CN112142809B - Uretidine dipropionate phosphoramidate compound, pharmaceutical composition thereof, and preparation method and application thereof - Google Patents
Uretidine dipropionate phosphoramidate compound, pharmaceutical composition thereof, and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of medicines, and discloses a uridylic acid dipropionate phosphoramidate compound, a pharmaceutical composition thereof, a preparation method and an application thereof, in particular to an application of the compound for treating hepatitis C. Experiments prove that the compound has the activity of inhibiting HCV virus replication, and the uridylic acid dipropionate phosphoramidate compound has the advantages of higher in vitro activity, larger development coefficient and the like compared with the current hepatitis C treatment drug sofosbuvir, and can be used for developing the hepatitis C treatment drug.
Description
Technical Field
The invention belongs to the technical field of medicines, and relates to a uridylic acid dipropionate phosphoramidate compound, a pharmaceutical composition, a preparation method and an application thereof, which are suitable for treating flaviviridae virus infection, in particular to treating hepatitis C.
Background
The world health organization estimates that there are 3-4 million new patients with Hepatitis C Virus (HCV) annually, over 2 billion infected people worldwide, and over 1000 million people in china, where HCV is a virus of the hepacivirus family flaviviridae.
Chronic hepatitis c virus infection is mild to inflammation, severe to cirrhosis and liver cancer. And various complications can occur when the hepatitis C cirrhosis is in the decompensation stage, such as ascites abdominal infection, upper gastrointestinal hemorrhage, hepatic encephalopathy, hepatorenal syndrome, hepatic failure and the like.
The pathogenesis of hepatitis C is not clear, and when HCV is replicated in hepatocytes to cause structural and functional changes of the hepatocytes or interfere with protein synthesis of the hepatocytes, degeneration and necrosis of the hepatocytes can be caused, which indicates that HCV directly damages the liver to cause pathogenesis.
Since the HCV genome is similar in structural and phenotypic characteristics to human flaviviruses and pestiviruses, it is classified as a flaviviridae HCV. Viruses of the flaviviridae family include at least three distinct genera: pestiviruses (pestiviruses), which cause disease in cattle and pigs; flaviviruses (flavivruses), which are the major causes of diseases such as dengue and yellow fever; and hepaciviruses (hepaciviruses), the only member of which is HCV. The flavivirus genus included more than 68 members, grouped based on serological relationship. Clinical symptoms vary and include fever, encephalitis, and hemorrhagic fever. Flaviviruses of global interest in relation to human disease include dengue hemorrhagic fever virus (DHF), yellow fever virus, shock syndrome virus and japanese encephalitis virus. Hepatitis c virus is a positive-stranded RNA virus that surrounds a lipid-containing envelope, with spikes, outside the nucleocapsid.
The first treatment for HCV infection was interferon and ribavirin combination therapy, to which only 50% of the patients responded, with interferon having significant side effects such as flu-like symptoms, weight loss and fatigue weakness, and interferon and ribavirin combination therapy producing considerable side effects including hemolysis, anemia and fatigue.
The hepatitis C drug Sofosbuvir (trade name: Sovaldi, common name: Sofosbuvir) from Gilidide Inc. was approved by the U.S. Food and Drug Administration (FDA) for the treatment of adult patients with chronic hepatitis C (Hepatitis C) by 6.12.3.2013. Sofosbuvir is the first approved drug for the full oral treatment regimen for hepatitis C, and eliminates the need for the traditional injection of the drug Interferon (IFN) when used in the treatment of chronic hepatitis C of a specific genotype (type 2, type 3).
However, because sofosbuvir has low in vivo bioavailability and requires a large dosage of a drug, and patients with hepatitis c need to be treated for a long time, the problems of viral resistance and long-term safety are not negligible, and therefore, the development of new therapeutic drugs for HCV infection with high bioavailability, longer half-life and high drug efficacy is still an urgent clinical need.
Disclosure of Invention
The invention aims to further modify the structure of the uridylic acid dipropionate phosphoramidate compound so as to obtain a novel uridylic acid dipropionate phosphoramidate analogue with lower toxicity and higher anti-Hepatitis C Virus (HCV) activity, and lays a foundation for further research and development of antiviral application of the compound.
