CN112194694B - Urodylate phenylpropionate phosphoramidate compound, pharmaceutical composition thereof, and preparation method and application thereof - Google Patents
Urodylate phenylpropionate 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 bis-phenylpropionate based phosphoramidate compound, a pharmaceutical composition thereof, a preparation method and application thereof, in particular to application of the compound for treating hepatitis C. Experiments prove that the uridylic acid double-phenylpropionate amino phosphate compound has the activity of inhibiting HCV virus replication, has the advantages of high in-vitro activity, large development coefficient and the like compared with the conventional medicine sofosbuvir for treating hepatitis C, and can be used for developing the medicine for treating hepatitis C.
Description
Technical Field
The invention belongs to the technical field of medicines, and relates to a uridylic acid bis-phenylpropionate based 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 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 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 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 double-phenylpropionate radical phosphoramidate compound so as to obtain a novel uridylic acid double-phenylpropionate radical 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 uridylic acid double-phenylpropionate radical phosphoramidate compound.
In order to solve the problems, the technical scheme adopted by the invention is as follows:
the invention provides a uridylic acid bis-phenylpropionate phosphoramidate compound or pharmaceutically acceptable salt thereof, wherein the structural formula of the uridylic acid bis-phenylpropionate phosphoramidate compound is (Ia):
wherein:
Rcany one selected from alkyl groups having 1 to 12 carbon atoms.
The invention further provides the uridylic acid bis-phenylpropionate based 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 bis-phenylpropionate based phosphoramidate compound or pharmaceutically acceptable salt thereof of the invention is RcSelected from isopropyl, ethyl, isobutyl, neopentyl, n-butyl, cyclohexyl, tert-butyl or 2-ethylbutyl, wherein the uridine is preferably uridineThe structural formula of the acid bis-phenylpropionate based phosphoramidate compound is selected from one of the following structural formulas:
the invention relates to pharmaceutically acceptable salts of uridylic acid bis-phenylpropionate amido phosphate compounds.
The pharmaceutical composition comprises the uridylic acid bis-phenylpropionate based phosphoramidate compound or 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 bis-phenylpropionate based phosphoramidate compound comprises the following steps:
the synthetic route of the preparation method is shown in figure 1:
wherein: rcAny one selected from alkyl groups having 1 to 12 carbon atoms.
Under the alkaline condition, after the amino acid ester hydrochloride reacts with phosphorus oxychloride serving as a phosphorylation reagent, pentafluorophenol is added to react to obtain a compound FP 3737; FP3737 was then reacted with nucleoside SOF at-20 ℃ to-80 ℃ to give compound (Ia).
The uridylic acid bis-phenylpropionate based phosphoramidate compound or the pharmaceutically acceptable salt thereof is applied to the preparation of the medicine 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 for becoming a medicament for treating hepatitis C, which are determined by a detection mechanism, and the following are specific:
in vitro screening for anti-HCV activity, EC50 of compound SOF370370, SOF371371, SOF374374 or SOF379379 was 2-5 times or more higher than sofosbuvir (positive control), and the selection coefficient for biological activity SI was 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 preparation method of uridylic acid bis-phenylpropionate based phosphoramidate compound of 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) -2-phenylethyl) amino) phosphoryl ] -L-phenylalanine isopropyl ester (FP 370370).
Phosphorus oxychloride (5g, 3.04mL, 32.6mmol) and acetonitrile (200mL) are added into a reaction bottle, the reaction bottle is cooled to-70 ℃, HA370(14.331g, 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 completed, the reaction bottle is heated to 0 ℃ and reacts 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 to separate an organic phase, the mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and the residue was separated by a silica gel column (0 to 30% ethyl acetate/hexane) to give 6.1g of a white solid, and the solid was recrystallized from 10% t-butyl methyl ether/hexane to give FP370370(3.1g) as a white solid.
1H NMR(400MHz，CDCl3)δ(ppm)：1.17-1.38(12H，m，4×CH3)，3.73-4.04(6H，m，2×CH2and 2 × NCH), 4.28-4.44(2H, m, 2 × NH), 4.89-5.06(2H, m, 2 × COOCH), 7.05-7.39(10H, m, hydrogen on both benzene rings).
Example 2
Synthesis of N- [ (pentafluorophenoxy) (((S) -1- (ethoxycarbonyl) -2-phenylethyl) amino) phosphoryl ] -L-phenylalanine ethyl ester (FP 371371).
