Methods for treating infections caused by viruses of the Arenaviridae and Coronaviridae families

Abstract

Methods of treating Arenaviridae and Coronaviridae viral infections by administering nucleosides of Formula I and prodrugs thereof, wherein the 1' position of the nucleoside sugar is substituted. The provided compounds, compositions and methods are particularly useful for treating Lassa virus and Junin virus infections.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 219,302, filed September 16, 2015, and U.S. Provisional Application No. 62 / 239,696, filed October 9, 2015. The above applications are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention generally relates to methods and compounds for treating infections with viruses belonging to the Arenaviridae family, and more particularly to methods and nucleosides and prodrugs thereof for treating Lassa virus and Junin virus. The present invention generally relates to methods and compounds for treating infections with viruses belonging to the Coronaviridae family, and more particularly to methods and nucleosides and prodrugs thereof for treating SARS virus and MERS virus. Background Art

[0004] Lassa virus is a segmented, negative-sense RNA virus belonging to the Arenaviridae family. Arenaviruses are further subdivided into the Old World and New World virus complexes based on serological cross-reactivity, phylogenetic relationships, and geographic distribution (Wulff, 1978; Bowen, 1997). The New World arenavirus complex includes viruses circulating in North America (i.e., Whitewater Arroyo (WWAV), Tamiami (TAMV), and Bell Canyon (BCNV) viruses) and South America (i.e., Tacaribe (TACV), Junín (JUNV), Machupo (MACV), Guanarito (GTOV), and Sabia (SABV) viruses). The Old World complex includes arenaviruses circulating in Africa, Europe, and Asia (i.e., lymphocytic ganglioneuronal meningitis (LCMV) and Lassa (LASV) viruses). LCMV is distributed worldwide due to its association with the globally migratory Mus domesticus and M. musculus (Salazar-Bravo, 2002). The geographic distribution of arenaviruses is otherwise restricted by the range of reservoir rodent species. The reservoir hosts of LASV are rodents of the genus Mastomys, which are endemic in sub-Saharan Africa (Salazar-Bravo, 2002). At least seven arenaviruses are known to cause severe hemorrhagic fever in humans, including LASV, JUNV, MACV, GTOV, and SABV, which are endemic in West Africa, Argentina, Bolivia, Venezuela, and Brazil, respectively. Recently, Lujo (LUJV) and Chapare (CHAPV) viruses have been described, originating in Zambia and Bolivia (Breise, 2009; Delgado, 2008).

[0005] Lassa virus (LASV) is endemic to West Africa, infecting an estimated 300,000–500,000 people annually (McCormick, 1987). Transmission occurs through contact with infected rodents (Mastomys natalensis) or virus-contaminated rodent feces, and human-to-human transmission, particularly in hospital settings, has been documented (McCormick, 1987). Illness caused by LASV ranges from subclinical infection to mild to severe hemorrhagic fever associated with multiorgan failure. The mortality rate associated with LASV infection is variable, ranging from approximately 2% to 15% of hospitalized cases and exceeding 50% in some outbreaks (McCormick, 1987; Fisher-Hoch, 1995). Despite the high incidence and associated morbidity and mortality, there are no approved treatments for LASV infection in humans. Maintenance therapy and early administration of ribavirin are the current standard of care.

[0006] LASV initially infects monocytes, macrophages, and dendritic cells and disseminates systemically, producing a primary viremia that leads to internal organ infection. Viral replication causes elevated levels of inflammatory cytokines and the development of coagulopathy, leading to vascular leakage, hypovolemic shock, and multiorgan failure (Hensley, 2011).

[0007] Arenavirus replication is catalyzed by the L polymerase protein, which utilizes a viral RNA template consisting of genomic RNA encapsidated by the viral nucleocapsid protein (NP) and containing the viral ribonucleoprotein (RNP) (Buchmeier, 2007). Replication begins upon viral entry into the host cell, where the L polymerase, associated with the viral RNP, initiates transcription from a genomic promoter located 3' to each of the genomic RNA segments, L and S. A primary transcription event results in the synthesis of antigenomic mRNAs encoding NP and L polymerase from the S and L segments. Transcription terminates distally to a stem-loop (SL) structure within the intergenic region (IGR). Arenaviruses utilize a capping strategy to acquire the cap structure of cellular mRNAs to facilitate translation. Capping is mediated by the endonuclease activity of the L polymerase, which is co-produced by the cap-binding activity of the NP to produce capped, non-polyadenylated mRNA. Subsequently, the L polymerase adopts replicase mode and moves through the IGR to produce the full-length complementary antigenomic RNA (agRNA). This agRNA serves as a template for the synthesis of genomically encoded GPC and Z mRNAs from the S and L segments, respectively, and for the synthesis of full-length genomic RNA (gRNA) (Buchmeier, 2007; Franze-Fernandez, 1987; Meyer, 1993; Qi, 2010; Lelke, 2010; Morin, 2010).

[0008] Human coronaviruses, first identified in the mid-1960s, are common viruses that infect most people at some time in their lives, typically causing mild to moderate upper respiratory and gastrointestinal illnesses. The new coronavirus, called Middle East Respiratory Syndrome Coronavirus (MERS-CoV or MERS), was first reported in Saudi Arabia in 2012 and has since spread to several other countries. SARS-CoV, the coronavirus responsible for Severe Acute Respiratory Syndrome (SARS), was first identified in China in 2002 and caused a global outbreak in 2002 and 2003. Summary of the Invention

[0009] Methods and compounds are provided for treating infections caused by the Arenaviridae family of viruses.

[0010] Provided are methods for treating Arenaviridae infection in a human in need thereof, comprising administering a therapeutically effective amount of a compound of Formula I:

[0011]

[0012] or a pharmaceutically acceptable salt or ester thereof;

[0013] in:

[0014] Each R 1 is H or halogen;

[0015] Each R 2 、R 3 、R 4 or R 5 are independently H, OR a 、N(R a )2、N3、CN、NO2、S(O)nR a , halogen, (C1-C8)alkyl, (C4-C8)carbocyclylalkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)alkynyl, (C2-C8)substituted alkynyl;

[0016] Or any two R on adjacent carbon atoms 2 、R 3 、R 4 or R 5 together are -O(CO)O- or together with the ring carbon atoms to which they are attached form a double bond;

[0017] R 6 is OR a 、N(R a )2、N3、CN、NO2、S(O) n R a、-C(=O)R 11 、-C(=O)OR 11 、-C(=O)NR 11 R 12 、-C(=O)SR 11 、-S(O)R 11 、-S(O)2R 11 、-S(O)(OR 11 )、-S(O)2(OR 11 ), -SO2NR 11 R 12 , halogen, (C1-C8)alkyl, (C4-C8)carbocyclylalkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)alkynyl, (C2-C8)substituted alkynyl, or (C6-C8) 20 ) aryl(C1-C8)alkyl;

[0018] R 7 Selected from the group consisting of:

[0019] a) H, -C(=O)R 11 、-C(=O)OR 11 、-C(=O)NR 11 R 12 、-C(=O)SR 11 、-S(O)R 11 、-S(O)2R 11 、-S(O)(OR 11 )、-S(O)2(OR 11 ) or -SO2NR 11 R 12 ,

[0020] Each R 11 or R 12 (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl or (C6-C 20 ) aryl (C1-C8) alkyl groups are independently optionally substituted with one or more halogen, hydroxy, CN, N3, N(R a )2 OR a and wherein each of the (C1-C8) alkyl groups has one or more non-terminal carbon atoms that may be optionally substituted with -O-, -S- or -NR a -replace,

[0021] b)

[0022]

[0023] c)

[0024]

[0025] in:

[0026] R c is selected from phenyl, 1-naphthyl, 2-naphthyl,

[0027]

[0028] R d is H or CH3;

[0029] R e1 and R e2 Each is independently H, (C1-C6) alkyl or benzyl;

[0030] R f is selected from H, (C1-C8)alkyl, benzyl, (C3-C6)cycloalkyl and -CH2-(C3-C6)cycloalkyl;

[0031] R g Selected from (C1-C8)alkyl, -O-(C1-C8)alkyl, benzyl, -O-benzyl, -CH2-(C3-C6)cycloalkyl, -O-CH2-(C3-C6)cycloalkyl and CF3; and

[0032] n' is selected from 1, 2, 3 and 4; and

[0033] d) a group of the formula:

[0034]

[0035] in:

[0036] Q is O, S, NR, + N(O)(R), N(OR), + N(O)(OR) or N-NR2;

[0037] Z 1 and Z 2 Together for-Q 1 (C(R y )2)3Q 1 -;

[0038] in

[0039] Each Q 1 are independently O, S or NR; and

[0040] Each R y are independently H, F, Cl, Br, I, OH, R, -C(=Q 2 )R、-C(=Q 2)OR、-C(=Q 2 )N(R)2、-N(R)2、- + N(R)3, -SR, -S(O)R, -S(O)2R, -S(O)(OR), -S(O)2(OR), -OC(=Q 1 )R、-OC(=Q 2 )OR、-OC(=Q 2 )(N(R)2),-SC(=Q 2 )R、-SC(=Q 2 )OR、-SC(=Q 2 )(N(R)2),-N(R)C(=Q 2 )R、-N(R)C(=Q 2 )OR、-N(R)C(=Q 2 )N(R)2, -SO2NR2, -CN, -N3, -NO2, -OR or Z3; or, two R on the same carbon atom y together form a carbocyclic ring having 3 to 7 carbon atoms;

[0041] Each Q 2 are independently O, S, NR, + N(O)(R), N(OR), + N(O)(OR) or N-NR2; or

[0042] Z 1 and Z 2 are each independently a group of formula Ia:

[0043]

[0044] in:

[0045] Each Q 3 are independently a bond, O, CR2, NR, + N(O)(R), N(OR), + N(O)(OR), N-NR2, S, SS, S(O) or S(O)2;

[0046] M2 is 0, 1, or 2;

[0047] Each R x Independently R y Or the following:

[0048]

[0049] in:

[0050] Each of M1a, M1c and M1d is independently 0 or 1;

[0051] M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0052] Z 3 It's Z 4 or Z 5 ;

[0053] Z 4 is R, -C(Q 2 )R y 、-C(Q 2 )Z 5 、-SO2R y or -SO2Z 5 ;and

[0054] Z 5 is a carbocyclic or heterocyclic ring, wherein Z 5 Independently by 0 to 3 R y group substitution;

[0055] R 8 Halogen, NR 11 R 12 、N(R 11 )OR 11 NR 11 NR 11 R 12 ,N3,NO,NO2,CHO,CN,-CH(=NR 11 ),-CH=NNHR 11 、-CH=N(OR 11 )、-CH(OR 11 )2, -C(=O)NR 11 R 12 、-C(=S)NR 11 R 12 、-C(=O)OR 11 , (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C4-C8) carbocyclylalkyl, (C6-C 20 ) optionally substituted aryl, optionally substituted heteroaryl, -C(=O)(C1-C8)alkyl, -S(O) n (C1-C8)alkyl, (C6-C 20 )aryl(C1-C8)alkyl, OR 11 or SR 11 ;

[0056] Each R 9 or R 10 are independently H, halogen, NR 11 R 12 、N(R11 )OR 11 NR 11 NR 11 R 12 ,N3,NO,NO2,CHO,CN,-CH(=NR 11 ),-CH=NHNR 11 、-CH=N(OR 11 )、-CH(OR 11 )2, -C(=O)NR 11 R 12 、-C(=S)NR 11 R 12 、-C(=O)OR 11 、R 11 , OR 11 or SR 11 ;

[0057] Each R 11 or R 12 are independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C4-C8)carbocyclylalkyl, (C6-C 20 ) optionally substituted aryl, optionally substituted heteroaryl, -C(=O)(C1-C8)alkyl, -S(O) n (C1-C8)alkyl or (C6-C 20 ) aryl(C1-C8)alkyl; or R 11 and R 12 Together with the nitrogen to which they are attached, they form a 3-7 membered heterocyclic ring, wherein any carbon atom of the heterocyclic ring may be optionally replaced by -O-, -S- or -NR a -replace;

[0058] Each R a are independently H, (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C6-C 20 )aryl(C1-C8)alkyl, (C4-C8)carbocyclylalkyl, -C(=O)R, -C(=O)OR, -C(=O)NR2, -C(=O)SR, -S(O)R, -S(O)2R, -S(O)(OR), -S(O)2(OR) or -SO2NR2; wherein,

[0059] Each R is independently H, (C1-C8) alkyl, (C1-C8) substituted alkyl, (C2-C8) alkenyl, (C2-C8) substituted alkenyl, (C2-C8) alkynyl, (C2-C8) substituted alkynyl, (C6-C8) 20 )aryl, (C6-C 20 ) substituted aryl, (C2-C20 ) heterocyclic group, (C2-C 20 ) substituted heterocyclic group, (C6-C 20 ) aryl (C1-C8) alkyl or substituted (C6-C 20 ) aryl(C1-C8)alkyl;

[0060] each n is independently 0, 1 or 2; and

[0061] Each R 2 、R 3 、R 5 、R 6 、R 11 or R 12 Each (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl or (C6-C 20 ) aryl (C1-C8) alkyl groups are independently optionally substituted with one or more halogen, hydroxy, CN, N3, N(R a )2 OR a and wherein each of the (C1-C8) alkyl groups has one or more non-terminal carbon atoms that may be optionally substituted with -O-, -S- or -NR a -replace.

