CN112194680A - Nucleotide phosphoramidate compound, pharmaceutical composition thereof, and preparation method and application thereof - Google Patents
Nucleotide 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 nucleotide phosphoramidate compound, a pharmaceutical composition thereof, a preparation method and application thereof, in particular to application of the nucleotide phosphoramidate compound in treating neocoronary pneumonia. Experiments prove that the compound has the activity of inhibiting the replication of 2019-nCoV, and meanwhile, the nucleotide phosphoramidate compound has the advantages of high in-vitro activity, large development coefficient and the like compared with the existing medicine Redexi Wevir for treating the new coronary pneumonia, and can be used for developing the medicine for treating the new coronary pneumonia.
Description
Technical Field
The invention belongs to the technical field of medicines, and relates to a nucleotide phosphoramidate compound, a pharmaceutical composition thereof, a preparation method and an application thereof, which are suitable for treating coronavirus infection, in particular to treating new coronary pneumonia.
Background
The impact of a coronavirus (2019-nCoV) pandemic on the world is enormous: the latest data published by the world health organization of 8 months and 31 days in tile 8 months and 31 days (reporter Liuqu) in Xinhua Commitment show that 25118689 cases of globally accumulated newly-crown confirmed diagnosis cases and 844312 cases of death cases.
Coronaviruses belong phylogenetically to the order of the nested viruses (Nidovirales) the family of Coronaviridae (Coronaviridae) the genus coronaviruses (Coronavirus). A total of 7 human-infectable coronaviruses are currently found, HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-CoV and 2019-nCoV, respectively.
At present, dozens of new vaccines of coronavirus (2019-nCoV) are researched in the world, and some vaccines enter a phase III clinical test.
The medicines in the research which have the inhibiting effect on the new coronavirus (2019-nCoV) in the world are just started at present, the medicines in the research of Reid Wevir of the Jilide science company in the United states have weak inhibiting effect on the new coronavirus (2019-nCoV) (on Vero E6 cells, the half effective concentration EC50 of Reid Wevir on the 2019-nCoV is 0.77 mu M (micromole per liter), the half cytotoxic concentration CC50 is more than 100 mu M (micromole per liter), and the selection index SI is more than 129. however, the novel coronavirus (2019-nCoV) has extremely strong transmission capability, so that the measures of traffic limitation and the like cannot block the spread of diseases, the possible long-standing problems of the novel coronavirus (2019-nCoV) cannot be ignored, therefore, on one hand, the promotion of the new inactivated coronavirus vaccine to be marketed is accelerated, and on the other hand, the development of a high-efficiency low-toxicity therapeutic drug for inhibiting the infection of the new coronavirus (2019-nCoV) is still an urgent clinical need.
Disclosure of Invention
The invention aims to further modify the structure of a nucleotide phosphoramidate compound so as to obtain a novel nucleotide phosphoramidate compound analogue with lower toxicity and higher activity of resisting novel coronavirus (2019-nCoV), 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 nucleotide phosphoramidate compound or a pharmaceutically acceptable salt thereof, wherein the structural formula of the nucleotide phosphoramidate compound is (Ia):
wherein:
Rais methyl or cyano;
Rbis hydroxy or fluoro;
Rcany one selected from alkyl groups having 1 to 12 carbon atoms.
The invention further provides the nucleotide phosphoramidate compound or a pharmaceutically acceptable salt thereof, wherein the amino acid ester linked to the phosphorus atom is in the S configuration and has the structural formula (Ib):
wherein:
Rais methyl or cyano;
Rbis hydroxy or fluoro;
Rcany one selected from alkyl groups having 1 to 12 carbon atoms.
The present invention still further provides the nucleotide phosphoramidate compound or pharmaceutically acceptable salt thereof, wherein the phosphorus atom is a chiral phosphorus atom, preferably S(P)Configuration or R(P)One or two of the configurations, the structural formula is one or two selected from the following (Ic1) or (Ic 2):
wherein:
Rais methyl or cyano;
Rbis hydroxy or fluoro;
Rcany one selected from alkyl groups having 1 to 12 carbon atoms.
The nucleotide phosphoramidate compound of the present invention or a pharmaceutically acceptable salt thereof, wherein R iscSelected from any one of isopropyl, ethyl, isobutyl, neopentyl, n-butyl, cyclohexyl, tert-butyl or 2-ethylbutyl, and the structural formula of the nucleotide phosphoramidate compound is selected from one of the following structural formulas:
the pharmaceutical composition comprises the nucleotide 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, palivizumab, mevizumab, RSV-IGIV (respiratory syncytial virus-intravenous immunoglobulin), MEDI-557 (a respiratory syncytial virus treatment medicament from MedImmune), A-60444 (a respiratory virus treatment medicament active component), MDT-637 (a respiratory virus treatment medicament, cas of the compound: 235106-62-4) or BMS-433771 (a respiratory virus treatment medicament, cas of the compound: 543700-68-1).