In order to solve the problems, the technical scheme adopted by the invention is as follows:
the invention provides a uridylic acid dipropionate phosphoramidate compound or a pharmaceutically acceptable salt thereof, wherein the structural formula of the uridylic acid dipropionate phosphoramidate compound is (Ia):
wherein:
Rcany one selected from alkyl groups having 1 to 12 carbon atoms.
The invention further provides the uridylic acid dipropionate phosphoramidate compound or a pharmaceutically acceptable salt thereof, wherein the amino acid ester connected with the phosphorus atom has an S configuration, and the structural formula is (Ib):
wherein:
Rcany one selected from alkyl groups having 1 to 12 carbon atoms.
The uridylic acid dipropionate phosphoramidate compound or the pharmaceutically acceptable salt thereof in the invention is characterized in that Rc is selected from any one of isopropyl, ethyl, isobutyl, neopentyl, n-butyl, cyclohexyl, tert-butyl or 2-ethylbutyl, wherein the structural formula of the uridylic acid dipropionate phosphoramidate compound is selected from one of the following structural formulas:
the invention also provides a pharmaceutically acceptable salt of the uridylic acid dipropionate phosphoramidate compound.
The pharmaceutical composition comprises the uridylic acid dipropionate phosphoramidate compound or the pharmaceutically acceptable salt thereof, and an auxiliary material, wherein the auxiliary material is a pharmaceutically acceptable carrier or excipient.
The pharmaceutical composition further comprises at least one of Ribavirin (Ribavirin), interferon, hepatitis C NS3 protease inhibitor, HCV reverse transcriptase NS5B non-nucleoside inhibitor, HCV reverse transcriptase NS5B nucleoside inhibitor, NS5A inhibitor, synergist of NS5A inhibitor, entry inhibitor, cyclosporin immunosuppressant, NS4A antagonist, NS4B inhibitor or cyclophilin inhibitor.
The preparation method of the uridylic acid dipropionate phosphoramidate compound comprises the following steps: under the alkaline condition, after the amino acid ester hydrochloride reacts with phosphorus oxychloride serving as a phosphorylation reagent, pentafluorophenol is added for reaction to obtain a compound FP 33; FP33 was then reacted with nucleoside SOF at-20 ℃ to-80 ℃ to give compound (Ia).
The synthetic route of the preparation method is shown in figure 1:
wherein: rc is any one selected from alkyl groups with carbon number of 1-12.
The uridylic acid dipropionate phosphoramidate compound or the pharmaceutically acceptable salt thereof provided by the invention can be applied to the preparation of medicines for treating human Flaviviridae virus infection.
The use of the present invention, wherein the human flaviviridae viral infection is a human HCV viral infection.
The invention also provides application of the pharmaceutical composition in preparing a medicament for resisting human Flaviviridae virus infection, which is characterized in that the Flaviviridae virus is HCV virus.
The invention has the beneficial effects that:
the compound of the invention has excellent properties required by being a medicament for treating hepatitis C through detection mechanism determination, and the specific properties are as follows:
in the in vitro anti-HCV activity screening, EC50 of compound SOF3030, SOF3131, SOF3434 or SOF3939 is 2-5 times or more higher than sofosbuvir (positive control), and the biological activity selection coefficient SI is 2-5 times or more higher than sofosbuvir (positive control).
This indicates that: the compound can effectively inhibit HCV infection at a cellular level, and is expected to be a medicament for treating HCV infection.
Drawings
FIG. 1 is a schematic diagram of the synthetic route of the process for preparing uridylic acid dipropionate phosphoramidate compounds according to the present invention.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The present invention will be further illustrated by the following examples, but the present invention is not limited to these examples. The reagents and starting materials used in the examples of the invention were all commercially available.
Example 1
Synthesis of N- [ (pentafluorophenoxy) (((S) -1- (isopropoxycarbonyl) ethyl) amino) phosphoryl ] -L-alanine isopropyl ester (FP 3030).