[0062] In another embodiment, the method comprises administering to a mammal in need thereof a therapeutically effective amount of a racemate, enantiomer, diastereomer, tautomer, polymorph, pseudopolymorph, amorphous form, hydrate or solvate of a compound of Formula I, or a pharmaceutically acceptable salt or ester thereof.

[0063] In another embodiment, the method comprises treating an Arenaviridae infection in a human in need thereof by administering a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt or ester thereof.

[0064] In another embodiment, the method comprises treating Lassa virus infection in a human in need thereof by administering a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt or ester thereof.

[0065] In another embodiment, the method comprises treating Junin virus infection in a human in need thereof by administering a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt or ester thereof.

[0066] In another embodiment, a method of treating an Arenaviridae infection in a human in need thereof comprises administering a therapeutically effective amount of a pharmaceutical composition comprising an effective amount of a compound of Formula I or a pharmaceutically acceptable salt or ester thereof and a pharmaceutically acceptable diluent or carrier.

[0067] In another embodiment, a method of treating an Arenaviridae infection in a human in need thereof comprises administering a therapeutically effective amount of a pharmaceutical composition comprising an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt or ester thereof, and at least one additional therapeutic agent.

[0068] In another embodiment, the method comprises administering a therapeutically effective amount of a combination comprising: a) a first pharmaceutical composition comprising a compound of Formula I or a pharmaceutically acceptable salt, solvate, or ester thereof; and b) a second pharmaceutical composition comprising at least one additional therapeutic agent active against an infectious Arenaviridae virus.

[0069] In another embodiment, the present application provides a method for inhibiting Arenaviridae RNA-dependent RNA polymerase, comprising contacting cells infected with Arenaviridae viruses with an effective amount of a compound of Formula I or a pharmaceutically acceptable salt, solvate and / or ester thereof.

[0070] In another embodiment, there is provided the use of a compound of Formula I or a pharmaceutically acceptable salt, solvate and / or ester thereof for treating a viral infection caused by an Arenaviridae virus.

[0071] Provided is a method of treating a Coronaviridae infection in a human in need thereof, comprising administering a therapeutically effective amount of a compound of formula I:

[0072]

[0073] or a pharmaceutically acceptable salt or ester thereof;

[0074] in:

[0075] Each R 1 is H or halogen;

[0076] Each R 2 、R 3 、R 4 or R 5 are independently H, OR a 、N(R a )2、N3、CN、NO2、S(O)nR a , halogen, (C1-C8)alkyl, (C4-C8)carbocyclylalkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)alkynyl, (C2-C8)substituted alkynyl;

[0077] Or any two R on adjacent carbon atoms 2 、R 3 、R 4 or R 5together are -O(CO)O- or together with the ring carbon atoms to which they are attached form a double bond;

[0078] R 6 is OR a 、N(R a )2、N3、CN、NO2、S(O) n R a 、-C(=O)R 11 、-C(=O)OR 11 、-C(=O)NR 11 R 12 、-C(=O)SR 11 、-S(O)R 11 、-S(O)2R 11 、-S(O)(OR 11 )、-S(O)2(OR 11 ), -SO2NR 11 R 12 , halogen, (C1-C8)alkyl, (C4-C8)carbocyclylalkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)alkynyl, (C2-C8)substituted alkynyl, or (C6-C8) 20 ) aryl(C1-C8)alkyl;

[0079] R 7 Selected from the group consisting of:

[0080] a) H, -C(=O)R 11 、-C(=O)OR 11 、-C(=O)NR 11 R 12 、-C(=O)SR 11 、-S(O)R 11 、-S(O)2R 11 、-S(O)(OR 11 )、-S(O)2(OR 11 ) or -SO2NR 11 R 12 ,

[0081] Each R 11 or R 12 (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl or (C6-C 20 ) aryl (C1-C8) alkyl groups are independently optionally substituted with one or more halogen, hydroxy, CN, N3, N(R a )2 OR aand wherein each of the (C1-C8) alkyl groups has one or more non-terminal carbon atoms that may be optionally substituted with -O-, -S- or -NR a -replace,

[0082] b)

[0083]

[0084] c)

[0085]

[0086] in:

[0087] R c is selected from phenyl, 1-naphthyl, 2-naphthyl,

[0088]

[0089] R d is H or CH3;

[0090] R e1 and R e2 Each is independently H, (C1-C6) alkyl or benzyl;

[0091] R f is selected from H, (C1-C8)alkyl, benzyl, (C3-C6)cycloalkyl and -CH2-(C3-C6)cycloalkyl;

[0092] R g Selected from (C1-C8)alkyl, -O-(C1-C8)alkyl, benzyl, -O-benzyl, -CH2-(C3-C6)cycloalkyl, -O-CH2-(C3-C6)cycloalkyl and CF3; and

[0093] n' is selected from 1, 2, 3 and 4; and

[0094] d) a group of the formula:

[0095]

[0096] in:

[0097] Q is O, S, NR, + N(O)(R), N(OR), + N(O)(OR) or N-NR 2 ;

[0098] Z 1 and Z 2 Together for-Q 1 (C(R y )2)3Q 1 -;

[0099] in

[0100] Each Q 1 are independently O, S or NR; and

[0101] Each R y are independently H, F, Cl, Br, I, OH, R, -C(=Q 2 )R、-C(=Q 2 )OR、-C(=Q 2 )N(R)2、-N(R)2、- + N(R)3, -SR, -S(O)R, -S(O)2R, -S(O)(OR), -S(O)2(OR), -OC(=Q 1 )R、-OC(=Q 2 )OR、-OC(=Q 2 )(N(R)2),-SC(=Q 2 )R、-SC(=Q 2 )OR、-SC(=Q 2 )(N(R)2),-N(R)C(=Q 2 )R、-N(R)C(=Q 2 )OR、-N(R)C(=Q 2 )N(R)2, -SO2NR2, -CN, -N3, -NO2, -OR or Z3; or, two R on the same carbon atom y together form a carbocyclic ring having 3 to 7 carbon atoms;

[0102] Each Q 2 are independently O, S, NR, + N(O)(R), N(OR), + N(O)(OR) or N-NR2; or

[0103] Z 1 and Z 2 are each independently a group of formula Ia:

[0104]

[0105] in:

[0106] Each Q 3 are independently a bond, O, CR2, NR, + N(O)(R), N(OR), + N(O)(OR), N-NR2, S, SS, S(O) or S(O)2;

[0107] M2 is 0, 1, or 2;

[0108] Each R x Independently R y Or the following:

[0109]

[0110] in:

[0111] Each of M1a, M1c and M1d is independently 0 or 1;

[0112] M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0113] Z 3 It's Z 4 or Z 5 ;

[0114] Z 4 is R, -C(Q 2 )R y 、-C(Q 2 )Z 5 、-SO2R y or -SO2Z 5 ;and

[0115] Z 5 is a carbocyclic or heterocyclic ring, wherein Z 5 Independently by 0 to 3 R y group substitution;

[0116] R 8 Halogen, NR 11 R 12 、N(R 11 )OR 11 NR 11 NR 11 R 12 ,N3,NO,NO2,CHO,CN,-CH(=NR 11 ),-CH=NNHR 11 、-CH=N(OR 11 )、-CH(OR 11 )2, -C(=O)NR 11 R 12 、-C(=S)NR 11 R 12 、-C(=O)OR 11 , (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C4-C8) carbocyclylalkyl, (C6-C 20 ) optionally substituted aryl, optionally substituted heteroaryl, -C(=O)(C1-C8)alkyl, -S(O) n(C1-C8)alkyl, (C6-C 20 )aryl(C1-C8)alkyl, OR 11 or SR 11 ;

[0117] Each R 9 or R 10 are independently H, halogen, NR 11 R 12 、N(R 11 )OR 11 NR 11 NR 11 R 12 ,N3,NO,NO2,CHO,CN,-CH(=NR 11 ),-CH=NHNR 11 、-CH=N(OR 11 )、-CH(OR 11 )2, -C(=O)NR 11 R 12 、-C(=S)NR 11 R 12 、-C(=O)OR 11 、R 11 , OR 11 or SR 11 ;

[0118] Each R 11 or R 12 are independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C4-C8)carbocyclylalkyl, (C6-C 20 ) optionally substituted aryl, optionally substituted heteroaryl, -C(=O)(C1-C8)alkyl, -S(O) n (C1-C8)alkyl or (C6-C 20 ) aryl(C1-C8)alkyl; or R 11 and R 12 Together with the nitrogen to which they are attached, they form a 3-7 membered heterocyclic ring, wherein any carbon atom of the heterocyclic ring may be optionally replaced by -O-, -S- or -NR a -replace;

[0119] Each R a are independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C6-C 20)aryl(C1-C8)alkyl, (C4-C8)carbocyclylalkyl, -C(=O)R, -C(=O)OR, -C(=O)NR2, -C(=O)SR, -S(O)R, -S(O)2R, -S(O)(OR), -S(O)2(OR) or -SO2NR2; wherein,

[0120] Each R is independently H, (C1-C8) alkyl, (C1-C8) substituted alkyl, (C2-C8) alkenyl, (C2-C8) substituted alkenyl, (C2-C8) alkynyl, (C2-C8) substituted alkynyl, (C6-C8) 20 )aryl, (C6-C 20 ) substituted aryl, (C2-C 20 ) heterocyclic group, (C2-C 20 ) substituted heterocyclic group, (C6-C 20 ) aryl (C1-C8) alkyl or substituted (C6-C 20 ) aryl(C1-C8)alkyl;

[0121] each n is independently 0, 1 or 2; and

[0122] Each R 2 、R 3 、R 5 、R 6 、R 11 or R 12 Each (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl or (C6-C 20 ) aryl (C1-C8) alkyl groups are independently optionally substituted with one or more halogen, hydroxy, CN, N3, N(R a )2 OR a and wherein each of the (C1-C8) alkyl groups has one or more non-terminal carbon atoms that may be optionally substituted with -O-, -S- or -NR a -replace.