The preparation method of the nucleotide phosphoramidate compound comprises the following steps:
under the alkaline condition, after the alkoxy carbonyloxyalkyl alcohol reacts with phosphorus oxychloride which is a phosphorylation reagent, amino acid ester hydrochloride and pentafluorophenol are added to react to obtain a compound FP23, and the compound FP23 reacts with nucleoside R at the temperature of between 20 ℃ below zero and 80 ℃ below zero to obtain a compound (Ia).
The synthetic route of the preparation method is shown in figure 1:
wherein:
Rais methyl or cyano;
Rbis hydroxy or fluoro;
Rcany one selected from alkyl groups having 1 to 12 carbon atoms.
The application of the nucleotide phosphoramidate compound or the pharmaceutically acceptable salt thereof in preparing the medicine for treating human coronavirus infection is provided.
The application of the invention, wherein the human coronavirus infection comprises human SARS virus infection, human MERS virus infection or human 2019-nCoV virus infection.
The invention discloses application of a pharmaceutical composition in preparing a medicament for resisting human coronavirus infection, wherein the coronavirus comprises SARS virus, MERS virus or 2019-nCoV virus.
The invention has the beneficial effects that:
the compound has excellent properties required by being a medicament for treating the new coronary pneumonia, which is determined by a detection mechanism and specifically comprises the following components:
in an in vitro assay for 2019-nCoV activity, the EC for Compound RED2371-1, RED2371-2, RED2374-1 or RED2374-250Is 2-5 times of the Rudexilvir (positive control), and the biological activity selection coefficient SI is 2-5 times of the Rudexilvir (positive control).
This indicates that: the compound can effectively inhibit 2019-nCoV infection at a cellular level, and is expected to become a medicament for treating 2019-nCoV infection.
Drawings
FIG. 1 is a schematic diagram of the synthetic route of the preparation method of the nucleotide 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 (R) -N- [ (pentafluorophenoxy) (isopropoxycarbonyloxymethoxy) phosphoryl ] -L-phenylalanine isopropyl ester (FP2370-1) and (S) -N- [ (pentafluorophenoxy) (isopropoxycarbonyloxymethoxy) phosphoryl ] -L-phenylalanine isopropyl ester (FP 2370-2).
Phosphorus oxychloride (5g, 3.04mL, 32.6mmol) and acetonitrile (200mL) were added to the reaction flask, cooled to-70 ℃, a solution of K2(7.16g, 32.6mmol) and triethylamine (3.3g, 4.53mL, 32.6mmol) in acetonitrile (60mL) was slowly added dropwise, and after the addition was complete, the temperature was slowly raised to room temperature, and the reaction was allowed to proceed overnight. Cooling the mixture to 0 deg.C, adding HA370(4.93g, 29.4mmol), cooling to-70 deg.C, adding triethylamine (7.3g, 10mL, 72mmol) in acetonitrile 60mL, heating to 0 deg.C, reacting for 3 hours, adding pentafluorophenol (5.4g, 29.4mmol) and triethylamine (7.3g, 10mL, 72mmol) in acetonitrile 60mL dropwise to the solution, stirring at 0 deg.C for 1 hour, heating to room temperature, stirring overnight, adding 100 mL dichloromethane and 100 mL water, separating out the organic phase, drying with anhydrous sodium sulfate, concentrating under reduced pressure, separating the residue with silica gel column (0-30% ethyl acetate/hexane) to obtain 8.1g white solid, recrystallizing with 10% t-butyl methyl ether/hexane to obtain white solid FP2370-2(3.1g), separating the mother liquor with silica gel column (50% ethyl acetate/hexane) to obtain FP2370-1(2.6g) and FP2370-2 (0.236 g), the purity of both FP2370-2 and FP2370-1 is more than 99%.
Nuclear magnetic hydrogen spectrum data of FP 2370-1:1H NMR(400MHz，CDCl3)δ(ppm)：1.14-1.42(12H， m，4×CH3)，3.71-4.07(3H，m，CH2 and NCH)，4.29-4.45(1H，m，NH)，4.84-5.10(2H， m，2×COOCH)，5.54-5.74(2H，m，OCH2o), 7.04-7.38(5H, m, hydrogen on benzene ring).
31P NMR(162MHz,CDCl3)δ-1.58。
ESI-MS:[M+H]570.4。
Nuclear magnetic hydrogen spectrum data of FP 2370-2:1H NMR(400MHz，CDCl3)δ(ppm)：1.16-1.44(12H， m，4×CH3)，3.75-4.09(3H，m，CH2 and NCH)，4.30-4.46(1H，m，NH)，4.86-5.09(2H， m，2×COOCH)，5.55-5.77(2H，m，OCH2o), 7.01-7.39(5H, m, hydrogen on benzene ring).