FP371371 was synthesized by a similar synthesis method to that of example 1.
Nuclear magnetic hydrogen spectrum data of FP 371371:1H NMR(400MHz，CDCl3)δ(ppm)：1.17-1.36(6H，m，2×CH3)，3.75-3.94(8H，m，2×CH2，2×NH and 2×NCH)，4.12-4.40(4H，m，2×COOCH2) 7.05-7.38(10H, m, hydrogen on both benzene rings).
LCMS-ESI+(m/z):615.5(M+H)。
Example 3
Synthesis of N- [ (pentafluorophenoxy) (((S) -1- (isobutoxycarbonyl) -2-phenylethyl) amino) phosphoryl ] -L-phenylalanine isobutyl ester (FP 372372).
FP372372 was synthesized by a similar synthesis method to example 1.
Nuclear magnetic hydrogen spectrum data of FP 372372:1H NMR(400MHz，CDCl3)δ(ppm)：0.86-1.15(12H，m，4×CH3)，2.34-2.50(2H，m，2×CH)，3.76-3.95(8H，m，2×CH2，2×NH and 2×NCH)，4.04-4.39(4H，m，2×COOCH2) 7.07-7.38(10H, m, hydrogen on both benzene rings). LCMS-ESI+(m/z):671.6(M+H)。
Example 4
Synthesis of N- [ (pentafluorophenoxy) (((S) -1- (neopentyloxycarbonyl) -2-phenylethyl) amino) phosphoryl ] -L-phenylalanine neopentyl ester (FP 373373).
FP373373 was synthesized in a similar manner to example 1
1H NMR(400MHz，CDCl3)δ(ppm)：0.84-1.17(18H，m，6×CH3)，3.73-3.95(8H，m，2×CH2，2×NH and 2×NCH)，4.07-4.38(4H，m，2×COOCH2) 7.00-7.39(10H, m, hydrogen on both benzene rings).
Example 5
Synthesis of N- [ (pentafluorophenoxy) (((S) -1- (N-butyloxycarbonyl) -2-phenylethyl) amino) phosphoryl ] -L-phenylalanine N-butyl ester (FP 374374).
FP374374 was synthesized in a similar manner to example 1
1H NMR(400MHz，CDCl3)δ(ppm)：0.85-1.13(6H，m，2×CH3)，1.19-1.41(4H，m，2×CH2)，1.56-1.70(4H，m，2×CH2)，3.91-4.08(8H，m，2×CH2，2×NH and 2×NCH)，4.20-4.39(4H，m，2×COOCH2) 7.04-7.37(10H, m, two benzene rings)Hydrogen as above).
Example 6
Synthesis of N- [ (pentafluorophenoxy) (((S) -1- (tert-butyloxycarbonyl) -2-phenylethyl) amino) phosphoryl ] -L-phenylalanine tert-butyl ester (FP 378378).
FP378378 was synthesized by a similar synthesis method to example 1.
Nuclear magnetic hydrogen spectrum data of FP 378378:1H NMR(400MHz，CDCl3)δ(ppm)：0.83-1.16(18H，m，6×CH3)，3.75-3.96(8H，m，2×CH22 × NH and 2 × NCH), 7.05-7.39(10H, m, hydrogen on both benzene rings).
LCMS-ESI+(m/z):671.6(M+H)。
Example 7
Synthesis of N- [ (pentafluorophenoxy) (((S) -1- (2-ethylbutyloxycarbonyl) -2-phenylethyl) amino) phosphoryl ] -L-phenylalanine-2-ethylbutyl ester (FP 379379).
FP379379 was synthesized by a similar synthesis method to example 1.
Nuclear magnetic hydrogen spectrum data of FP 379379:1H NMR(400MHz，CDCl3)δ(ppm)：0.88-1.16(12H，m，4×CH3)，1.20-1.37(8H，m，4×CH2)，1.97-2.15(2H，m，2×CH)，3.77-3.98(8H，m，2×CH2，2×NH and 2×NCH)，4.00-4.32(4H，m，2×COOCH2) 7.05-7.40(10H, m, hydrogen on both benzene rings).
LCMS-ESI+(m/z):727.7(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) -2-phenylethylamino) ] phosphate (SOF 370370).
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 FP370370(1.03g, 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 SOF370370(381mg) as a white solid product in 53% yield.