[0123] In another embodiment, the method comprises administering to a mammal in need thereof a therapeutically effective amount of a racemate, enantiomer, diastereomer, tautomer, polymorph, pseudopolymorph, amorphous form, hydrate or solvate of a compound of Formula I, or a pharmaceutically acceptable salt or ester thereof.

[0124] In another embodiment, the method comprises treating a Coronaviridae infection in a human in need thereof by administering a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt or ester thereof.

[0125] In another embodiment, the method comprises treating MERS virus infection in a human in need thereof by administering a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt or ester thereof.

[0126] In another embodiment, the method comprises treating SARS virus infection in a human in need thereof by administering a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt or ester thereof.

[0127] In another embodiment, a method of treating a Coronaviridae infection in a human in need thereof comprises administering a therapeutically effective amount of a pharmaceutical composition comprising an effective amount of a compound of Formula I or a pharmaceutically acceptable salt or ester thereof and a pharmaceutically acceptable diluent or carrier.

[0128] In another embodiment, a method of treating a Coronaviridae infection in a human in need thereof comprises administering a therapeutically effective amount of a pharmaceutical composition comprising an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt or ester thereof, and at least one additional therapeutic agent.

[0129] In another embodiment, the method comprises administering a therapeutically effective amount of a combination pharmaceutical composition comprising: a) a first pharmaceutical composition comprising a compound of Formula I or a pharmaceutically acceptable salt, solvate, or ester thereof; and b) a second pharmaceutical composition comprising at least one additional therapeutic agent active against a contagious Coronaviridae virus.

[0130] In another embodiment, the present application provides a method for inhibiting Coronaviridae RNA-dependent RNA polymerase, comprising contacting a cell infected with a Coronaviridae virus with an effective amount of a compound of Formula I or a pharmaceutically acceptable salt, solvate and / or ester thereof.

[0131] In another embodiment, there is provided a use of a compound of Formula I or a pharmaceutically acceptable salt, solvate and / or ester thereof for treating a viral infection caused by a Coronaviridae virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] Figure 1 : Changes in body weight of mice treated with vehicle and compound 32 after infection;

[0133] Figure 2A and 2B : Viral loads in lung tissues of vehicle- and compound 32-treated mice on days 2 and 5 post-infection;

[0134] Figure 3A -F: Whole-body plethysmogram of mice infected with SARS-CoV;

[0135] Figure 4A : Changes in body weight of monkeys treated with vehicle and compound 32 after infection;

[0136] Figure 4B : Changes in body temperature of monkeys treated with vehicle and compound 32 after infection;

[0137] Figure 4C : Changes in respiratory rate after infection in monkeys treated with vehicle and compound 32;

[0138] Figure 5 : Tissue viral RNA concentrations of the treated groups. Viral loads were measured by qRT-PCR. Detailed Description of the Invention

[0140] I. Definition

[0141] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings:

[0142] When a trade name is used herein, Applicants intend to include independently both the trade name product and the active pharmaceutical ingredient of the trade name product.

[0143] As used herein, "a compound of the present invention" or "a compound of formula I" refers to a compound of formula I or a pharmaceutically acceptable salt thereof. Similarly, with respect to an isolatable intermediate, the phrase "a compound of formula (number)" refers to a compound of that formula and a pharmaceutically acceptable salt thereof.

[0144] "Alkyl" is a hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms. For example, an alkyl group may have 1 to 20 carbon atoms (i.e., C1-C 20alkyl), 1-8 carbon atoms (i.e., C1-C8 alkyl), or 1-6 carbon atoms (i.e., C1-C6 alkyl). Examples of suitable alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, isopropyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, isobutyl, -CH2CH(CH3)2), 2-butyl (s-Bu, sec-butyl, -CH(CH3)2). )CH2CH3), 2-methyl-2-propyl (t-Bu, tert-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH 2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(C H3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3 and octyl (-(CH2)7CH3).

[0145] "Alkoxy" refers to a group having the formula -O-alkyl, wherein the alkyl group as defined above is attached to the parent molecule through an oxygen atom. The alkyl portion of the alkoxy group may have 1 to 20 carbon atoms (i.e., C1-C 20 Alkoxy), 1-12 carbon atoms (ie C1-C 12 Suitable alkoxy groups include, but are not limited to, methoxy (-O-CH or -OMe), ethoxy (-OCHCH or -OEt), tert-butoxy (-OC(CH) or -OtBu), and the like.

[0146] "Haloalkyl" is an alkyl group as defined above wherein one or more of the hydrogen atoms of the alkyl group is replaced by a halogen atom. The alkyl portion of a haloalkyl group may have 1 to 20 carbon atoms (i.e., C1-C 20 Haloalkyl), 1-12 carbon atoms (i.e. C1-C 12 -haloalkyl) or 1-6 carbon atoms (i.e. C1-C6 alkyl). Examples of suitable haloalkyl groups include, but are not limited to, -CF3, -CHF2, -CFH2, -CH2CF3, and the like.

[0147] An "alkenyl" group is a group containing at least one site of unsaturation (ie, a carbon-carbon sp 2 For example, an alkenyl group may have 2 to 20 carbon atoms (i.e., C2-C 20 Suitable alkenyl groups include, but are not limited to, ethylene or vinyl (-CH=CH), allyl (-CHCH=CH), cyclopentenyl (-C5H7), and 5-hexenyl (-CHCHCHCHCH=CH).

[0148] "Alkynyl" is a hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms with at least one site of unsaturation (i.e., a carbon-carbon sp triple bond). For example, an alkynyl group can have 2 to 20 carbon atoms (i.e., C2-C 20 The alkynyl radicals are 2 to 8 carbon atoms (i.e., C≡C≡alkynyl), 2 to 8 carbon atoms (i.e., C≡C≡alkynyl), or 2 to 6 carbon atoms (i.e., C≡C≡alkynyl). Examples of suitable alkynyl radicals include, but are not limited to, ethynyl (—C≡CH≡), propynyl (—CH≡CH≡), and the like.

[0149] "Alkylene" refers to a saturated, branched, straight-chain, or cyclic hydrocarbon radical having two monovalent radical centers derived by removing two hydrogen atoms from the same or two different carbon atoms of a parent alkane. For example, an alkylene radical may have 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. Typical alkylene radicals include, but are not limited to, methylene (-CH2-), 1,1-ethyl (-CH(CH3)-), 1,2-ethyl (-CH2CH2-), 1,1-propyl (-CH(CH2CH3)-), 1,2-propyl (-CH2CH(CH3)-), 1,3-propyl (-CH2CH2CH2-), 1,4-butyl (-CH2CH2CH2CH2-), and the like.

[0150] "Alkenylene" refers to an unsaturated, branched, straight-chain, or cyclic hydrocarbon radical having two monovalent radical centers derived by removing two hydrogen atoms from the same or two different carbon atoms of a parent alkene. For example, an alkenylene radical can have 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. Typical alkenylene radicals include, but are not limited to, 1,2-ethylene (-CH=CH-).

[0151] "Alkynylene" refers to an unsaturated branched or straight chain or cyclic hydrocarbon radical having two monovalent radical centers derived by removing two hydrogen atoms from the same or two different carbon atoms of a parent alkyne. For example, an alkynylene radical can have 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. Typical alkynylene radicals include, but are not limited to, ethynylene (-C≡C-), propynylene (-CH≡C-), and 4-pentynylene (-CH≡CH≡C-).

[0152] "Amino" generally refers to a nitrogen radical, which can be considered a derivative of ammonia, having the formula -N(X)2, where each "X" is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, etc. The hybridization of the nitrogen is approximately sp 3 . Non-limiting types of amino groups include -NH2, -N(alkyl)2, -NH(alkyl), -N(carbocyclyl)2, -NH(carbocyclyl), -N(heterocyclyl), -NH(heterocyclyl), -N(aryl), -NH(aryl), -N(alkyl)(aryl), -N(alkyl)(heterocyclyl), -N(carbocyclyl)(heterocyclyl), -N(aryl)(heteroaryl), -N(alkyl)(heteroaryl), etc. The term "alkylamino" refers to an amino group substituted with at least one alkyl group. Non-limiting examples of amino groups include -NH2, -NH(CH3), -N(CH3)2, -NH(CH2CH3), -N(CH2CH3)2, -NH(phenyl), -N(phenyl), -NH(benzyl), -N(benzyl)2, etc. Substituted alkylamino groups generally refer to alkylamino groups as defined above, in which at least one substituted alkyl group as defined herein is attached to the amino nitrogen atom. Non-limiting examples of substituted alkylamino groups include -NH(alkylene-C(O)-OH), -NH(alkylene-C(O)-O-alkyl), -N(alkylene-C(O)-OH)2, -N(alkylene-C(O)-O-alkyl)2, and the like.

[0153] "Aryl" refers to an aromatic hydrocarbon radical derived by removing a hydrogen atom from a single carbon atom of a parent aromatic ring system. For example, an aryl group can have 6-20 carbon atoms, 6-14 carbon atoms, or 6-10 carbon atoms. Typical aryl groups include, but are not limited to, radicals derived from benzene (e.g., phenyl), substituted benzenes, naphthalene, anthracene, biphenyl, and the like.

[0154] "Arylalkyl" refers to an acyclic alkyl group in which the carbon atom (usually a terminal or sp 3 One of the hydrogen atoms bonded to the alkyl group (carbon atom) is replaced by an aryl group. Typical arylalkyl groups include but are not limited to benzyl, 2-phenylethane-1-yl, naphthylmethyl, 2-naphthylethane-1-yl, naphthylbenzyl, 2-naphthylethane-1-yl etc. Arylalkyl groups can comprise 7 to 20 carbon atoms, for example, the alkyl moiety is 1 to 6 carbon atoms and the aryl moiety is 6 to 14 carbon atoms.

[0155] "Arylalkenyl" refers to an acyclic alkenyl group in which the carbon atom (typically a terminal or sp 3 Carbon atoms, but also sp 2 In some embodiments, the arylalkenyl group comprises an aryl group having 8 to 20 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, and an aryl group having 6 to 14 carbon atoms.

[0156] "Arylalkynyl" refers to an acyclic alkynyl radical in which the carbon atom (typically a terminal or sp 3 In some embodiments, the alkynyl group is substituted with an aryl group. In some embodiments, the alkynyl group is substituted with an aryl group. In some embodiments, the alkynyl group is substituted with an aryl group. In some embodiments, the alkynyl group is substituted with an aryl group. In some embodiments, the alkynyl group is substituted with an aryl group. In some embodiments, the alkynyl group is substituted with an aryl group.

[0157] The term "substituted" with respect to alkyl, alkylene, aryl, arylalkyl, alkoxy, heterocyclyl, heteroaryl, carbocyclyl, and the like, for example, "substituted alkyl," "substituted alkylene," "substituted aryl," "substituted arylalkyl," "substituted heterocyclyl," and "substituted carbocyclyl" refers to alkyl, alkylene, aryl, arylalkyl, heterocyclyl, carbocyclyl, respectively, wherein one or more hydrogen atoms are independently replaced by a non-hydrogen substituent. Typical substituents include, but are not limited to, -X, -R b 、-O - ,=O,-OR b 、-SR b 、-S-、-NR b 2. -N + R b 3. =NR b , -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, -NHC(=O)R b 、-OC(=O)R b 、-NHC(=O)NRb 2. -S(=O)2-, -S=O)2OH, -S(=O)2R b 、-OS(=O)2OR b 、-S(=O)2NR b 2. -S(=O)R b 、-OP(=)(OR b )2、-P(=O)(OR b )2、-P(=O)(O - )2、-P(=O)(OH)2、-P(O)(OR b )(O - ),-C(=O)R b 、-C(=O)X、-C(S)R b 、-C(O)OR b 、-C(O)O - 、-C(S)OR b 、-C(O)SR b 、-C(S)SR b 、-C(O)NR b 2. -C(S)NR b 2. -C(=NR b )NR b 2, wherein each X is independently a halogen: F, Cl, Br or I; and each R b is independently H, alkyl, aryl, arylalkyl, heterocycle or protecting group or prodrug moiety.Alkylene, alkenylene and alkynylene can also be substituted similarly.Unless otherwise indicated, when the term "substituted" is used together with a group having two or more parts capable of substitution, such as arylalkyl, the substituent can be connected to the aryl moiety, the alkyl moiety or both.