31P NMR(162MHz,CDCl3)δ-1.82。
ESI-MS:[M+H]570.4
Example 2
Synthesis of (R) -ethyl-N- [ (pentafluorophenoxy) (isopropoxycarbonyloxymethoxy) phosphoryl ] -L-phenylalanine (FP2371-1) and (S) -ethyl-N- [ (pentafluorophenoxy) (isopropoxycarbonyloxymethoxy) phosphoryl ] -L-phenylalanine (FP 2371-2).
FP2371-1 and FP2371-2 were synthesized by a similar synthesis method to that of example 1.
Nuclear magnetic hydrogen spectrum data of FP 2371-1:1H NMR(400MHz，CDCl3)δ(ppm)：1.26-1.40(9H， m，3×CH3)，3.77-3.97(4H，m，CH2，NH and NCH)，4.05-4.39(2H，m，COOCH2)， 4.85-4.97(1H，m，COOCH)，5.55-5.72(2H，m，OCH2o), 7.06-7.38(5H, m, hydrogen on benzene ring).
31P NMR(162MHz,CDCl3)δ-1.72。
Nuclear magnetic hydrogen spectrum data of FP 2371-2:1H NMR(400MHz，CDCl3)δ(ppm)：1.28-1.42(9H， m，3×CH3)，3.90-4.04(4H，m，CH2，NH and NCH)，4.16-4.38(2H，m，COOCH2)， 4.88-5.05(1H，m，COOCH)，5.55-5.77(2H，m，OCH2o), 7.07-7.36(5H, m, hydrogen on benzene ring).
31P NMR(162MHz,CDCl3)δ-1.99。
ESI-MS:[M+H]556.4
Example 3
Synthesis of (R) -N- [ (pentafluorophenoxy) (isopropoxycarbonyloxymethoxy) phosphoryl ] -L-phenylalanine isobutyl ester (FP2372-1) and (S) -N- [ (pentafluorophenoxy) (isopropoxycarbonyloxymethoxy) phosphoryl ] -L-phenylalanine isobutyl ester (FP 2372-2).
FP2372-1 and FP2372-2 were synthesized by a similar synthesis method to that of example 1.
Nuclear magnetic hydrogen spectrum data of FP 2372-1:1H NMR(400MHz，CDCl3)δ(ppm)：0.90-1.04(6H， m，2×CH3)，1.27-1.37(6H，m，2×CH3)，2.33-2.51(1H，m，CH)，3.75-3.96(4H， m，CH2，NH and NCH)，4.09-4.35(2H，m，COOCH2)，4.83-4.98(1H，m，COOCH)， 5.56-5.74(2H，m，OCH2o), 7.04-7.35(5H, m, hydrogen on benzene ring).
31P NMR(162MHz,CDCl3)δ-1.66。
Nuclear magnetic hydrogen spectrum data of FP 2372-2:1H NMR(400MHz，CDCl3)δ(ppm)：0.92-1.06(6H， m，2×CH3)，1.29-1.39(6H，m，2×CH3)，2.37-2.58(1H，m，CH)，3.88-3.99(4H， m，CH2，NH and NCH)，4.12-4.33(2H，m，COOCH2)，4.87-5.03(1H，m，COOCH)， 5.55-5.76(2H，m，OCH2o), 7.00-7.37(5H, m, hydrogen on benzene ring).
31P NMR(162MHz,CDCl3)δ-1.97。
ESI-MS:[M+H]584.4。
Example 4
Synthesis of (R) -N- [ (pentafluorophenoxy) (isopropoxycarbonyloxymethoxy) phosphoryl ] -L-phenylalanine neopentyl ester (FP2373-1) and (S) -N- [ (pentafluorophenoxy) (isopropoxycarbonyloxymethoxy) phosphoryl ] -L-phenylalanine neopentyl ester (FP 2373-2).
FP2373-1 and FP2373-2 were synthesized by a similar synthesis method to that of example 1.
Nuclear magnetic hydrogen spectrum data of FP 2373-1:1H NMR(400MHz，CDCl3)δ(ppm)：0.94(9H，s， 3×CH3)，1.26-1.36(6H，m，2×CH3)，3.76-3.95(4H，m，CH2，NH and NCH)， 4.08-4.39(2H，m，COOCH2)，4.86-4.97(1H，m，COOCH)，5.57-5.73(2H，m，OCH2o), 7.05-7.37(5H, m, hydrogen on benzene ring).
31P NMR(162MHz,CDCl3)δ-1.75。
Nuclear magnetic hydrogen spectrum data of FP 2373-2:1H NMR(400MHz，CDCl3)δ(ppm)：0.95(9H，s， 3×CH3)，1.28-1.37(6H，m，2×CH3)，3.90-3.99(4H，m，CH2，NH and NCH)， 4.11-4.38(2H，m，COOCH2)，4.87-5.01(1H，m，COOCH)，5.59-5.76(2H，m，OCH2o), 7.00-7.36(5H, m, hydrogen on benzene ring).