Nuclear magnetic hydrogen spectroscopy data of SOF 370370:1H NMR(400MHz，CDCl3)δ(ppm)：1.20-1.48(15H，m，5×CH3)，②3.25-3.53(6H，m，2×CH2and 2 × NCH), 4.10 to 4.27(1H, m, H at the 3 ' -position of the sugar ring), 4.27(1H, brs, OH at the 3 ' -position of the sugar ring), 4.33 to 4.45(3H, m, 2 × PNH and H at the 4 ' -position of the sugar ring), 4.50 to 4.61(2H, m, H at the 5 ' -position of the sugar ring), 4.83 to 5.08(2H, m, 2 × COOCH), 5.54 to 5.72(1H, m, H at the 5-position of the pyrimidine ring), 6.16(1H, s, H at the 1 ' -position of the sugar ring), 7.05 to 7.37(11H, m, H at the 6-position of the pyrimidine ring and H at both benzene rings), 9.78(1H, s, NH at the 3-position of the pyrimidine ring).
LCMS-ESI+(m/z):719.7(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) -2-phenylethylamino) ] phosphate (SOF 371371).
SOF371371 was synthesized by a similar synthesis method to that of example 8.
Nuclear magnetic hydrogen spectrum data of SOF 371371:1H NMR(400MHz，CDCl3)δ(ppm)：1.16-1.34(6H，m，2×CH3) 1.36-1.59(3H, s, CH at the 2' -position of the sugar ring)3)，3.77-3.96(8H，m，2×CH2，2×NH and 2×NCH)，4.10-4.44(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, 4.52-4.64(2H, m, H at the 5 ' -position of the sugar ring), 5.58-5.78(1H, m, H at the 5-position of the pyrimidine ring), 6.16(1H, s, H at the 1 ' -position of the sugar ring), 7.06-7.39(11H, m, H at the 6-position of the pyrimidine ring, hydrogen on both benzene rings), 9.78(1H, s, NH at the 3-position of the pyrimidine ring).
LCMS-ESI+(m/z):691.7(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) -2-phenylethylamino) ] phosphate (SOF 372372).
SOF372372 was synthesized by a similar synthesis method to that of example 8.
Nuclear magnetic hydrogen spectrum data of SOF 372372:1H NMR(400MHz，CDCl3)δ(ppm)：0.82-1.15(12H，m，4×CH3) 1.35-1.58(3H, s, CH at the 2' -position of the sugar ring)3)，2.32-2.51(2H，m，2×CH)，3.74-3.98(8H，m，2×CH2，2×NH and 2×NCH)，4.06-4.44(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),4.50-4.66(2H, m, H at the 5 '-position of the sugar ring), 5.55-5.78(1H, m, H at the 5-position of the pyrimidine ring), 6.18(1H, s, H at the 1' -position of the sugar ring), 7.05-7.38(11H, m, H at the 6-position of the pyrimidine ring, hydrogen on both benzene rings), 9.76(1H, s, NH at the 3-position of the pyrimidine ring). LCMS-ESI+(m/z):747.7(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) -2-phenylethylamino) ] phosphate (SOF 373373).
SOF373373 was synthesized by a similar synthesis method to that of example 8.
Nuclear magnetic hydrogen spectrum data of SOF 373373:1H NMR(400MHz，CDCl3)δ(ppm)：0.85-1.06(18H，m，6×CH3) 1.26(3H, s, CH at the 3' -position of the sugar ring)3)，3.27-3.52(4H，d，2×CH2) 3.84-3.96(4H, m, 2 XPNH and 2 XPNH), 4.11-4.40(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) 4.50-4.64(2H, m, H at the 5 '-position of the sugar ring), 5.52-5.73(1H, m, H at the 5-position of the pyrimidine ring), 6.16(1H, s, H at the 1' -position of the sugar ring), 7.06-7.36(11H, m, H at the 6-position of the pyrimidine ring and H on both phenyl rings), 9.77(1H, s, NH at the 3-position of the pyrimidine ring).
LCMS-ESI+(m/z):775.8(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) -2-phenylethylamino) ] phosphate (SOF 374374).
SOF374374 was synthesized by a similar synthesis method to that of example 8.