[0158] "Prodrug" is defined in the pharmaceutical field as a biologically inactive derivative of a drug that is converted into a biologically active parent drug according to certain chemical or enzymatic pathways after administration to the human body.

[0159] Those skilled in the art will recognize that the substituents and other moieties of the compounds of Formula I-IV should be selected to provide compounds that are sufficiently stable to provide pharmaceutically useful compounds that can be formulated into acceptable stable pharmaceutical compositions. Compounds of Formula I-IV having such stability are considered to fall within the scope of the present invention.

[0160] "Heteroalkyl" refers to an alkyl group in which one or more carbon atoms are replaced by a heteroatom, such as O, N, or S. For example, if a carbon atom of an alkyl group attached to the parent molecule is replaced by a heteroatom (e.g., O, N, or S), the resulting heteroalkyl group is an alkoxy group (e.g., -OCH3, etc.), an amine (e.g., -NHCH3, -N(CH3)2, etc.), or a thioalkyl group (e.g., -SCH3), respectively. If a non-terminal carbon atom of an alkyl group not attached to the parent molecule is replaced by a heteroatom (e.g., O, N, or S), the resulting heteroalkyl group is an alkyl ether (e.g., -CH2CH2-O-CH3, etc.), an alkylamine (e.g., -CH2NHCH3, -CH2N(CH3)2, etc.), or a thioalkyl ether (e.g., -CH2-S-CH3), respectively. If a terminal carbon atom of an alkyl group is replaced by a heteroatom (e.g., O, N, or S), the resulting heteroalkyl group is a hydroxyalkyl group (e.g., -CH2CH2-OH), an aminoalkyl group (e.g., -CH2NH2), or an alkylthiol group (e.g., -CH2CH2-SH), respectively. The heteroalkyl group may have, for example, 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. A C1-C6 heteroalkyl group refers to a heteroalkyl group having 1 to 6 carbon atoms.

[0161] As used herein, "heterocycle" or "heterocyclyl" includes, for example but not limited to, those in Paquette, Leo A.; Principles ofModern Heterocyclic Chemistry (WA Benjamin, New York, 1968), especially chapters 1, 3, 4, 6, 7, and 9; The Chemistry of Heterocyclic Compounds,A Series of Monographs (John Wiley & Sons, New York, 1950 to present), in particular volumes 13, 14, 16, 19 and 28; and those heterocycles described in J. Am. Chem. Soc. (1960) 82:5566. In a specific embodiment of the invention, "heterocycle" includes "carbocycle" as defined herein, in which one or more (e.g., 1, 2, 3 or 4) carbon atoms have been replaced by heteroatoms (e.g., O, N or S). The term "heterocycle" or "heterocyclyl" includes saturated rings, partially unsaturated rings and aromatic rings (i.e., heteroaromatic rings). Substituted heterocyclyls include, for example, heterocycles substituted with any substituent disclosed herein, including carbonyl. Non-limiting examples of carbonyl-substituted heterocyclyls are:

[0162]

[0163] Examples of heterocycles include, for example and without limitation, pyridyl, dihydropyridyl, tetrahydropyridyl (piperidyl), thiazolyl, tetrahydrothiophenyl, thiooxytetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthyl, indolyl, indolinyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidinonyl, pyrrolinyl, tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,5,2-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, anthranyl , phenoxathiyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolyl, isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, isatinoyl and bis-tetrahydrofuranyl:

[0164]

[0165] By way of example, and not limitation, the carbon-bonded heterocycle is bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole, position 2, 4, or 5 of an oxazole, imidazole, or thiazole, position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of an aziridine, position 2, 3, 4, 5, 6, 7, or 8 of a quinoline, or position 1, 3, 4, 5, 6, 7, or 8 of an isoquinoline. More typically, the carbon-bonded heterocycle includes 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

[0166] By way of example and not limitation, nitrogen-bonded heterocycles are bonded at the following positions: aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, position 1, isoindole or isoindole, position 2, morpholine, and position 9 of carbazole or β-carboline. More typically, nitrogen-bonded heterocycles include 1-aziridinyl, 1-azetidinyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

[0167] "Heterocyclylalkyl" refers to a non-cyclic alkyl group in which the carbon atom (typically a terminal or sp 3 One of the hydrogen atoms bonded to a carbon atom is replaced by a heterocyclyl group (i.e., a heterocyclyl-alkylene-moiety). Typical heterocyclylalkyl groups include, but are not limited to, heterocyclyl-CH2-, 2-(heterocyclyl)ethane-1-yl, and the like, wherein the "heterocyclyl" moiety includes any of the heterocyclyl groups described above, including Principles of Modern Heterocyclic Chemistry Those described in. It will also be understood by those skilled in the art that heterocyclic radical can be connected to the alkyl part of heterocyclic radical alkyl by carbon-carbon bond or carbon-heteroatom bond, provided that the resulting group is chemically stable.Heterocyclic radical alkyl comprises 3 to 20 carbon atoms, for example, the alkyl part of arylalkyl is 1 to 6 carbon atoms and heterocyclic radical part is 2 to 14 carbon atoms.The example of heterocyclic radical alkyl includes for example but not limited to 5-yuan sulfur-containing, oxygen and / or nitrogen heterocycle, such as thiazolylmethyl, 2-thiazolylethane-1-base, imidazolylmethyl, oxadiazolylmethyl, thiadiazolylmethyl etc., 6-yuan sulfur-containing, oxygen and / or nitrogen heterocycle, such as piperidinylmethyl, piperazinylmethyl, morpholinylmethyl, pyridinylmethyl, pyridizylmethyl, pyrimidinylmethyl, pyrazinylmethyl etc.

[0168] "Heterocyclylalkenyl" refers to a non-cyclic alkenyl group in which the carbon atom (typically a terminal or sp 3 Carbon atoms, but also sp 2 One of the hydrogen atoms bonded to a carbon atom is replaced by a heterocyclyl group (i.e., a heterocyclyl-alkenylene-moiety). The heterocyclyl portion of the heterocyclylalkenyl group includes any heterocyclyl group described herein, including Principles ofModern Heterocyclic Chemistry The heterocyclylalkenyl group may be any of the alkenyl groups described herein, and the alkenyl portion of the heterocyclylalkenyl group includes any alkenyl group disclosed herein. It will also be understood by those skilled in the art that the heterocyclyl group may be attached to the alkenyl portion of the heterocyclylalkenyl group via a carbon-carbon bond or a carbon-heteroatom bond, provided that the resulting group is chemically stable. The heterocyclylalkenyl group contains 4 to 20 carbon atoms, for example, the alkenyl portion of the heterocyclylalkenyl group is 2 to 6 carbon atoms and the heterocyclyl portion is 2 to 14 carbon atoms.

[0169] "Heterocyclylalkynyl" refers to a non-cyclic alkynyl group in which the 3 One of the hydrogen atoms of the heterocyclyl group (i.e., a heterocyclyl-alkynylene-moiety) is replaced by a sp carbon atom. The heterocyclyl portion of the heterocyclylalkynyl group includes any heterocyclyl group described herein, including Principles of Modern Heterocyclic Chemistry and the alkynyl moiety of the heterocyclylalkynyl group includes any alkynyl group disclosed herein. It will also be understood by those skilled in the art that the heterocyclyl group can be attached to the alkynyl moiety of the heterocyclylalkynyl group by a carbon-carbon bond or a carbon-heteroatom bond, provided that the resulting group is chemically stable. The heterocyclylalkynyl group contains 4 to 20 carbon atoms, for example, the alkynyl moiety of the heterocyclylalkynyl group is 2 to 6 carbon atoms and the heterocyclyl moiety is 2 to 14 carbon atoms.

[0170] "Heteroaryl" refers to an aromatic heterocyclic radical having at least one heteroatom in the ring. Non-limiting examples of suitable heteroatoms that may be included in the aromatic ring include oxygen, sulfur, and nitrogen. Non-limiting examples of heteroaryl rings include all those aromatic rings listed in the definition of "heterocyclic radical", including pyridyl, pyrrolyl, oxazolyl, indolyl, isoindolyl, purinyl, furyl, thienyl, benzofuranyl, benzothienyl, carbazolyl, imidazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, quinolyl, isoquinolyl, pyridazinyl, pyrimidinyl, pyrazinyl, etc.

[0171] "Carbocycle" or "carbocyclyl" refers to a saturated (i.e., cycloalkyl), partially unsaturated (e.g., cycloalkenyl, cycloalkadienyl, etc.), or aromatic ring having 3 to 7 carbon atoms as a monocyclic ring, 7 to 12 carbon atoms as a bicyclic ring, and up to about 20 carbon atoms as a polycyclic ring. Monocyclic carbocycles have 3 to 7 ring atoms, more typically 5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, for example, arranged as a bicyclic [4,5], [5,5], [5,6], or [6,6] system, or 9 or 10 ring atoms arranged as a bicyclic [5,6] or [6,6] system, or a spiro-fused ring. Non-limiting examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl and phenyl. Non-limiting examples of bicyclic carbocycles include naphthyl, tetralin and decalin.

[0172] "Carbocyclylalkyl" refers to a non-cyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom is replaced by a carbocyclyl group as described herein. Typical, but non-limiting, examples of carbocyclylalkyl include cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclopentylmethyl, and cyclohexylmethyl.

[0173] "Arylheteroalkyl" refers to a heteroalkyl group as defined herein in which a hydrogen atom (which may be attached to a carbon atom or a heteroatom) has been replaced by an aryl group as defined herein. The aryl group may be bonded to a carbon atom of the heteroalkyl group, or to a heteroatom of the heteroalkyl group, provided that the resulting arylheteroalkyl group provides a chemically stable moiety. For example, an arylheteroalkyl group may have the formula -alkylene-O-aryl, -alkylene-O-alkylene-aryl, -alkylene-NH-aryl, -alkylene-NH-alkylene-aryl, -alkylene-S-aryl, -alkylene-S-alkylene-aryl, etc. Additionally, any alkylene moiety in the above formula may be further substituted by any substituent as defined or exemplified herein.

[0174] "Heteroarylalkyl" refers to an alkyl group as defined herein in which a hydrogen atom has been replaced by a heteroaryl group as defined herein. Non-limiting examples of heteroarylalkyl include -CH2-pyridyl, -CH2-pyrrolyl, -CH2-oxazolyl, -CH2-indolyl, -CH2-isoindolyl, -CH2-purinyl, -CH2-furyl, -CH2-thienyl, -CH2-benzofuranyl, -CH2-benzothienyl, -CH2-carbazolyl, -CH2-imidazolyl, -CH2-thiazolyl, -CH2-isoxazolyl, -CH2-pyrazolyl, -CH2-isothiazolyl, -CH2-quinolyl, -CH2-isoquinolyl, -CH2-pyridazinyl, -CH2-pyrimidinyl, -CH2-pyrazinyl, -CH(CH3)-pyridyl, -CH(CH3)-pyrrolyl, -CH(CH3)- -CH(CH3)-oxazolyl, -CH(CH3)-indolyl, -CH(CH3)-isoindolyl, -CH(CH3)-purinyl, -CH(CH3)-furyl, -CH(CH3)-thienyl, -CH(CH3)-benzofuranyl, -CH(CH3)-benzothienyl, -CH(CH3)-carbazolyl, -CH(CH3)-imidazolyl, -CH(CH3)-thiazolyl, -CH(CH3)-isoxazolyl, -CH(CH3)-pyrazolyl, -CH(CH3)-isothiazolyl, -CH(CH3)-quinolyl, -CH(CH3)-isoquinolyl, -CH(CH3)-pyridazinyl, -CH(CH3)-pyrimidinyl, -CH(CH3)-pyrazinyl, and the like.