31P NMR(162MHz,CDCl3)δ-1.97。
ESI-MS:[M+H]598.5
Example 5
Synthesis of (R) -N- [ (pentafluorophenoxy) (isopropoxycarbonyloxymethoxy) phosphoryl ] -L-phenylalanine N-butyl ester (FP2374-1) and (S) -N- [ (pentafluorophenoxy) (isopropoxycarbonyloxymethoxy) phosphoryl ] -L-phenylalanine N-butyl ester (FP 2374-2).
FP2374-1 and FP2374-2 were synthesized in a similar manner to example 1.
Nuclear magnetic hydrogen spectrum data of FP 2374-1:1H NMR(400MHz，CDCl3)δ(ppm)：0.93(3H，t， CH3)，1.18-1.44(8H，m，2×CH3 and CH2)，1.55-1.67(2H，m，CH2)，3.96-4.07(4H， m，CH2，NH and NCH)，4.08-4.41(2H，m，COOCH2)，4.85-4.98(1H，m，COOCH)， 5.53-5.72(2H，m，OCH2o), 7.03-7.37(5H, m, hydrogen on benzene ring).
31P NMR(162MHz,CDCl3)δ-1.48。
Nuclear magnetic hydrogen spectrum data of FP 2374-2:1H NMR(400MHz，CDCl3)δ(ppm)：0.94(3H，t， CH3)，1.20-1.46(8H，m，2×CH3 and CH2)，1.57-1.69(2H，m，CH2)，3.98-4.08(4H， m，CH2，NH and NCH)，4.21-4.39(2H，m，COOCH2)，4.87-5.01(1H，m，COOCH)， 5.55-5.73(2H，m，OCH2o), 7.04-7.37(5H, m, hydrogen on benzene ring).
31P NMR(162MHz,CDCl3)δ-1.71。
ESI-MS:[M+H]584.4
Example 6
Synthesis of (R) -N- [ (pentafluorophenoxy) (isopropoxycarbonyloxymethoxy) phosphoryl ] -L-phenylalanine tert-butyl ester (FP2378-1) and (S) -N- [ (pentafluorophenoxy) (isopropoxycarbonyloxymethoxy) phosphoryl ] -L-phenylalanine iso-tert-butyl ester (FP 2378-2).
FP2378-1 and FP2378-2 were synthesized in a similar manner to example 1.
Nuclear magnetic hydrogen spectrum data of FP 2378-1:1H NMR(400MHz，CDCl3)δ(ppm)：1.25-1.34(15H， m，5×CH3)，3.79-3.96(4H，m，CH2，NH and NCH)，4.85-4.98(1H，m，COOCH)， 5.58-5.73(2H，m，OCH2o), 7.07-7.37(5H, m, hydrogen on benzene ring).
31P NMR(162MHz,CDCl3)δ-1.81。
Nuclear magnetic hydrogen spectrum data of FP 2378-2:1H NMR(400MHz，CDCl3)δ(ppm)：1.26-1.39(15H， m，5×CH3)，3.86-3.97(4H，m，CH2，NH and NCH)，4.87-5.02(1H，m，COOCH)， 5.59-5.77(2H，m，OCH2o), 7.05-7.39(5H, m, hydrogen on benzene ring).
31P NMR(162MHz,CDCl3)δ-1.99。
ESI-MS:[M+H]584.4
Example 7
Synthesis of (R) -N- [ (pentafluorophenoxy) (isopropoxycarbonyloxymethoxy) phosphoryl ] -L-phenylalanine-2-ethylbutyl ester (FP2379-1) and (S) -N- [ (pentafluorophenoxy) (isopropoxycarbonyloxymethoxy) phosphoryl ] -L-phenylalanine-2-ethylbutyl ester (FP 2379-2).
FP2379-1 and FP2379-2 were synthesized in a similar manner to example 1.
Nuclear magnetic hydrogen spectrum data of FP 2379-1:1H NMR(400MHz，CDCl3)δ(ppm)：0.90-1.06(6H， m，2×CH3)，1.25-1.38(10H，m，2×CH2，2×CH3)，1.97-2.17(1H，m，CH)，3.78-3.92(4H， m，CH2，NH and NCH)，4.06-4.33(2H，m，COOCH2)，4.87-4.95(1H，m，COOCH)， 5.55-5.72(2H，m，OCH2o), 7.10-7.33(5H, m, hydrogen on benzene ring).