Phosphorus oxychloride (5g, 3.04mL, 32.6mmol) and acetonitrile (200mL) are added into a reaction bottle, the reaction bottle is cooled to-70 ℃, HA30(9.86g, 58.8mmol) is slowly added, the reaction bottle is cooled to-70 ℃, triethylamine (7.3g, 10mL, 72mmol) solution in acetonitrile (60 mL) is dropwise added, after the addition is finished, the temperature is raised to 0 ℃, the reaction bottle is reacted for 3 hours, pentafluorophenol (5.4g, 29.4mmol) and triethylamine (7.3g, 10mL, 72mmol) solution in acetonitrile (60 mL) are dropwise added into the solution, after stirring at 0 ℃ for 1 hour, the mixture was warmed to room temperature and stirred overnight, 100 ml of methylene chloride and 100 ml of water were added, the organic phase was separated, dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was separated by a silica gel column (0-30% ethyl acetate/hexane) to give 5.5g of a white solid, which was recrystallized from 10% t-butyl methyl ether/hexane to give FP3030(2.8g) as a white solid.
1H NMR(400MHz，CDCl3)δ(ppm)：1.17-1.38(18H，m，6×CH3)，3.74-4.01(2H，m，2×NCH)，4.28-4.43(2H，m，2×NH)，4.89-5.08(2H，m，2×COOCH)。
Example 2
Synthesis of N- [ (pentafluorophenoxy) (((S) -1- (ethoxycarbonyl) -2-ethyl) amino) phosphoryl ] -L-alanine ethyl ester (FP 3131).
FP3131 was synthesized in a similar manner to example 1.
Nuclear magnetic hydrogen spectrum data of FP 3131:1H NMR(400MHz，CDCl3)δ(ppm)：1.20-1.41(12H，m，4×CH3)，3.80-3.96(4H，m，2×NH and 2×NCH)，4.14-4.38(4H，m，2×COOCH2)。
LCMS-ESI+(m/z):463.3(M+H)。
example 3
Synthesis of N- [ (pentafluorophenoxy) (((S) -1- (isobutoxycarbonyl) -2-ethyl) amino) phosphoryl ] -L-alanine isobutyl ester (FP 3232).
FP3232 was synthesized by a similar synthesis method to example 1.
Nuclear magnetic hydrogen spectrum data of FP 3232:1H NMR(400MHz，CDCl3)δ(ppm)：0.94-1.33(18H，m，6×CH3)，2.38-2.50(2H，m，2×CH)，3.78-3.94(4H，m，2×NH and 2×NCH)，4.06-4.37(4H，m，2×COOCH2)。
LCMS-ESI+(m/z):519.4(M+H)。
example 4
Synthesis of N- [ (pentafluorophenoxy) (((S) -1- (neopentyloxycarbonyl) ethyl) amino) phosphoryl ] -L-alanine neopentyl ester (FP 3333).
FP3333 Synthesis in a similar manner to example 1
1H NMR(400MHz，CDCl3)δ(ppm)：0.85-1.16(24H，m，8×CH3)，3.74-3.92(4H，m，2×NH and 2×NCH)，4.07-4.38(4H，m，2×COOCH2)。
Example 5
Synthesis of N- [ (pentafluorophenoxy) (((S) -1- (N-butoxycarbonyl) ethyl) amino) phosphoryl ] -L-alanine N-butyl ester (FP 3434).
FP3434 Synthesis in a similar manner to example 1
1H NMR(400MHz，CDCl3)δ(ppm)：0.86-1.12(6H，m，2×CH3)，1.17-1.39(10H，m，2×CH3 and 2×CH2)，1.57-1.70(4H，m，2×CH2)，3.91-4.10(4H，m，2×NH and 2×NCH)，4.20-4.39(4H，m，2×COOCH2)。
Example 6
Synthesis of tert-butyl N- [ (pentafluorophenoxy) (((S) -1- (tert-butyloxycarbonyl) -2-ethyl) amino) phosphoryl ] -L-alanine (FP 3838).
FP3838 was synthesized in a similar manner to example 1.
Nuclear magnetic hydrogen spectrum data of FP 3838:1H NMR(400MHz，CDCl3)δ(ppm)：0.84-1.12(18H，m，6×CH3)，1.19-1.38(6H，m，2×CH3)，3.77-3.98(4H，m，2×NH and 2×NCH)。
LCMS-ESI+(m/z):519.4(M+H)。
example 7
Synthesis of N- [ (pentafluorophenoxy) (((S) -1- (2-ethylbutyloxycarbonyl) -2-ethyl) amino) phosphoryl ] -L-alanine-2-ethylbutyl ester (FP 3939).