[0175] The term "optionally substituted" with respect to a particular moiety (e.g., optionally substituted aryl) of a compound of Formula I-IV means wherein all substituents are hydrogen or wherein one or more hydrogens of the moiety may be replaced with a substituent such as listed under the definition of "substituted".

[0176] With respect to a particular moiety of a compound of Formula I-IV (e.g., the carbon atom of the (C1-C8)alkyl group may be optionally replaced by -O-, -S-, or -NR a-substituted) means that one or more methylene groups of the (C1-C8)alkyl group may be replaced by 0, 1, 2 or more specified groups (e.g., -O-, -S- or -NR a -)replace.

[0177] The term "non-terminal carbon atom" with respect to an alkyl, alkenyl, alkynyl, alkylene, alkenylene, or alkynylene moiety refers to a carbon atom between the first carbon atom of the moiety and the last carbon atom of the moiety. Thus, by way of example and not limitation, the C* atom in an alkyl moiety -CH2(C*)H2(C*)H2CH3 or an alkylene moiety -CH2(C*)H2(C*)H2CH2- would be considered a non-terminal carbon atom.

[0178] Some Q and Q1 substitutes are nitrogen oxides such as + N(O)(R) or + N(O)(OR). These nitrogen oxides, as shown here attached to a carbon atom, can also be represented by charge-separated groups, e.g.

[0179]

[0180] and are intended to be equivalent to the above-described representatives for the purposes of describing the present invention.

[0181] "Linker" or "linker" refers to a chemical moiety comprising a covalent bond or a chain of atoms. Linkers include repeating units of alkoxy groups (e.g., polyethyleneoxy, PEG, polymethyleneoxy) and repeating units of alkylamino groups (e.g., polyethyleneamino, Jeffamine TM ); and diacids and amides, including succinates, succinamides, diglycolates, malonates, and hexanamides.

[0182] Terms such as "oxygen-linked," "nitrogen-linked," "carbon-linked," "sulfur-linked," or "phosphorus-linked" mean that if the bond between the two moieties can be formed using more than one atom type in the moieties, the bond formed between the moieties is through the designated atom. For example, a nitrogen-linked amino acid will be linked through the nitrogen atom of the amino acid, rather than through an oxygen or carbon atom of the amino acid.

[0183] In some embodiments of the compound of Formula I-IV, Z 1 or Z 2In one or more of the groups independently being nitrogen-linked naturally occurring α-amino acid esters. Examples of naturally occurring amino acids include isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, selenocysteine, serine, tyrosine, arginine, histidine, ornithine, and taurine. Esters of these amino acids include any of those described for the substituent R, particularly those wherein R is an optionally substituted (C1-C8) alkyl.

[0184] The term "purine" or "pyrimidine" bases include, but are not limited to, adenine, N 6 -alkylpurine, N 6 -acylpurine (wherein acyl is C(O)(alkyl, aryl, alkylaryl or arylalkyl), N 6 -Benzylpurine, N 6 -halopurine, N 6 -vinylpurine, N 6 -acetylene purine, N 6 -acylpurine, N 6 -Hydroxyalkylpurine, N 6 -allylaminopurine, N 6 -Thioallylpurine, N 2 -alkylpurine, N 2 -alkyl-6-thiopurine, thymine, cytosine, 5-fluorocytosine, 5-methylcytosine, 6-azapyrimidine including 6-azacytosine, 2- and / or 4-mercaptopyrimidine, uracil, 5-halouracil including 5-fluorouracil, C 5 -alkylpyrimidine, C 5 -Benzylpyrimidine, C 5 -halogenated pyrimidine, C 5 -vinylpyrimidine, C 5 -ethynylpyrimidine, C 5 -acylpyrimidine, C 5 -Hydroxyalkylpurine, C 5 -acylaminopyrimidine, C 5 -cyanopyrimidine, C 5 -5-iodopyrimidine, C 6 -iodo-pyrimidine, C 5 -Br-vinylpyrimidine, C 6 -Br-ethynylpyrimidine, C 5 -nitropyrimidine, C 5 -amino-pyrimidine, N 2 -alkylpurine, N 2-alkyl-6-thiopurine, 5-azacytidine base, 5-azauracil base, triazolopyridinyl, imidazopyridinyl, pyrrolopyrimidinyl and pyrazolopyrimidinyl. Purine bases include but are not limited to guanine, adenine, hypoxanthine, 2,6-diaminopurine and 6-chloropurine. The purine and pyrimidine bases of Formula I-III are connected to ribose or its analogs through the nitrogen atom of the base. The functional oxygen and nitrogen groups on the base can be protected when necessary or desired. Suitable protecting groups are well known to those skilled in the art and include trimethylsilyl, dimethylhexylsilyl, tert-butyldimethylsilyl and tert-butyldiphenylsilyl, trityl, alkyl and acyl groups such as acetyl and propionyl, methylsulfonyl and p-toluenesulfonyl.

[0185] Unless otherwise indicated, the carbon atoms of the compounds of Formulas I-IV are intended to have a valence of four. In some chemical structure representations, where a carbon atom does not have a sufficient number of variables attached to produce a valence of four, the remaining carbon substituents required to provide the valence of four should be considered to be hydrogen. For example,

[0186]

[0187] Have the same meaning.

[0188] "Protecting group" refers to a portion of a compound that masks or changes the properties of a functional group or the properties of the entire compound. The chemical substructures of protecting groups vary greatly. One function of a protecting group is to serve as an intermediate for synthesizing a parent drug. Chemical protecting groups and protection / deprotection strategies are well known in the art. See "Protective Groups in Organic Chemistry", Theodora W. Greene (John Wiley & Sons, Inc., New York, 1991). Protecting groups are often used to mask the reactivity of certain functional groups to help improve the efficiency of desired chemical reactions, for example, to make and break chemical bonds in an orderly and planned manner. In addition to the reactivity of protected functional groups, the protection of a compound's functional groups can change other physical properties, such as polarity, lipophilicity (hydrophobicity), and other properties that can be measured by common analytical tools. Chemically protected intermediates themselves may be biologically active or inactive. "Hydroxy protecting groups" refer to those protecting groups used to protect hydroxyl groups (-OH).

[0189] Protected compounds may also show changes, in some cases, optimized external and internal properties, such as resistance to enzyme degradation or chelation by cell membrane and. During this period, protected compounds with expected therapeutic effects may be referred to as prodrugs. Another function of blocking group is to convert the parent drug into a prodrug, thus releasing the parent drug when converting the prodrug in vivo. Because active prodrugs may be more effectively absorbed than the parent drug, prodrugs may have larger effectiveness in vivo than the parent drug. In vitro, in the case of chemical intermediates, or in vivo, in the case of prodrugs, removing blocking groups. For chemical intermediates, the product obtained after the deprotection, for example, alcohol, is physiologically acceptable and not particularly important, although usually if the product is pharmacologically harmless, then more ideal.

[0190] The term “chiral” refers to molecules that have the property of being non-superimposable on their mirror image partner, whereas the term “achiral” refers to molecules that are superimposable on their mirror image partner.

[0191] The term "stereoisomers" refers to compounds that have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

[0192] "Diastereomers" refer to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, reactivity, and biological properties. For example, when R 7 yes And Z 1 and Z 2 When different, the compounds of formula I-IV may have a chiral phosphorus atom. 1 or Z 2 When at least one of them also has a chiral center, for example, Z 1 or Z 2 When the chiral, naturally occurring α-amino acid ester is a nitrogen-linked chiral, naturally occurring α-amino acid ester, the compounds of Formulas I-IV will exist as diastereoisomers due to the presence of two chiral centers in the molecule. All such diastereoisomers and their uses described herein are included in the present invention. Mixtures of diastereoisomers can be separated under high-resolution analytical procedures such as electrophoresis, crystallization, and / or chromatography. Diastereoisomers can have different physical properties, such as, but not limited to, solubility, chemical stability, and crystallinity, and can also have different biological properties, such as, but not limited to, enzymatic stability, absorption, and metabolic stability.

[0193] "Enantiomers" refer to two stereoisomers of a compound that are non-superimposable mirror images of one another.

[0194] The modifier "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (eg, includes the degree of error associated with measurement of the particular quantity).

[0195] As used herein, unless otherwise indicated, the term "treat" means to reverse, alleviate, inhibit the progress of, or prevent the disorder or condition to which such term applies, or one or more symptoms of such condition or condition. As used herein, the term "treat" refers to the act of treating, as "treating" is defined immediately above.

[0196] As used herein, the term "therapeutically effective amount" is the amount of a compound of Formula I-IV present in a composition as described herein that is necessary to provide the desired level of drug in the secretions and tissues of the airways and lungs, or, when such a composition is administered by a selected route of administration, to produce the desired physiological response or desired biological effect in the bloodstream of the individual to be treated. The exact amount will depend on many factors, such as the specific compound of Formula I-IV, the specific activity of the composition, the delivery device used, the physical properties of the composition, its intended use, and patient considerations, such as the severity of the disease state, patient cooperation, etc., and can be readily determined by one skilled in the art based on the information provided herein.

[0197] The term "normal saline" refers to an aqueous solution containing 0.9% (w / v) NaCl.

[0198] The term "hypertonic saline" refers to an aqueous solution containing greater than 0.9% (w / v) NaCl. For example, 3% hypertonic saline would contain 3% (w / v) NaCl.

[0199] "Forming a reaction mixture" refers to the process of contacting at least two different substances so that they mix together and can react. However, it should be understood that the resulting reaction product can be produced directly from the reaction between the added reagents or from intermediates from one or more added reagents that can be produced in the reaction mixture.

[0200] "Coupling agent" refers to a reagent capable of coupling two different compounds. The coupling agent can be catalytic or stoichiometric. For example, the coupling agent can be a lithium-based coupling agent or a magnesium-based coupling agent such as a Grignard reagent. Exemplary coupling agents include, but are not limited to, n-BuLi, MgCl2, iPrMgCl, tBuMgCl, PhMgCl, or combinations thereof.

[0201] "Silane" refers to a silicon-containing group having the formula SiR4, where each R group can be an alkyl, alkenyl, cycloalkyl, phenyl, or other silicon-containing group. When a silane is attached to another compound, the silane is called a "silyl group" and has the formula -SiR3.

[0202] "Halosilane" refers to a silane having at least one halogen group attached to a silicon atom. Representative halosilanes have the formula halogen-SiR3, where each R group can be an alkyl, alkenyl, cycloalkyl, phenyl, or other silicon-containing group. Specific halosilanes include Cl-Si(CH3)3 and Cl-Si(CH3)2CH2CH2Si(CH3)2-Cl.

[0203] "Non-nucleophilic base" refers to an electron donor, a Lewis base, such as a nitrogen base including triethylamine, diisopropylethylamine, N,N-diethylaniline, pyridine, 2,6-lutidine, 2,4,6-collidine, 4-dimethylaminopyridine and quinuclidine.

[0204] "Leaving group" refers to a group that maintains the bonded electron pair during heterolytic bond cleavage. For example, a leaving group is easily displaced during a nucleophilic displacement reaction. Suitable leaving groups include, but are not limited to, chloride, bromide, methanesulfonate, toluenesulfonate, triflate, 4-nitrobenzenesulfonate, 4-chlorobenzenesulfonate, 4-nitrophenoxy, pentafluorophenoxy, and the like. Those skilled in the art will recognize other leaving groups that can be used in the present invention.

