33? C. or 37? C. for 6-7 days. Cell viability in each well was then detected with CCK-8.
[0219] Determination of inhibitory activity of the compound on the replication of porcine epidemic diarrhea virus (PEDV) by Fluorescent quantitative PCR: Vero cells were digested and passaged, adjusted the cell density to 1?10.sup.5/mL with cell growth medium, inoculated in a 96 well plate with 100 ?L/well, and placed in a 37? C., 5% CO.sub.2 incubator to incubate for 24 hours. The 96 well plate was taken out with the medium in the wells be discarded, washed three times with 1?PBS, spin-dried, added with a mixed solution of the compound (10 concentration points) and the virus (0.01 MOI per well) in each well, with 8 replicate wells for each concentration, and incubated in a 37? C., 5% CO.sub.2 incubator, setting virus control and cell control at the same time. After 36 hours, the cell samples were collected, and the changes of virus content in different treatment groups were measured by fluorescent quantitative PCR, and the EC.sub.50 of the compound was calculated.
TABLE-US-00002 TABLE 2 Inhibitory activity against novel coronavirus (SARS-COV-2) and human coronavirus OC43 (HCoV OC43) SARS-COV-2 (Vero) HCoV OC43 (Huh7) Compound Structure (free state) EC.sub.50 (?M) CC.sub.50 (?M) EC.sub.50 (?M) CC.sub.50 (?M) NHC
[0220] Table 3: Inhibitory Activity Against Respiratory Syncytial Virus (RSV) and Influenza Virus
TABLE-US-00003 TABLE 3 inhibitory activity against respiratory syncytial virus (RSV) and influenza virus RSV (HEp-2) H3N2 (MDCK) Compound Structure (free state) EC.sub.50 (?M) CC.sub.50 (?M) EC.sub.50 (?M) CC.sub.50 (?M) NHC
TABLE-US-00004 TABLE 4 Inhibitory activity against replication of Porcine Epidemic Diarrhea Virus (PEDV), Zika Virus (Zika) and Dengue Virus (DENV) DENV (Vero) PEDV (Vero) Zika (Huh7) Inhibition rate Compound Structure EC.sub.50 (?M) EC.sub.50 (?M) at 5 ?M NHC
Test Example 3: In Vivo Pharmacokinetic Evaluation in Rats
[0221] A total of 18 male SD rats were divided into intravenous group (IV) and intragastric administration group (PO). They were fasted for 12 h before the experiment (the intravenous group was not fasted), had free access to water, and ate uniformly 4 h after administration. The dosages of B1, B2 and B6 were 76 ?mol/kg (n=3) for intragastric administration and 38 ?mol/kg (n=3) for intravenous injection, and the administration vehicle is DMSO/EtOH/PEG400/0.9% NaCl (5/5/40/50, v/v/v/v). 0.2-0.3 mL of blood was collected from the jugular vein 5 min (intravenous group only), 0.25 h, 0.5 h, 1.0 h, 2.0 h, 4.0 h, 6.0 h, 8.0 h and 24 h after administration, placed in a sodium heparin anticoagulant tube, mixed gently and centrifuged at 2000 g for 10 minutes, and the plasma was separated and stored in a ?70? C. refrigerator for testing. The concentration of the nucleoside metabolite in plasma was determined by LC-MS-MS, and the pharmacokinetic parameters were calculated.
TABLE-US-00005 TABLE 5 In vivo pharmacokinetic parameters of nucleoside metabolites in rats for single oral administration (76 ?mol/kg) and injection administration (38 ?mol/kg) of B1, B2 and B6 Compound B1 B1 B2 B2 B6 B6 PK parameters (po) (iv) (po) (iv) (po) (iv) T.sub.max(h) 4.0 2.0 2.67 C.sub.max(ng/ml) 1122 2590 3930 4543 2410 2933 T.sub.1/2(h) 6.03 6.24 5.47 6.60 4.93 5.31 AUC(0-t) 11638 9820 29836 18081 18472 13874 (h*ng/ml) AUC(0-?) 12359 10385 31111 19265 19051 14345 (h*ng/ml) F % 59% 83% 67%
Test Example 4: In Vivo Pharmacokinetic Evaluation in Cynomolgus Monkeys
[0222] A total of 6 cynomolgus monkeys were given a single intragastric administration of A1, A2 and A6 (0.35 mmol/kg, n=2), and 10 blood samples were collected from each monkey within 48 hours after administration for LC-MS/MS analysis, to detect the concentrations of nucleoside metabolites NHC and Al and calculate conventional pharmacokinetic parameters, and the data were summarized.