31P NMR(162MHz,CDCl3)δ-1.62。
Nuclear magnetic hydrogen spectrum data of FP 2379-2:1H NMR(400MHz，CDCl3)δ(ppm)：0.92-1.07(6H， m，2×CH3)，1.26-1.39(10H，m，2×CH2，2×CH3)，1.98-2.19(1H，m，CH)， 3.85-3.99(4H，m，CH2，NH and NCH)，4.12-4.37(2H，m，COOCH2)，4.84-5.05(1H， m，COOCH)，5.53-5.76(2H，m，OCH2o), 7.08 to 7.35(5H, m, hydrogen on benzene ring).
31P NMR(162MHz,CDCl3)δ-1.87。
ESI-MS:[M+H]612.5。
Example 8
Synthesis of (2S) -2- (((R) - ((((2R, 3S,4R,5R) -5- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) (isopropoxycarbonyloxymethoxy) phosphoryl) amino) isopropyl benzoate (RED 2370-1).
Preparation of nucleoside RED is described in patent application No. 201180035776.1 and patent application No. 201510615482.6.
A50 mL flask was charged with nucleoside RED (291.3mg,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 FP2370-2(0.911g, 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 RED2370-1(311mg), a white solid product, in 46% yield.
Nuclear magnetic hydrogen spectrum data of RED 2370-1:1H NMR(400MHz，CDCl3)δ(ppm)：1.21-1.38(12H， m，4×CH3)，3.27-3.50(3H，m，CH2and NCH), 3.81-4.11(2H, m, H at the 2 '-position of the sugar ring and H at the 3' -position), 4.27(2H, brs, OH at the 2 '-position of the sugar ring and OH at the 3' -position), 4.34-4.45(2H, m, PNH and H at the 4 '-position of the sugar ring), 4.47-4.58(2H, m, H at the 5' -position of the sugar ring), 4.84-5.07(2H, m, 2x COOCH), 5.58-5.72(4H, m, H and OCH at the pyrrole ring)2O), 6.18 (NH in position 5 of the 2H, s, triazine ring2) 7.07-7.41(5H, m, H on the phenyl ring), 9.15(1H, s, H on the 3-position of the triazine ring).
31P NMR(162MHz,CDCl3)δ7.3；
LCMS-ESI+(m/z):677.6(M+H)。
Example 9 RED2371
Synthesis of ethyl (2S) -2- (((S) - ((((2R, 3S,4R,5R) -5- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) (isopropoxycarbonyloxymethoxy) phosphoryl) amino) phenylpropionate (RED 2371-2).
RED2371-2 was synthesized by a similar synthetic method as example 8.
Nuclear magnetic hydrogen spectrum data of RED 2371-2:1H NMR(400MHz，CDCl3)δ(ppm)：1.25-1.43(9H， m，3×CH3)，3.85-4.17(6H，m，CH2NH and NCH, H at the 2 '-position and H at the 3' -position of the sugar ring), 4.20-4.42(5H, m, COOCH)2OH at the 2 '-position of the sugar ring, OH at the 3' -position, H at the 4 '-position of the sugar ring), 4.46 to 4.59(2H, m, H at the 5' -position of the sugar ring), 4.85 to 5.03(1H, m, COOCH), 5.55 to 5.74(4H, m, OCH)2O, H on the pyrrole ring), 6.17(2H, s, NH in position 5 of the triazine ring2) 7.08-7.32(5H, m, hydrogen on benzene ring), 9.14(1H, m, H on triazine ring 3 position).
31P NMR(162MHz,CDCl3)δ9.1；
LCMS-ESI+(m/z):663.6(M+H)。
Example 10 RED2372
Synthesis of (2S) -2- (((S) - ((((2R, 3S,4R,5R) -5- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) (isopropoxycarbonyloxymethoxy) phosphoryl) amino) isobutyl phenylpropionate (RED 2372-2).
RED2372-2 was synthesized by a similar synthetic method as example 8.
Nuclear magnetic hydrogen spectrum data of RED 2372-2:1H NMR(400MHz，CDCl3)δ(ppm)：0.88-1.04(6H， m，2×CH3)，1.23-1.37(6H，m，2×CH3)，2.33-2.45(1H，mCH), 3.70-4.15(6H, m, H at the 2 '-position of the sugar ring, H, CH at the 3' -position2NH and NCH), 4.19-4.43(5H, m, OH at the 2 ' -position of the sugar ring, OH at the 3 ' -position, H and COOCH at the 4 ' -position of the sugar ring2) 4.46-4.59(2H, m, H at the 5' -position of the sugar ring), 4.85-4.98(1H, m, COOCH), 5.58-5.74(4H, m, H and OCH at the pyrrole ring)2O), 6.16 (NH in position 5 of the 2H, s, triazine ring)2) 7.05-7.29(5H, m, hydrogen on benzene ring), 9.18(1H, m, H on triazine ring 3 position).