[0205] "Deprotecting agent" refers to any reagent capable of removing a protecting group. The deprotecting agent will depend on the type of protecting group used. Representative deprotecting agents are known in the art and can be found in Protective Groups in Organic Chemistry, Peter G.M. Wuts and Theodora W. Greene, 4th edition, 2006.

[0206] II. Compounds of the Invention

[0207] Reference will now be made in detail to certain embodiments of the present invention, examples of which are illustrated in the accompanying description, structures, and formulae. Although the present invention will be described in conjunction with the enumerated embodiments, it should be understood that they are not intended to limit the invention to those embodiments. On the contrary, the present invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the present invention.

[0208] Provided are methods for treating Arenaviridae infection in a human in need thereof, comprising administering a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt or ester thereof:

[0209]

[0210] in:

[0211] Each R 1 is H or halogen;

[0212] Each R2 、R 3 、R 4 or R 5 are independently H, OR a 、N(R a )2、N3、CN、NO2、S(O) n R a , halogen, (C1-C8)alkyl, (C4-C8)carbocyclylalkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)alkynyl or (C2-C8)substituted alkynyl;

[0213] Or any two R on adjacent carbon atoms 2 、R 3 、R 4 or R 5 together are -O(CO)O- or together with the ring carbon atoms to which they are attached form a double bond;

[0214] R 6 is OR a 、N(R a )2、N3、CN、NO2、S(O) n R a 、-C(=O)R 11 、-C(=O)OR 11 、-C(=O)NR 11 R 12 、-C(=O)SR 11 、-S(O)R 11 、-S(O)2R 11 、-S(O)(OR 11 )、-S(O)2(OR 11 ), -SO2NR 11 R 12 , halogen, (C1-C8)alkyl, (C4-C8)carbocyclylalkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)alkynyl, (C2-C8)substituted alkynyl, or (C6-C8) 20 )aryl(C1-C8)alkyl;

[0215] R 7 Selected from the group consisting of:

[0216] a) H, -C(=O)R 11 、-C(=O)OR 11 、-C(=O)NR 11 R 12 、-C(=O)SR 11 、-S(O)R11 、-S(O)2R 11 、-S(O)(OR 11 )、-S(O)2(OR 11 ) or -SO2NR 11 R 12 ,

[0217] Each R 11 or R 12 (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl or (C6-C 20 ) aryl (C1-C8) alkyl groups are independently optionally substituted with one or more halogen, hydroxy, CN, N3, N(R a )2 OR a and wherein each of the (C1-C8) alkyl groups has one or more non-terminal carbon atoms that may be optionally substituted with -O-, -S- or -NR a -replace,

[0218] b)

[0219]

[0220] c)

[0221]

[0222] in:

[0223] R c is selected from phenyl, 1-naphthyl, 2-naphthyl,

[0224]

[0225] R d is H or CH3;

[0226] R e1 and R e2 Each is independently H, (C1-C6) alkyl or benzyl;

[0227] R f is selected from H, (C1-C8)alkyl, benzyl, (C3-C6)cycloalkyl and -CH2-(C3-C6)cycloalkyl;

[0228] R g Selected from (C1-C8)alkyl, -O-(C1-C8)alkyl, benzyl, -O-benzyl, -CH2-(C3-C6)cycloalkyl, -O-CH2-(C3-C6)cycloalkyl and CF3; and

[0229] n' is selected from 1, 2, 3 and 4; and

[0230] d) a group of the formula:

[0231]

[0232] in:

[0233] Q is O, S, NR, + N(O)(R), N(OR), + N(O)(OR) or N-NR2;

[0234] Z 1 and Z 2 Together for-Q 1 (C(R y )2)3Q 1 -;

[0235] in

[0236] Each Q 1 are independently O, S or NR; and

[0237] Each R y are independently H, F, Cl, Br, I, OH, R, -C(=Q 2 )R、-C(=Q 2 )OR、-C(=Q 2 )N(R)2、-N(R)2、- + N(R)3, -SR, -S(O)R, -S(O)2R, -S(O)(OR), -S(O)2(OR), -OC(=Q 1 )R、-OC(=Q 2 )OR、-OC(=Q 2 )(N(R)2),-SC(=Q 2 )R、-SC(=Q 2 )OR、-SC(=Q 2 )(N(R)2),-N(R)C(=Q 2 )R、-N(R)C(=Q 2 )OR、-N(R)C(=Q 2 )N(R)2, -SO2NR2, -CN, -N3, -NO2, -OR or Z3; or two R on the same carbon atom y together form a carbocyclic ring having 3 to 7 carbon atoms;

[0238] Each Q 2 are independently O, S, NR, + N(O)(R), N(OR), + N(O)(OR) or N-NR2; or

[0239] Z1 and Z 2 are each independently a group of formula Ia:

[0240]

[0241] in:

[0242] Each Q 3 are independently a bond, O, CR2, NR, + N(O)(R), N(OR), + N(O)(OR), N-NR2, S, SS, S(O) or S(O)2;

[0243] M2 is 0, 1, or 2;

[0244] Each R x Independently R y Or the following:

[0245]

[0246] in:

[0247] Each of M1a, M1c and M1d is independently 0 or 1;

[0248] M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0249] Z 3 It's Z 4 or Z 5 ;

[0250] Z 4 is R, -C(Q 2 )R y 、-C(Q 2 )Z 5 、-SO2R y or -SO2Z 5 ;and

[0251] Z 5 is a carbocyclic or heterocyclic ring, wherein Z 5 Independently by 0 to 3 R y group substitution;

[0252] R 8 Halogen, NR 11 R 12 、N(R 11 )OR 11 NR 11 NR 11 R 12 ,N3,NO,NO2,CHO,CN,-CH(=NR11 ),-CH=NNHR 11 、-CH=N(OR 11 )、-CH(OR 11 )2, -C(=O)NR 11 R 12 、-C(=S)NR 11 R 12 、-C(=O)OR 11 , (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C4-C8) carbocyclylalkyl, (C6-C 20 ) optionally substituted aryl, optionally substituted heteroaryl, -C(=O)(C1-C8)alkyl, -S(O) n (C1-C8)alkyl, (C6-C 20 )aryl(C1-C8)alkyl, OR 11 or SR 11 ;

[0253] Each R 9 or R 10 are independently H, halogen, NR 11 R 12 、N(R 11 )OR 11 NR 11 NR 11 R 12 ,N3,NO,NO2,CHO,CN,-CH(=NR 11 ),-CH=NHNR 11 、-CH=N(OR 11 )、-CH(OR 11 )2, -C(=O)NR 11 R 12 、-C(=S)NR 11 R 12 、-C(=O)OR 11 、R 11 , OR 11 or SR 11 ;

[0254] Each R 11 or R 12 are independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C4-C8)carbocyclylalkyl, (C6-C 20 ) optionally substituted aryl, optionally substituted heteroaryl, -C(=O)(C1-C8)alkyl, -S(O) n (C1-C8)alkyl or (C6-C 20 ) aryl(C1-C8)alkyl; or R11 and R 12 Together with the nitrogen to which they are attached, they form a 3-7 membered heterocyclic ring, wherein any carbon atom of the heterocyclic ring may be optionally replaced by -O-, -S- or -NR a -replace;

[0255] Each R a are independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C6-C 20 )aryl(C1-C8)alkyl, (C4-C8)carbocyclylalkyl, -C(=O)R, -C(=O)OR, -C(=O)NR2, -C(=O)SR, -S(O)R, -S(O)2R, -S(O)(OR), -S(O)2(OR) or -SO2NR2; wherein,

[0256] Each R is independently H, (C1-C8) alkyl, (C1-C8) substituted alkyl, (C2-C8) alkenyl, (C2-C8) substituted alkenyl, (C2-C8) alkynyl, (C2-C8) substituted alkynyl, (C6-C8) 20 )aryl, (C6-C 20 ) substituted aryl, (C2-C 20 ) heterocyclic group, (C2-C 20 ) substituted heterocyclic group, (C6-C 20 ) aryl (C1-C8) alkyl or substituted (C6-C 20 )aryl(C1-C8)alkyl;

[0257] each n is independently 0, 1 or 2; and

[0258] Each R 2 、R 3 、R 5 、R 6 、R 11 or R 12 Each (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl or (C6-C 20 ) aryl (C1-C8) alkyl groups are independently optionally substituted with one or more halogen, hydroxy, CN, N3, N(R a )2 OR a and wherein each of the (C1-C8) alkyl groups has one or more non-terminal carbon atoms that may be optionally substituted with -O-, -S- or -NR a -replace.

[0259] In another embodiment, a method of treating an Arenaviridae infection in a human in need thereof is provided, comprising administering a therapeutically effective amount of a compound of Formula I represented by Formula II, or a pharmaceutically acceptable salt or ester thereof:

[0260]

[0261] in,

[0262] R 1 、R 3 、R 5 、R 7 、R 8 and R 9 As defined above for Formula I:

[0263] Each R 2 is OR a or halogen; and

[0264] R 6 is OR a 、N(R a )2、N3、CN、S(O) n R a 、-C(=O)R 11 、-C(=O)OR 11 、-C(=O)NR 11 R 12 、-C(=O)SR 11 、-S(O)R 11 、-S(O)2R 11 、-S(O)(OR 11 )、-S(O)2(OR 11 ), -SO2NR 11 R 12 , halogen, (C1-C8)alkyl, (C4-C8)carbocyclylalkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)alkynyl or (C2-C8)substituted alkynyl.

[0265] In one embodiment of the method of treating Arenaviridae infections by administering a compound of Formula II, R 1 is H. In another aspect of this embodiment, R of Formula II 6 is N3, CN, halogen, (C1-C8) alkyl, (C1-C8) substituted alkyl, (C2-C8) alkenyl, (C2-C8) substituted alkenyl, (C2-C8) alkynyl or (C2-C8) substituted alkynyl. In another aspect of this embodiment, R of Formula II 6 is CN, methyl, vinyl or ethynyl. In another aspect of this embodiment, R of formula II6 In another aspect of this embodiment, R of Formula II 6 In another aspect of this embodiment, R of Formula II 5 is H. In another aspect of this embodiment, R of Formula II 2 OR a In another aspect of this embodiment, R of Formula II 2 In another aspect of this embodiment, R 2 is F. In another aspect of this embodiment, R of Formula II 3 is OR a In another aspect of this embodiment, R of Formula II 3 is OH, -OC(=O)R 11 OR-OC(=O)OR 11 In another aspect of this embodiment, R of Formula II 3 In another aspect of this embodiment, R 8 It is NR 11 R 12 In another aspect of this embodiment, R of Formula II 8 In another aspect of this embodiment, R 8 is OR 11 In another aspect of this embodiment, R of Formula II 8 In another aspect of this embodiment, R 9 is H. In another aspect of this embodiment, R of Formula II 9 NR 11 R 12 In another aspect of this embodiment, R of Formula II 9 In another aspect of this embodiment, R 7 H, -C(=O)R 11 、-C(=O)OR 11 or

[0266]

[0267] In another aspect of this embodiment, R of Formula II 7 is H. In another aspect of this embodiment, R of Formula II 7 for

[0268]

[0269] In another embodiment of the method of treating an Arenaviridae infection comprising administering a compound of Formula II, the Arenaviridae infection is caused by an Arenaviridae virus. In another aspect of this embodiment, the Arenaviridae virus is Lassa virus or Junin virus. In another aspect of this embodiment, the Arenaviridae virus is Lassa virus. In another aspect of this embodiment, the Arenaviridae virus is Junin virus. In another aspect of this embodiment, the Arenaviridae virus is caused by a Lassa virus caused by a strain selected from the group consisting of Josiah, NL, z148, Macenta, AV, and CSF.