Nuclear magnetic hydrogen spectrum data of SOF 374374:1H NMR(400MHz，CDCl3)δ(ppm)：0.88-1.03(6H，m，2×CH3)，1.20-1.41(7H，m，CH3 and 2×CH2)，1.58-1.70(4H，m，2×CH2)，3.31-3.54(4H，m，2×CH2) 3.92-4.06(4H, m, 2 XPNH and 2 XPNH), 4.13-4.46(7H, m, OH at the 3 '-position of the sugar ring, H at the 4' -position of the sugar ring and 2 XPOCOH)2) 4.50-4.66(2H, m, H at the 5 '-position of the sugar ring), 5.55-5.75(1H, m, H at the 5-position of the pyrimidine ring), 6.19(1H, s, H at the 1' -position of the sugar ring), 7.09-7.41(11H, m, H at the 6-position of the pyrimidine ring and H on both phenyl rings), 9.78(1H, s, NH at the 3-position of the pyrimidine ring).
LCMS-ESI+(m/z):747.7(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) -2-phenylethylamino) ] phosphate (SOF 378378).
SOF378378 was synthesized by a similar synthesis method to that of example 8
Nuclear magnetic hydrogen spectroscopy data of SOF 378378:1H NMR(400MHz，CDCl3)δ(ppm)：0.85-1.16(18H，m，6×CH3) 1.36-1.55(3H, s, CH at the 2' -position of the sugar ring)3)，3.77-3.96(8H，m，2×CH22 × NH and 2 × NCH), 4.10-4.46(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), 4.50-4.66(2H, m, H at the 5 ' -position of the sugar ring), 5.57-5.78(1H, m, H at the 5-position of the pyrimidine ring), 6.18(1H, s, H at the 1 ' -position of the sugar ring), 7.08-7.39(11H, m, hydrogen on both benzene rings, 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):747.7(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) -2-phenylethylamino) ] phosphate (SOF 379379).
SOF379379 was synthesized by a similar synthesis method to that of example 8.
Nuclear magnetic hydrogen spectrum data of SOF 379379:1H NMR(400MHz，CDCl3)δ(ppm)：0.86-1.16(12H，m，4×CH3)，1.22-1.35(8H，m，4×CH2) 1.39-1.58(3H, s, CH at the 2' -position of the sugar ring)3)，1.98-2.16(2H，m，2×CH)，3.74-3.98(8H，m，2×CH2，2×NH and 2×NCH)，4.10-4.46(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), 4.50 to 4.68(2H, m, H at the 5 ' -position of the sugar ring), 5.55 to 5.78(1H, m, H at the 5-position of the pyrimidine ring), 6.16(1H, s, H at the 1 ' -position of the sugar ring), 7.07 to 7.42(11H, m, H at the 6-position of the pyrimidine ring, hydrogen on both benzene rings), 9.78(1H, s, NH at the 3-position of the pyrimidine ring).
LCMS-ESI+(m/z):803.9(M+H)。
Example 15
Biological evaluation
1. Detection of antiviral Activity of the Urodylate phosphoramidate Compounds of the present invention in the HCV replicon (HCVpp) System
HCV replicon assay procedure
General procedure Huh-7 derived cell lines (ZLuc) bearing HCV genotype 1b replicon and luciferase reporter genes were grown in Dulbecco's Modified Eagle Medium (DMEM) 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). By using lipid/histone based transfectionZluc cells were transiently transfected with human carboxylesterase 1(CES 1). 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 (7)
1. A uridylic acid bis-phenylpropionate phosphoramidate compound or a pharmaceutically acceptable salt thereof, wherein the structural formula of the uridylic acid bis-phenylpropionate phosphoramidate compound is selected from one of the following structural formulas:
2. a pharmaceutically acceptable salt of the uridylic acid bis-phenylpropionate based phosphoramidate compound of claim 1.
3. The pharmaceutical composition is characterized by comprising the uridylic acid bis-phenylpropionate based phosphoramidate compound or the pharmaceutically acceptable salt thereof according to claim 1, and an auxiliary material, wherein the auxiliary material is a pharmaceutically acceptable carrier or excipient.
4. The pharmaceutical composition of claim 3, further comprising at least one of ribavirin, interferon, a hepatitis C NS3 protease inhibitor, a HCV reverse transcriptase NS5B non-nucleoside inhibitor, a HCV reverse transcriptase NS5B nucleoside inhibitor, a NS5A inhibitor, and a potentiator of a NS5A inhibitor, a cyclosporin immunosuppressant, a NS4A antagonist, a NS4B inhibitor, or a cyclophilin inhibitor.
5. Use of the uridylic acid bis-phenylpropionate based phosphoramidate compound of claim 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of a human flaviviridae infection.
6. The use according to claim 5, wherein the human Flaviviridae viral infection is a human HCV viral infection.
7. 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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