31P NMR(162MHz,CDCl3)δ7.9；
LCMS-ESI+(m/z):691.7(M+H)。
Example 11
Synthesis of (2S) -2- (((S) - ((((2R, 3S,4R,5R) -5- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) (isopropoxycarbonyloxymethoxy) phosphoryl) amino) benzenepropanoic acid neopentyl ester (RED 2373-2).
RED2373-2 was synthesized by a similar synthetic method as in example 8.
Nuclear magnetic hydrogen spectrum data of RED 2373-2:1H NMR(400MHz，CDCl3)δ(ppm)：0.89-1.00(9H， m，3×CH3)，1.25-1.37(6H，m，2×CH3)，3.29-3.54(2H，d，CH2) 3.82-3.99(4H, m, H at the 2 ' -position of the sugar ring, H at the 3 ' -position of the sugar ring, PNH, NCH), 4.17-4.40(5H, m, OH at the 2 ' -position of the sugar ring, OH at the 3 ' -position, H and COOCH at the 4 ' -position of the sugar ring2) 4.47-4.59(2H, m, H at the 5' -position of the sugar ring), 4.85-4.98(1H, m, COOCH), 5.58-5.76(4H, m, H at the pyrrole ring, OCH)2O), 6.19 (NH in position 5 of the 2H, s, triazine ring)2) 7.07-7.44(5H, m, H on the phenyl ring), 9.16(1H, s, H on the 3-position of the triazine ring).
31P NMR(162MHz,CDCl3)δ7.8；
LCMS-ESI+(m/z):705.7(M+H)。
Example 12
Synthesis of n-butyl (2S) -2- (((S) - ((((2R, 3S,4R,5R) -5- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) (isopropoxycarbonyloxymethoxy) phosphoryl) amino) phenylpropionate (RED 2374-2).
RED2374-2 was synthesized by a similar synthetic method as example 8.
Nuclear magnetic hydrogen spectrum data of RED 2374-2:1H NMR(400MHz，CDCl3)δ(ppm)：0.97(3H，t， CH3)，1.20-1.45(8H，m，2×CH3andCH2)，1.58-1.73(2H，m，CH2)，3.31-3.50(2H， d，CH2) 3.88-4.04(4H, m, H at the 2 ' -position of the sugar ring, H at the 3 ' -position of the sugar ring, PNH and NCH), 4.12-4.43(5H, m, OH at the 2 ' -position of the sugar ring, OH at the 3 ' -position, H and COOCH at the 4 ' -position of the sugar ring2) 4.45-4.59(2H, m, H at the 5' -position of the sugar ring), 4.88-5.01(1H, m, COOCH), 5.57-5.79(4H, m, Hand OCH at the pyrrole ring)2O), 6.18 (NH in position 5 of the 2H, s, triazine ring2) 7.09-7.42(5H, m, H on the phenyl ring), 9.14(1H, s, H on the 3-position of the triazine ring).
31P NMR(162MHz,CDCl3)δ6.5；
LCMS-ESI+(m/z):691.7(M+H)。
Example 13
Synthesis of tert-butyl (2S) -2- (((R) - ((((2R, 3S,4R,5R) -5- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) (isopropoxycarbonyloxymethoxy) phosphoryl) amino) benzoate (RED 2378-1).
RED2378-1 was synthesized by a similar synthetic method as in example 8
Nuclear magnetic hydrogen spectrum data of RED 2378-1:1H NMR(400MHz，CDCl3)δ(ppm)：1.23-1.39 (15H，m，5×CH3) 3.80-4.17(6H, m, H at the 2 '-position of the sugar ring, H, CH at the 3' -position2NH and NCH), 4.26 to 4.40(3H, m, OH at the 2 '-position of the sugar ring, OH at the 3' -position, H at the 4 '-position of the sugar ring), 4.45 to 4.58(2H, m, H at the 5' -position of the sugar ring), 4.88 to 5.00(1H, m, COOCH), 5.57 to 5.75(4H, m, H and OCH at the pyrrole ring)2O), 6.17 (NH in position 5 of the 2H, s, triazine ring2) 7.04-7.33(5H, m, hydrogen on benzene ring), 9.17(1H, m, H on triazine ring 3 position).
31P NMR(162MHz,CDCl3)δ9.6；
LCMS-ESI+(m/z):691.7(M+H)。
Example 14
Synthesis of (2S) -2- (((S) - ((((2R, 3S,4R,5R) -5- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) (isopropoxycarbonyloxymethoxy) phosphoryl) amino) phenylpropionic acid-2-ethylbutyl ester (RED 2379-2).
RED2379-2 was synthesized by a similar synthetic method as example 8.