FP3939 was synthesized by a similar synthesis method to that of example 1.
Nuclear magnetic hydrogen spectrum data of FP 3939:1H NMR(400MHz，CDCl3)δ(ppm)：0.84-1.15(12H，m，4×CH3)，1.22-1.39(14H，m，2×CH3 and 4×CH2)，1.97-2.17(2H，m，2×CH)，3.75-3.99(4H，m，2×NH and 2×NCH)，4.03-4.30(4H，m，2×COOCH2)。
LCMS-ESI+(m/z):575.5(M+H)。
example 8
Synthesis of [ (((2R, 3R, 4R, 5R) -5- (2, 4(1H, 3H) -pyrimidinedione-1-yl) -4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methyl) bis ((S) -1- (isopropoxycarbonyl) ethylamino) ] phosphate (SOF 3030).
Preparation of nucleoside SOF refer to the preparation method of patent application No. 200880018024.2.
A50 mL flask was charged with nucleoside SOF (260.2mg,1mmol) and 5.0mL anhydrous THF, and the mixture was cooled to 0 ℃ in an ice-water bath. A1.0M in THF solution of tert-butylmagnesium chloride (3.0mL, 3.0mmol) was added dropwise, the reaction mixture stirred at 0 deg.C for 30min, followed by addition of a solution of phosphorus reagent FP3030(0.784g, 1.6mmol) in 5mL THF at 0 deg.C. The resulting clear reaction solution was warmed to room temperature, stirred for 20 hours, and then saturated NH was added4Cl (15mL), stirred for 5 min, the mixture was diluted with ethyl acetate (200mL), the organic phase was separated and the aqueous layer was extracted twice with ethyl acetate (30 mL). The combined organic layers were washed with water (30mL), saturated NaHCO3(2X30mL), brine (30mL) and Na2SO4After drying, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography on silica gel (0-10% methanol in dichloromethane) to give SOF3030(253mg), a white solid product, in 46% yield.
1H NMR(400MHz，CDCl3)δ(ppm)：1.21-1.43(21H，m，7×CH3) 3.31-3.54(2H, m, 2 XNCH), 4.10-4.29(1H, m, H at the 3 '-position of the sugar ring), 4.25(1H, brs, OH at the 3' -position of the sugar ring), 4.35-4.44(3H, m, 2 XPNH and H at the 4 '-position of the sugar ring), 4.50-4.66(2H, m, H at the 5' -position of the sugar ring), 4.87-5.09(2H, m, 2H, 2 XNCH)X COOCH), 5.54-5.77(1H, m, H at the 5-position of the pyrimidine ring), 6.16(1H, s, H at the 1' -position of the sugar ring), 7.16-7.28(1H, m, H at the 6-position of the pyrimidine ring), 9.77(1H, s, NH at the 3-position of the pyrimidine ring). LCMS-ESI+(m/z):567.5(M+H)。
Example 9
Synthesis of [ (((2R, 3R, 4R, 5R) -5- (2, 4(1H, 3H) -pyrimidinedione-1-yl) -4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methyl) bis ((S) -1- (ethoxycarbonyl) ethylamino) ] phosphate (SOF 3131).
SOF3131 was synthesized in a similar manner to example 8.
Nuclear magnetic hydrogen spectrum data of SOF 3131:1H NMR(400MHz，CDCl3)δ(ppm)：1.20-1.39(12H，m，4×CH3) 1.41-1.57(3H, s, CH at the 2' -position of the sugar ring)3)，3.80-3.96(4H，m，2×NH and 2×NCH)，
4.10-4.45(7H，m，2×COOCH2H at the 3 ' -position of the sugar ring, OH at the 3 ' -position of the sugar ring, H at the 4 ' -position of the sugar ring), 4.51 to 4.66(2H, m, H at the 5 ' -position of the sugar ring), 5.55 to 5.76(1H, m, H at the 5-position of the pyrimidine ring), 6.18(1H, s, H at the 1 ' -position of the sugar ring), 7.16 to 7.29(1H, m, H at the 6-position of the pyrimidine ring), 9.78(1H, s, NH at the 3-position of the pyrimidine ring).