TABLE-US-00006 TABLE 6 In vivo pharmacokinetic parameters of the nucleoside metabolite in monkeys for single intragastric administration (0.35 mmol/kg) of Molnupiravir (control, 5-isobutyrate prodrug of NHC), A1, A2 and A6 T.sub.1/2 T.sub.max C.sub.max AUC.sub.0-t Compound (h) (h) (ng/mL) (ng .Math. h/mL) Molnupiravir 0.50 1.50 2670 6337 A1 1.26 1.50 195 591 A2 0.63 1.00 3620 7435 A6 0.83 1.50 1209 2417
TABLE-US-00007 TABLE 7 In vivo pharmacokinetic parameters of A1 in monkeys for single intragastric administration (0.35 mmol/kg) of A1, A2 and A6 t.sub.1/2 T.sub.max C.sub.max AUC.sub.0-t Compound (h) (h) (ng/mL) (ng .Math. h/mL) A1 1.20 1.50 90.8 365 A2 0.88 1.00 684 1562 A6 0.91 1.50 326 818
Test Example 5
In Vivo Pharmacokinetic Evaluation in Rats
Experimental Method:
[0223] 6 Male SD rats for each compound were divided into 2 groups (gastric administration group and intravenous injection group), 3 rats in each group. The rates were fasted for 12 h before the experiment (the intravenous experiment group was not fasted), had free access to water, and ate uniformly 4 hours after the administration. The dosages of C22 and C23 were 16 mg/kg and 20 mg/kg for intragastric administration, respectively, and the administration vehicle was 5% DMSO+5% solutol+90% saline. The intravenous injection dosages were 4 mg/kg and 5 mg/kg respectively, and the administration vehicle was DMSO/EtOH/PEG300/0.9% NaCl (5/5/40/50, v/v/v/v). 0.2 mL of blood was collected from the jugular vein 5 min, 0.25 h, 0.5 h, 1.0 h, 2.0 h, 4.0 h, 6.0 h, 8.0 h and 24 h after the administration, placed in an EDTA-K2 test tube abd centrifuged at 11,000 rpm for 5 minutes, and the plasma was separated, and stored in a ?70? C. refrigerator for testing. The operation was under an ice water bath. The concentration of nucleoside metabolites in plasma was determined by LC-MS-MS, and the pharmacokinetic parameters were calculated.
TABLE-US-00008 TABLE 8 In vivo pharmacokinetic parameters of nucleoside metabolites of C22 and C23 in rats Compound C22 C22 C23 C23 PK parameters (po-16 mpk) (iv-4 mpk) (po-20 mpk) (iv-5 mpk) T.sub.max(h) 0.5 1.0 C.sub.max(ng/mL) 2217 2883 2933 3903 T.sub.1/2(h) 1.08 1.35 3.47 5.24 AUC(0-t) 7068 2348 11360 3954 (h*ng/mL) AUC(0-?) 7157 2398 11393 4024 (h*ng/mL) F % 75.3% 71.8% mpk: mg/kg body weight
[0224] The PK test in rats showed that when C22 and C23 were administered orally, the exposure of nucleoside metabolites was higher, and the bioavailability could reach 75.3% and 71.8% respectively.
[0225] According to the above test examples and the results of Tables 1-8, it can be seen that some compounds of the present application have high oral bioavailability and significant inhibitory activity against various viruses, and thus have good prospect in antiviral application.