Nuclear magnetic hydrogen spectrum data of RED 2379-2:1H NMR(400MHz，CDCl3)δ(ppm)：0.87-1.06(6H， m，2×CH3)，1.22-1.36(10H，m，2×CH2，2×CH3) 1.96-2.17(1H, m, CH), 3.78-4.08 (6H, m, H at the 2 '-position of the sugar ring, H, CH at the 3' -position2NH and NCH), 4.12-4.42(5H, m, OH at the 2 ' -position of the sugar ring, OH at the 3 ' -position, H and COOCH at the 4 ' -position of the sugar ring2) 4.46-4.59(2H, m, H at the 5' -position of the sugar ring), 4.86-4.95(1H, m, COOCH), 5.54-5.74(2H, m, H and OCH at the pyrrole ring)2O)，
6.17 (NH in position 5 of the 2H, s, triazine ring)2) 7.11-7.33(5H, m, hydrogen on benzene ring), 9.18(1H, m, H on triazine ring 3 position).
31P NMR(162MHz,CDCl3)δ8.6；
LCMS-ESI+(m/z):719.7(M+H)。
Example 15
Synthesis of (2S) -2- (((R) - ((((2R, 3R,4R,5R) -5- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-4-fluoro-3-hydroxytetrahydrofuran-2-yl) methoxy) (isopropoxycarbonyloxymethoxy) phosphoryl) amino) isopropyl benzoate (RNF 2370-1).
Preparation of nucleoside RNF is described by reference to the preparation methods of patent application No. 201180035776.1 and patent application No. 201510615482.6.
RNF2370-1 was synthesized by a similar method to that of example 8.
Nuclear magnetic hydrogen spectrum data of RNF 2370-1:1H NMR(400MHz，CDCl3)δ(ppm)：1.20-1.41(12H， m，4×CH3)，3.25-3.46(3H，m，CH2and NCH), 3.80 to 4.08(2H, m, H at the 2 ' -position of the sugar ring and H at the 3 ' -position), 4.26(1H, brs, OH at the 3 ' -position of the sugar ring), 4.33 to 4.45(2H, m, H at the 4 ' -position of the PNH and sugar ring), 4.47 to 4.58(2H, m, H at the 5 ' -position of the sugar ring), 4.86 to 5.09 (2H, m, 2 xcooch), 5.58 to 5.69(4H, m, H at the pyrrole ring and OCH)2O), 6.17 (NH in position 5 of the 2H, s, triazine ring2) 7.09-7.39(5H, m, H on the phenyl ring), 9.19(1H, s, H on the 3-position of the triazine ring).
31P NMR(162MHz,CDCl3)δ8.2；
LCMS-ESI+(m/z):679.6(M+H)。
Example 16
Synthesis of (2S) -2- (((S) - ((((2R, 3R,4R,5S) -5- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -4-fluoro-3-hydroxy-5-methyltetrahydrofuran-2-yl) methoxy) (isopropoxycarbonyloxymethoxy) phosphoryl) amino) phenylpropionic acid n-butyl ester (RCF 2374-2).
Preparation of nucleoside RCF was made by the methods of preparation referenced in patent application No. 201180035776.1 and patent application No. 201510615482.6.
RCF2374-2 was synthesized by a similar synthesis method to that of example 8.
Nuclear magnetic hydrogen spectrum data of RCF 2374-2:1H NMR(400MHz，CDCl3)δ(ppm)：0.98(3H，t， CH3)，1.22-1.46(8H，m，2×CH3andCH2)，1.56-1.74(5H，m，CH3andCH2)， 3.32-3.47(2H，d，CH2) 3.86-4.02(4H, m, H at the 2 '-position of the sugar ring, H at the 3' -position of the sugar ring, PNH and NCH), 4.11-4.40 (OH at the 4H, m, 3 '-position, H and COOCH at the 4' -position of the sugar ring)2) 4.43-4.57(2H, m, H at the 5' -position of the sugar ring), 4.86-5.02(1H, m, COOCH), 5.56-5.77(4H, m, Hand OCH at the pyrrole ring)2O), 6.19 (NH in position 5 of the 2H, s, triazine ring)2) 7.09-7.40(5H, m, H on the phenyl ring), 9.10(1H, s, H on the 3-position of the triazine ring).
31P NMR(162MHz,CDCl3)δ7.8；
LCMS-ESI+(m/z):682.6(M+H)。
Example 17
Synthesis of (2S) -2- (((S) - ((((2R, 3S,4R,5S) -5- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -3, 4-dihydroxy-5-methyltetrahydrofuran-2-yl) methoxy) (isopropoxycarbonyloxymethoxy) phosphoryl) amino) phenylpropionic acid-2-ethylbutyl ester (RCO 2379-2).
Preparation of nucleoside RCO is described in patent application No. 201180035776.1 and patent application No. 201510615482.6.
RCO2379-2 was synthesized by a similar synthesis method to that of example 8.