LCMS-ESI+(m/z):539.5(M+H)。
Example 10
Synthesis of [ (((2R, 3R, 4R, 5R) -5- (2, 4(1H, 3H) -pyrimidinedione-1-yl) -4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methyl) bis ((S) -1- (isobutoxycarbonyl) ethylamino) ] phosphate (SOF 3232).
SOF3232 was synthesized in a similar manner to example 8.
Of SOF3232Nuclear magnetic hydrogen spectrum data:1H NMR(400MHz，CDCl3)δ(ppm)：0.96-1.30(18H，m，6×CH3) 1.38-1.52(3H, s, CH at the 2' -position of the sugar ring)3) 2.38-2.51(2H, m, 2 XCH), 3.76-3.98(4H, m, 2 XCH and 2 XCH), 4.08-4.46(7H, m, H at the 3 ' -position of the sugar ring, OH at the 3 ' -position of the sugar ring, H at the 4 ' -position of the sugar ring, 2 XCOCH2) 4.51-4.65(2H, m, H at the 5 '-position of the sugar ring), 5.55-5.76(1H, m, H at the 5-position of the pyrimidine ring), 6.17(1H, s, H at the 1' -position of the sugar ring), 7.12-7.26(1H, m, H at the 6-position of the pyrimidine ring), 9.77(1H, s, NH at the 3-position of the pyrimidine ring).
LCMS-ESI+(m/z):595.6(M+H)。
Example 11
Synthesis of [ (((2R, 3R, 4R, 5R) -5- (2, 4(1H, 3H) -pyrimidinedione-1-yl) -4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methyl) bis ((S) -1- (neopentyloxycarbonyl) ethylamino) ] phosphate (SOF 3333).
SOF3333 was synthesized in a similar manner to the synthesis of example 8.
1H NMR(400MHz，CDCl3)δ(ppm)：0.89-1.06(18H，m，6×CH3)，1.20-1.43(9H，m，3×CH3) 3.84-3.97(4H, m, 2 XPNH and 2 XPNH), 4.11-4.44(7H, m, H at the 3 ' -position of the sugar ring, OH at the 3 ' -position of the sugar ring, H at the 4 ' -position of the sugar ring and 2 XPOCOH2) 4.50-4.63(2H, m, H at the 5 '-position of the sugar ring), 5.57-5.75(1H, m, H at the 5-position of the pyrimidine ring), 6.16(1H, s, H at the 1' -position of the sugar ring), 7.14-7.27(1H, m, H at the 6-position of the pyrimidine ring), 9.76(1H, s, NH at the 3-position of the pyrimidine ring).
LCMS-ESI+(m/z):623.6(M+H)。
Example 12
Synthesis of [ (((2R, 3R, 4R, 5R) -5- (2, 4(1H, 3H) -pyrimidinedione-1-yl) -4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methyl) bis ((S) -1- (n-butoxycarbonyl) ethylamino) ] phosphate (SOF 3434).
SOF3434 was synthesized in a similar manner to example 8.
1H NMR(400MHz，CDCl3)δ(ppm)：0.87-1.02(6H，m，2×CH3)，1.21-1.44(13H，m，3×CH3 and 2×CH2，1.59-1.70(4H，m，2×CH2) 3.88-4.08(4H, m, 2 XPNH and 2 XPNH), 4.10-4.27(5H, m, H and 2 XPOCOH at the 3' -position of the sugar ring)24.30(1H, brs, OH at the 3 '-position of the sugar ring), 4.33-4.47(1H, m, H at the 4' -position of the sugar ring), 4.52-4.67(2H, m, H at the 5 '-position of the sugar ring), 5.56-5.73(1H, m, H at the 5-position of the pyrimidine ring), 6.18(1H, s, H at the 1' -position of the sugar ring), 7.17-7.26(1H, m, H at the 6-position of the pyrimidine ring), 9.73(1H, s, NH at the 3-position of the pyrimidine ring). LCMS-ESI+(m/z):595.6(M+H)。
Example 13
Synthesis of [ (((2R, 3R, 4R, 5R) -5- (2, 4(1H, 3H) -pyrimidinedione-1-yl) -4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methyl) bis ((S) -1- (tert-butoxycarbonyl) ethylamino) ] phosphate (SOF 3838).