[0270] In another aspect of this embodiment, the Arenaviridae infection is caused by Allpahuayo virus (ALLV), Amapari virus (AMAV), Bear Canyon virus (BCNV), Catalina virus, Chapare virus, Cupiqui virus (CPXV), Dandenong virus, Flexal virus (FLEV), Gastrointestinal virus (GTOV), Ippy virus (IPPYV), Junin virus (JUNV), Kodoko virus, Lassa virus (LASV), Latin virus (LATV), Lymphocytic choriomeningitis virus (LCMV), Lujo virus, Machupo virus (MACV), Mobala virus (MOBV), Morogoro virus, Mopeia virus (MOPV), Oliveros virus (OLVV), Parana virus (PARV), Pichinde virus (PICV), Pinhal virus, Pirital virus (PIRV), Sabia virus (SABV), Skinner's box virus, Tacaribe virus (TCRV), Tamiami virus (TAMV), or Whitewater Arroyo virus (WWAV).

[0271] In another embodiment, a method of treating an Arenaviridae infection in a human in need thereof is provided, comprising administering a therapeutically effective amount of a compound of Formula I represented by Formula III, or a pharmaceutically acceptable salt or ester thereof:

[0272]

[0273] in,

[0274] R 6 、R 7 、R 8 and R 9 As defined above for Formula II;

[0275] Each R 2 is OR a or F; and

[0276] Each R 3 is ORa .

[0277] In one embodiment of the method of treating an Arenaviridae infection comprising administering a compound of Formula III, R 6 is N3, CN, halogen, (C1-C8) alkyl, (C1-C8) substituted alkyl, (C2-C8) alkenyl, (C2-C8) substituted alkenyl, (C2-C8) alkynyl or (C2-C8) substituted alkynyl. In another aspect of this embodiment, R of Formula III 6 is CN, methyl, vinyl or ethynyl. In another aspect of this embodiment, R of formula III 6 In another aspect of this embodiment, R of Formula III 6 In another aspect of this embodiment, R of formula III 2 is OR a In another aspect of this embodiment, R of Formula III 2 In another aspect of this embodiment, R of formula III 2 In another aspect of this embodiment, R of formula III 3 OH, -OC(=O)R 11 OR-OC(=O)OR 11 In another aspect of this embodiment, R of Formula III 3 In another aspect of this embodiment, R 8 NR 11 R 12 In another aspect of this embodiment, R of Formula III 8 In another aspect of this embodiment, R 8 is OR 11 In another aspect of this embodiment, R of Formula III 8 In another aspect of this embodiment, R of formula III 9 is H. In another aspect of this embodiment, R of Formula III 9 NR 11 R 12 In another aspect of this embodiment, R of Formula III 9 In another aspect of this embodiment, R 7 H, -C(=O)R 11 、-C(=O)OR 11 or

[0278]

[0279] In another aspect of this embodiment, R of Formula III7 is H. In another aspect of this embodiment, R of Formula III 7 for

[0280]

[0281] In another embodiment of the method of treating Arenaviridae infection comprising administering a compound of Formula III, R 6 is N3, CN, halogen, (C1-C8)alkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)alkynyl or (C2-C8)substituted alkynyl, and R 8 In another aspect of this embodiment, R 6 is CN, methyl, vinyl or ethynyl. In another aspect of this embodiment, R of formula III 6 In another aspect of this embodiment, R of Formula III 6 In another aspect of this embodiment, R of formula III 2 is OR a In another aspect of this embodiment, R of Formula III 2 OH, -OC(=O)R 11 OR-OC(=O)OR 11 In another aspect of this embodiment, R of Formula III 2 In another aspect of this embodiment, R of formula III 2 In another aspect of this embodiment, R of formula III 3 OH, -OC(=O)R 11 OR-OC(=O)OR 11 In another aspect of this embodiment, R of Formula III 3 In another aspect of this embodiment, R of formula III 9 is H. In another aspect of this embodiment, R of Formula III 9 NR 11 R 12 In another aspect of this embodiment, R of Formula III 9 In another aspect of this embodiment, R 7 H, -C(=O)R 11 、-C(=O)OR 11 or

[0282]

[0283] In another aspect of this embodiment, R of Formula III 7is H. In another aspect of this embodiment, R of Formula III 7 for

[0284]

[0285] In another embodiment of the method of treating Arenaviridae infection comprising administering a compound of Formula III, R 6 is CN, methyl, vinyl or ethynyl, R 8 is NH2, and R 9 is H. In another aspect of this embodiment, R of Formula III 6 In another aspect of this embodiment, R of Formula III 6 In another aspect of this embodiment, R of formula III 2 is OR a In another aspect of this embodiment, R of Formula III 2 OH, -OC(=O)R 11 OR-OC(=O)OR 11 In another aspect of this embodiment, R of Formula III 2 In another aspect of this embodiment, R of formula III 2 In another aspect of this embodiment, R of formula III 3 OH, -OC(=O)R 11 OR-OC(=O)OR 11 In another aspect of this embodiment, R of Formula III 3 In another aspect of this embodiment, R of formula III 7 H, -C(=O)R 11 、-C(=O)OR 11 or

[0286]

[0287] In another aspect of this embodiment, R 7 is H. In another aspect of this embodiment, R of Formula III 7 for

[0288]

[0289] In another embodiment of the method of treating an Arenaviridae infection comprising administering a compound of Formula III, the Arenaviridae infection is caused by an Arenaviridae virus. In another aspect of this embodiment, the Arenaviridae virus is Lassa virus or Junin virus. In another aspect of this embodiment, the Arenaviridae virus is Lassa virus. In another aspect of this embodiment, the Arenaviridae virus is Junin virus. In another aspect of this embodiment, the Arenaviridae virus is caused by a Lassa virus caused by a strain selected from the group consisting of Josiah, NL, z148, Macenta, AV, and CSF.

[0290] In another aspect of this embodiment, the Arenaviridae infection is caused by Allpahuayo virus (ALLV), Amapari virus (AMAV), Bear Canyon virus (BCNV), Catalina virus, Chapare virus, Cupiqui virus (CPXV), Dandenong virus, Flexal virus (FLEV), citrullinated virus (GTOV), Ippy virus (IPPYV), Junin virus (JUNV), Kodoko virus, Lassa virus (LASV), Latin virus (LATV), lymphocytic choriomeningitis virus (LCMV), Lujo virus, Machupo virus (MACV), Mobala virus (MOBV), Morogoro virus, Mopeia virus (MOPV), Oliveros virus (OLVV), Parana virus (PARV), Pichinde virus (PICV), Pinhal virus, Pirital virus (PIRV), Sabia virus (SABV), Skinner box virus, Tacaribe virus (TCRV), Tamiami virus (TAMV), or Whitewater Arroyo virus (WWAV).

[0291] In another embodiment, a method of treating an Arenaviridae infection in a human in need thereof is provided, comprising administering a therapeutically effective amount of a compound of Formula I represented by Formula IV, or a pharmaceutically acceptable salt or ester thereof:

[0292]

[0293] where R 7 As defined above for Formula I.

[0294] In another embodiment of the method of treating Arenaviridae infection, comprising administering a compound of formula IV, R 7 Can be H. In another embodiment of the method of treating Arenaviridae infection, comprising administering a compound of formula IV, R 7 is selected from the group a), b) or c) as defined for formula I.

[0295] In another embodiment of the method of treating Arenaviridae infection, comprising administering a compound of formula IV, R 7 for

[0296]

[0297] where Z 1 and Z 2 Each independently is a group having the following structure:

[0298]

[0299] And Z 3 It's Z 5 .

[0300] In another embodiment of the method of treating Arenaviridae infection, comprising administering a compound of formula IV, R 7 for

[0301]

[0302] where Z 1 and Z 2 Each independently is a group having the following structure:

[0303]

[0304] And Z 3 It's Z 5 .

[0305] In another embodiment of the method of treating Arenaviridae infection, comprising administering a compound of formula IV, R 7 for

[0306]

[0307] Each Q 3b is independently O or N(R). In another embodiment, each Q 3b is O and each R x Independently:

[0308]

[0309] where M12c is 1, 2, or 3 and each Q 3 is independently a bond, O, CR2 or S.

[0310] In some embodiments, R e1 and R e2 Each of R is independently H, C1-C6 alkyl or benzyl. e1 can be H, C1-C6 alkyl or benzyl, and Re2 Can be H or C1-C6 alkyl. In some embodiments, R e1 and R e2 Each of R and R is independently H or C1-C6 alkyl. e1 and R e2 R e1 can be H, methyl or benzyl, and R e2 Can be H or methyl. In some embodiments, R e1 can be H or methyl, and R e2 Can be H or methyl. In some embodiments, R e1 can be methyl, and R e2 Can be H or methyl. In some embodiments, R e1 can be H or benzyl, and R e2 It can be H or methyl.

[0311] In another embodiment of the method of treating Arenaviridae infection, comprising administering a compound of formula IV, R 7 yes

[0312]

[0313] In another embodiment of the method of treating Arenaviridae infection, comprising administering a compound of formula IV, R 7 for

[0314]

[0315]

[0316] In another embodiment of the method of treating Arenaviridae infection, comprising administering a compound of formula IV, R 7 for

[0317]

[0318] where R f is selected from the group consisting of H, C1-C8 alkyl, benzyl, C3-C6 cycloalkyl and -CH2-C3-C6 cycloalkyl. In another embodiment of the compound of formula IV, R f In another embodiment of the compound of formula IV, R f It is 2-ethylbutyl.

[0319] In another embodiment of the method of treating Arenaviridae infection, comprising administering a compound of formula IV, R 7 yes

[0320]

[0321] in,

[0322] R f is selected from H, C1-C8 alkyl, benzyl, C3-C6 cycloalkyl and -CH2-C3-C6 cycloalkyl; and

[0323] R g Selected from C1-C8 alkyl, -O-C1-C8 alkyl, benzyl, -O-benzyl, -CH2-C3-C6 cycloalkyl, -O-CH2-C3-C6 cycloalkyl and CF3.

[0324] In another embodiment of the method of treating Arenaviridae infection, comprising administering a compound of formula IV, R 7 for

[0325]

[0326] where R f is selected from the group consisting of H, C1-C8 alkyl, benzyl, C3-C6 cycloalkyl and -CH2-C3-C6 cycloalkyl. In another embodiment of the compound of formula IV, R f In another embodiment of the compound of formula IV, R f In another embodiment of the compound of formula IV, R f It is 2-ethylbutyl.

[0327] In another embodiment of the method of treating Arenaviridae infection, comprising administering a compound of formula IV, R 7 for:

[0328]

[0329] where R g In another embodiment of the compound of formula IV, Rf is C1-C8 alkyl. In another embodiment of the compound of formula IV, R f It is a C1-C6 alkyl group.

[0330] In another embodiment of the method of treating Arenaviridae infection, comprising administering a compound of formula IV, R 7 Selected from the following groups:

[0331]

[0332] In another embodiment of the method of treating Arenaviridae infection, comprising administering a compound of formula IV, R 7 for

[0333]

[0334]

[0335] In another embodiment of the method of treating Arenaviridae infection comprising administering a compound of Formula IV, Z 1 and Z 2 Each can be:

[0336]

[0337] In another embodiment, there is provided a method of treating an Arenaviridae infection in a human in need thereof, comprising administering a therapeutically effective amount of a compound of Formula I-IV, wherein R 11 or R 12 are independently H, (C1-C8)alkyl, (C1-C8)alkenyl, (C2-C8)alkynyl, (C4-C8)carbocyclylalkyl, optionally substituted aryl, optionally substituted heteroaryl, -C(=O)(C1-C8)alkyl, -S(O) n (C1-C8) alkyl or aryl (C1-C8) alkyl. In another embodiment, R 11 and R 12 Together with the nitrogen to which they are attached, they form a 3-7 membered heterocyclic ring, wherein any carbon atom of the heterocyclic ring may be optionally replaced by -O-, -S- or -NR a - instead. Thus, by way of example and not limitation, -NR 11 R 12 The group can be represented by the following heterocyclic rings:

[0338] wait.