Nuclear magnetic hydrogen spectrum data of RCO 2379-2:1H NMR(400MHz，CDCl3)δ(ppm)：0.87-1.07(6H， m，2×CH3)，1.21-1.36(10H，m，2×CH2，2×CH3)，1.50-1.64(3H，m，CH3) 1.96-2.16(1H, m, CH), 3.77-4.08(6H, m, H at the 2 '-position of the sugar ring, H, CH at the 3' -position2NH and NCH), 4.12-4.40(5H, m, OH at the 2 ' -position of the sugar ring, OH at the 3 ' -position, H and COOCH at the 4 ' -position of the sugar ring2) 4.45-4.59(2H, m, H at the 5' -position of the sugar ring), 4.84-4.96(1H, m, COOCH), 5.56-5.73(2H, m, H and OCH at the pyrrole ring)2O), 6.17 (NH in position 5 of the 2H, s, triazine ring2) 7.12-7.33(5H, m, hydrogen on benzene ring), 9.18(1H, m, H on triazine ring 3 position).
31P NMR(162MHz,CDCl3)δ7.2；
LCMS-ESI+(m/z):708.7(M+H)。
Example 18
Biological evaluation
In the test, we evaluated candidate nucleoside compounds and the positive control, Reidesivir (Remdesivir), against 2019-nCoV clinical isolates. First, cytotoxicity of candidate compounds in Vero E6 cells (ATCC-1586) was determined by CCK8 analysis. Vero E6 cells were then infected with nCoV-2019BeTaCoV/WIV04/20192 at a multiplicity of infection (MOI) of 0.05 in the presence of different concentrations of the test drug.
Table 1: in vitro antiviral activity and cytotoxicity assay of candidate Compounds against 2019-nCoV
Note: , + ++ refers to 1-100 nM; + represents 0.1-0.5. mu.M; + represents 0.5-1. mu.M.
While the present disclosure 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 a broad, potential interpretation 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 (10)
1. A nucleotide phosphoramidate compound or a pharmaceutically acceptable salt thereof, wherein the nucleotide phosphoramidate compound has the structural formula (Ia):
wherein:
Rais methyl or cyano;
Rbis hydroxy or fluoro;
Rcany one selected from alkyl groups having 1 to 12 carbon atoms.
2. The nucleotide phosphoramidate compound or pharmaceutically acceptable salt thereof of claim 1 wherein the amino acid ester attached to the phosphorus atom has the S configuration and the structural formula (Ib):
wherein:
Rais methyl or cyano;
Rbis hydroxy or fluoro;
Rcany one selected from alkyl groups having 1 to 12 carbon atoms.
3. The nucleotide phosphoramidate compound or pharmaceutically acceptable salt thereof of claim 2 wherein the phosphorus atom is a chiral phosphorus atom, preferably S(P)Configuration or R(P)One or two of the configurations, the structural formula is one or two selected from the following (Ic1) or (Ic 2):
wherein:
Rais methyl or cyano;
Rbis hydroxy or fluoro;
Rcany one selected from alkyl groups having 1 to 12 carbon atoms.
4. The nucleotide phosphoramidate compound or pharmaceutically acceptable salt thereof of claim 3 wherein R iscSelected from any one of isopropyl, ethyl, isobutyl, neopentyl, n-butyl, cyclohexyl, tert-butyl or 2-ethylbutyl, and the structural formula of the nucleotide phosphoramidate compound is selected from one of the following structural formulas:
5. a pharmaceutical composition comprising the nucleotide phosphoramidate compound of any of claims 1-4 or a pharmaceutically acceptable salt thereof, and an excipient, wherein the excipient is a pharmaceutically acceptable carrier or excipient.
6. The pharmaceutical composition of claim 5, further comprising at least one of ribavirin, palivizumab, mevizumab, RSV-IGIV, MEDI-557, a-60444, MDT-637, or BMS-433771.
7. A method for preparing a nucleotide phosphoramidate compound, comprising the steps of: under the alkaline condition, after the alkoxy carbonyloxyalkyl alcohol reacts with phosphorus oxychloride which is a phosphorylation reagent, amino acid ester hydrochloride and pentafluorophenol are added to react to obtain a compound FP23, and the compound FP23 reacts with nucleoside R at the temperature of between 20 ℃ below zero and 80 ℃ below zero to obtain a compound (Ia).
8. Use of a nucleotide phosphoramidate compound of any of claims 1-4 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of a human coronavirus infection.
9. The use of claim 8, wherein the human coronaviridae viral infection comprises a human SARS viral infection, a human MERS viral infection, or a human 2019-nCoV viral infection.
10. Use of a pharmaceutical composition according to any one of claims 5 to 6 for the manufacture of a medicament against infection by a virus of the family Coronaviridae in humans, wherein said virus of the family Coronaviridae comprises a SARS virus, a MERS virus or a 2019-nCoV virus.
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