SOF3838 was synthesized in a similar manner to example 8.
Nuclear magnetic hydrogen spectrum data of SOF 3838:1H NMR(400MHz，CDCl3)δ(ppm)：0.82-1.14(18H，m，6×CH3)，1.19-1.36(6H，m，2×CH3) 1.39-1.57(3H, s, CH at the 2' -position of the sugar ring)3) 3.75-3.96(4H, m, 2 XNH and 2 XNCH), 4.14-4.42(3H, m, H at the 3 ' -position of the sugar ring, OH at the 3 ' -position of the sugar ring, H at the 4 ' -position of the sugar ring), 4.52-4.66(2H, m, H at the 5 ' -position of the sugar ring), 5.59-5.77(1H, m, H at the 5-position of the pyrimidine ring), 6.16(1H, s, H at the 1 ' -position of the sugar ring)H) in the above range of 7.17 to 7.29(1H, m, H at the 6-position of the pyrimidine ring), 9.79(1H, s, NH at the 3-position of the pyrimidine ring).
LCMS-ESI+(m/z):595.6(M+H)。
Example 14
Synthesis of [ ((((2R, 3R, 4R, 5R) -5- (2, 4(1H, 3H) -pyrimidinedione-1-yl) -4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl) methyl) bis ((S) -1- (2-ethylbutyloxycarbonyl) ethylamino) ] phosphate (SOF 3939).
SOF3939 was synthesized in a similar manner to example 8.
Nuclear magnetic hydrogen spectrum data of SOF 3939:1H NMR(400MHz，CDCl3)δ(ppm)：0.87-1.18(12H，m，4×CH3)，1.20-1.36(14H，m，2×CH3 and 4×CH2) 1.39-1.57(3H, s, CH at the 2' -position of the sugar ring)3)，1.95-2.18(2H，m，2×CH)，3.73-3.97(4H，m，2×NH and 2×NCH)，4.05-4.44(7H，m，2×COOCH2H at the 3 ' -position of the sugar ring, OH at the 3 ' -position of the sugar ring, H at the 4 ' -position of the sugar ring), 4.52 to 4.66(2H, m, H at the 5 ' -position of the sugar ring), 5.59 to 5.78(1H, m, H at the 5-position of the pyrimidine ring), 6.19(1H, s, H at the 1 ' -position of the sugar ring), 7.16 to 7.29(1H, m, H at the 6-position of the pyrimidine ring), 9.78(1H, s, NH at the 3-position of the pyrimidine ring).
LCMS-ESI+(m/z):651.7(M+H)。
Example 15
Biological evaluation
1. Detection of antiviral Activity of Compounds of the present invention in HCV replicon (HCVpp) System
HCV replicon assay procedure
General procedure Huh-7 derived cell line (ZLuc) with HCV genotype 1b replicon and luciferase reporter Gene in Dulbecco's modified Eagle Medium (DME) supplemented with 10% fetal bovine serum, 2mM GlutaMAX, 1% MEM nonessential amino acids, 100IU/mL penicillin, 100. mu.g/mL streptomycin, and 0.5mg/mL (G418)M) is grown. Zluc cells were transiently transfected with human carboxylesterase 1(CES1) by using a lipid/histone based transfection procedure. 24 and 48 hours after transfection, expression of CES1 was confirmed by Western blotting (Western blot) using anti-CES 1 and anti-tag antibodies. For dose response testing, cells were seeded in 96-well plates at 7.5xl03 cells/well in a volume of 50 μ L and at 37 ℃/5% CO2And (4) incubating. Drug solutions were freshly prepared in Huh-7 medium as 2X stock solutions. 10 additional 5-fold dilutions were prepared from these stocks in DMEM without G418. At least 3 hours after seeding with the Huc cells, drug treatment was started by adding 50 μ Ι _ of drug dilution to the plates in duplicate. The final concentration of the drug ranges from 100nM to 0.0000512n μm. Cells were then incubated at 37 ℃/5% CO2And (4) incubating. Alternatively, compounds were tested at two concentrations (10nM and 100 nM). In all cases, Huh-7 (which does not carry an HCV replicon) was used as a negative control. Inhibition of HCV replication was measured by quantifying the photons emitted by the singlet oxidation of 5' -fluoroluciferin to oxyfluoroluciferin (oxyiuoroluteciferin) by firefly luciferase after 72 hours of incubation. To this end, the medium was removed from the plate by tapping and 50 microliters of ONE-glo luciferase assay reagent was added to each well. The plate was gently shaken for 3 minutes at room temperature and the luminescence measured on a Victor3V1420 multiple mark counter (PerkinElmer) with a 1 second read-out time using a 700nm cut-off filter. EC was calculated from the dose-response curve of the resulting best-fit equation, as determined by Microsoft Excel and XLFit4.1 software50The value is obtained.
For cytotoxicity evaluation, Zluc cells were treated with the above compounds, and cell viability was monitored by adding 20 μ L of assay solution to each well using CellTiter-Blue cell viability assay (Promega). The plates were then incubated at 37 deg.C/5% CO2And incubating for at least 3 hours. Fluorescence of the plates was detected in a Victor3V1420 multiple-marker register (Perkin Elmer) using excitation and emission wavelengths of 560 and 590nm, respectively, and the CC was determined using Microsoft Excel and XLFit4.1 software50The value is obtained.
2. The compounds provided in the table below were determined according to the replicon assay described above
, + ++ refers to 1-10 nM; + represents 10-100 nM; + represents 0.1-1. mu.M;
sofosbuvir was prepared according to reference j
While the present invention has been described in considerable detail and with particular reference to a few illustrative embodiments thereof, it is not intended to be limited to any such details or embodiments or any particular embodiments, but it is to be construed as effectively covering the intended scope of the disclosure by providing broad, potential interpretations of such claims in view of the prior art with reference to the appended claims. Furthermore, the foregoing describes the disclosure in terms of embodiments foreseen by the inventor for which an enabling description was available, notwithstanding that insubstantial modifications of the disclosure, not presently foreseen, may nonetheless represent equivalent modifications thereto.
Claims (8)
1. A uridylic acid dipropionate phosphoramidate compound or a pharmaceutically acceptable salt thereof, wherein the uridylic acid dipropionate phosphoramidate compound has a structural formula selected from one of the following structural formulae:
2. a pharmaceutically acceptable salt of the uridylic acid dipropionate phosphoramidate compound of claim 1.
3. A pharmaceutical composition comprising the uridylic acid dipropionate phosphoramidate compound according to claim 1 or a pharmaceutically acceptable salt thereof, and an excipient, wherein the excipient is a pharmaceutically acceptable carrier or excipient.
4. The pharmaceutical composition of claim 3, further comprising at least one of Ribavirin (Ribavirin), an interferon, a hepatitis C NS3 protease inhibitor, an HCV reverse transcriptase NS5B non-nucleoside inhibitor, an HCV reverse transcriptase NS5B nucleoside inhibitor, an NS5A inhibitor, and a potentiator of an NS5A inhibitor, an entry inhibitor, a cyclosporin immunosuppressant, an NS4A antagonist, an NS4B inhibitor, or a cyclophilin inhibitor.
5. A preparation method of uridylic acid dipropionate phosphoramidate compound is characterized by comprising the following steps: under the alkaline condition, after the amino acid ester hydrochloride reacts with phosphorus oxychloride serving as a phosphorylation reagent, pentafluorophenol is added for reaction to obtain a compound FP 33; reacting FP33 with nucleoside SOF at-20 deg.C to-80 deg.C to obtain compound (Ia); wherein the structural formula of the amino acid ester hydrochloride is shown in the specification
FP33 has a structural formula
The structural formula of nucleoside SOF is
The structural formula of the compound (Ia) is
The R iscIs n-butyl, ethyl or 2-ethyl-butyl.
6. Use of a uridylic acid dipropionate phosphoramidate compound according to any of claims 1-2 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of a human flaviviridae virus infection.
7. The use according to claim 6, wherein the human Flaviviridae viral infection is a human HCV viral infection.
8. Use of a pharmaceutical composition according to any one of claims 3 to 4 in the manufacture of a medicament for the treatment of human flaviviridae infections, wherein said flaviviridae is HCV.
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