[0339] In another embodiment, there is provided a method of treating an Arenaviridae infection in a human in need thereof, comprising administering a therapeutically effective amount of a compound of Formula I-IV, wherein each R 3 、R 4 、R 5 、R 6 、R 11 or R 12 is independently (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl or aryl(C1-C8)alkyl, wherein said (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl or aryl(C1-C8)alkyl is independently optionally substituted by one or more halogen, hydroxyl, CN, N3, N(R a )2 ORa Therefore, by way of example and not limitation, R 3 、R 4 、R 5 、R 6 、R 11 or R 12 It may represent groups such as -CH(NH2)CH3, -CH(OH)CH2CH3, -CH(NH2)CH(CH3)2, -CH2CF3, -(CH2)2CH(N3)CH3, -(CH2)6NH2, and the like.

[0340] In another embodiment, there is provided a method of treating an Arenaviridae infection in a human in need thereof, comprising administering a therapeutically effective amount of a compound of Formula I-IV, wherein R 3 、R 4 、R 5 、R 6 、R 11 or R 12 is (C1-C8)alkyl, wherein one or more non-terminal carbon atoms of each of said (C1-C8)alkyl groups may be optionally replaced by -O-, -S- or -NR a - instead. Thus, by way of example and not limitation, R 3 、R 4 、R 5 、R 6 、R 11 or R 12 It may represent groups such as -CH2OCH3, -CH2OCH2CH3, -CH2OCH(CH3)2, -CH2SCH3, -(CH2)6OCH3, -(CH2)6N(CH3)2, and the like.

[0341] In another embodiment, the method of treating an Arenaviridae infection comprises administering a compound of Formula I, which is

[0342]

[0343] or a pharmaceutically acceptable salt or ester thereof.

[0344] In another embodiment, the method of treating an Arenaviridae infection comprises administering a compound of Formula I, which is

[0345]

[0346]

[0347] or a pharmaceutically acceptable salt or ester thereof.

[0348] In another embodiment, the method of treating an Arenaviridae infection comprises administering a compound of formula IV, which is:

[0349]

[0350]

[0351] or a pharmaceutically acceptable salt or ester thereof.

[0352] In another embodiment, the method of treating an Arenaviridae infection comprises administering a compound of formula IV, which is:

[0353]

[0354]

[0355] or a pharmaceutically acceptable salt or ester thereof.

[0356] In another embodiment of the method of treating Arenaviridae infection, the method comprises administering a compound of Formula I-IV, which is

[0357]

[0358]

[0359] or a pharmaceutically acceptable salt or ester thereof.

[0360] In another embodiment of the method of treating Arenaviridae infection, comprising administering a compound of Formula I-IV, said compound being

[0361]

[0362] or a pharmaceutically acceptable salt or ester thereof.

[0363] Provided is a method of treating a Coronaviridae infection in a human in need thereof, comprising administering a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt or ester thereof:

[0364]

[0365] in:

[0366] Each R 1 is H or halogen;

[0367] Each R 2 、R 3 、R 4 or R 5 are independently H, OR a 、N(R a)2、N3、CN、NO2、S(O) n R a , halogen, (C1-C8)alkyl, (C4-C8)carbocyclylalkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)alkynyl, (C2-C8)substituted alkynyl;

[0368] Or any two R on adjacent carbon atoms 2 、R 3 、R 4 or R 5 together are -O(CO)O- or together with the ring carbon atoms to which they are attached form a double bond;

[0369] R 6 is OR a 、N(R a )2、N3、CN、NO2、S(O) n R a 、-C(=O)R 11 、-C(=O)OR 11 、-C(=O)NR 11 R 12 、-C(=O)SR 11 、-S(O)R 11 、-S(O)2R 11 、-S(O)(OR 11 )、-S(O)2(OR 11 ), -SO2NR 11 R 12 , halogen, (C1-C8)alkyl, (C4-C8)carbocyclylalkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)alkynyl, (C2-C8)substituted alkynyl, or (C6-C8) 20 )aryl(C1-C8)alkyl;

[0370] R 7 Selected from the group consisting of:

[0371] a) H, -C(=O)R 11 、-C(=O)OR 11 、-C(=O)NR 11 R 12 、-C(=O)SR 11 、-S(O)R 11 、-S(O)2R 11 、-S(O)(OR 11 )、-S(O)2(OR 11 ) or -SO2NR11 R 12 ,

[0372] Each R 11 or R 12 (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl or (C6-C 20 ) aryl (C1-C8) alkyl groups are independently optionally substituted with one or more halogen, hydroxy, CN, N3, N(R a )2 OR a and wherein each of the (C1-C8) alkyl groups has one or more non-terminal carbon atoms that may be optionally substituted with -O-, -S- or -NR a -replace,

[0373] b)

[0374]

[0375] c)

[0376]

[0377] in:

[0378] R c is selected from phenyl, 1-naphthyl, 2-naphthyl,

[0379]

[0380] R d is H or CH3;

[0381] R e1 and R e2 Each is independently H, (C1-C6) alkyl or benzyl;

[0382] R f is selected from H, (C1-C8)alkyl, benzyl, (C3-C6)cycloalkyl and -CH2-(C3-C6)cycloalkyl;

[0383] R g Selected from (C1-C8)alkyl, -O-(C1-C8)alkyl, benzyl, -O-benzyl, -CH2-(C3-C6)cycloalkyl, -O-CH2-(C3-C6)cycloalkyl and CF3; and

[0384] n' is selected from 1, 2, 3 and 4; and

[0385] d) a group of the formula:

[0386]

[0387] in:

[0388] Q is O, S, NR, + N(O)(R), N(OR), + N(O)(OR) or N-NR2;

[0389] Z 1 and Z 2 Together for-Q 1 (C(R y )2)3Q 1 -;

[0390] in

[0391] Each Q 1 are independently O, S or NR; and

[0392] Each R y are independently H, F, Cl, Br, I, OH, R, -C(=Q 2 )R、-C(=Q 2 )OR、-C(=Q 2 )N(R)2、-N(R)2、- + N(R)3, -SR, -S(O)R, -S(O)2R, -S(O)(OR), -S(O)2(OR), -OC(=Q 1 )R、-OC(=Q 2 )OR、-OC(=Q 2 )(N(R)2),-SC(=Q 2 )R、-SC(=Q 2 )OR、-SC(=Q 2 )(N(R)2), -N(R)C(=Q2)R, -N(R)C(=Q 2 )OR、-N(R)C(=Q 2 )N(R)2, -SO2NR2, -CN, -N3, -NO2, -OR or Z3; or two R on the same carbon atom y together form a carbocyclic ring having 3 to 7 carbon atoms;

[0393] Each Q 2 are independently O, S, NR, + N(O)(R), N(OR), + N(O)(OR) or N-NR2; or

[0394] Z 1 and Z 2 are each independently a group of formula Ia:

[0395]

[0396] in:

[0397] Each Q 3 are independently a bond, O, CR2, NR, + N(O)(R), N(OR), + N(O)(OR), N-NR2, S, SS, S(O) or S(O)2;

[0398] M2 is 0, 1, or 2;

[0399] Each R x Independently R y Or the following:

[0400]

[0401] in:

[0402] Each of M1a, M1c and M1d is independently 0 or 1;

[0403] M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0404] Z 3 It's Z 4 or Z 5 ;

[0405] Z 4 is R, -C(Q 2 )R y 、-C(Q 2 )Z 5 、-SO2R y or -SO2Z 5 ;and

[0406] Z 5 is a carbocyclic or heterocyclic ring, wherein Z 5 Independently by 0 to 3 R y group substitution;

[0407] R 8 Halogen, NR 11 R 12 、N(R 11 )OR 11 NR 11 NR 11 R 12 ,N3,NO,NO2,CHO,CN,-CH(=NR 11 ),-CH=NNHR 11 、-CH=N(OR 11 )、-CH(OR 11 )2, -C(=O)NR 11 R 12、-C(=S)NR 11 R 12 、-C(=O)OR 11 , (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C4-C8) carbocyclylalkyl, (C6-C 20 ) optionally substituted aryl, optionally substituted heteroaryl, -C(=O)(C1-C8)alkyl, -S(O) n (C1-C8)alkyl, (C6-C 20 )aryl(C1-C8)alkyl, OR 11 or SR 11 ;

[0408] Each R 9 or R 10 are independently H, halogen, NR 11 R 12 、N(R 11 )OR 11 NR 11 NR 11 R 1 2. N3, NO, NO2, CHO, CN, -CH (=NR 11 ),-CH=NHNR 11 、-CH=N(OR 11 )、-CH(OR 11 )2, -C(=O)NR 11 R 12 、-C(=S)NR 11 R 12 、-C(=O)OR 11 、R 11 , OR 11 or SR 11 ;

[0409] Each R 11 or R 12 are independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C4-C8)carbocyclylalkyl, (C6-C 20 ) optionally substituted aryl, optionally substituted heteroaryl, -C(=O)(C1-C8)alkyl, -S(O) n (C1-C8)alkyl or (C6-C 20 ) aryl(C1-C8)alkyl; or R 11 and R 12 Together with the nitrogen to which they are attached, they form a 3-7 membered heterocyclic ring, wherein any carbon atom of the heterocyclic ring may be optionally replaced by -O-, -S- or -NR a -replace;

[0410] Each R a are independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C6-C 20 )aryl(C1-C8)alkyl, (C4-C8)carbocyclylalkyl, -C(=O)R, -C(=O)OR, -C(=O)NR2, -C(=O)SR, -S(O)R, -S(O)2R, -S(O)(OR), -S(O)2(OR) or -SO2NR2; wherein,

[0411] Each R is independently H, (C1-C8) alkyl, (C1-C8) substituted alkyl, (C2-C8) alkenyl, (C2-C8) substituted alkenyl, (C2-C8) alkynyl, (C2-C8) substituted alkynyl, (C6-C8) 20 )aryl, (C6-C 20 ) substituted aryl, (C2-C 20 ) heterocyclic group, (C2-C 20 ) substituted heterocyclic group, (C6-C 20 ) aryl (C1-C8) alkyl or substituted (C6-C 20 ) aryl(C1-C8)alkyl;

[0412] each n is independently 0, 1 or 2; and

[0413] Each R 2 、R 3 、R 5 、R 6 、R 11 or R 12 Each (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl or (C6-C 20 ) aryl (C1-C8) alkyl groups are independently optionally substituted with one or more halogen, hydroxy, CN, N3, N(R a )2 OR a ; and wherein each of the one or more non-terminal carbon atoms of the (C1-C8) alkyl group may be optionally replaced by -O-, -S- or -NR a -replace.

[0414] In another embodiment, a method of treating a Coronaviridae infection in a human in need thereof is provided, comprising administering a therapeutically effective amount of a compound of Formula I represented by Formula II, or a pharmaceutically acceptable salt or ester thereof:

[0415]

[0416] in,

[0417] R 1、R 3 、R 5 、R 7 、R 8 and R 9 As defined above for Formula I:

[0418] Each R 2 is OR a or halogen; and

[0419] R 6 is OR a 、N(R a )2、N3、CN、S(O) n R a 、-C(=O)R 11 、-C(=O)OR 11 、-C(=O)NR 11 R 12 、-C(=O)SR 11 、-S(O)R 11 、-S(O)2R 11 、-S(O)(OR 11 )、-S(O)2(OR 11 ), -SO2NR 11 R 12 , halogen, (C1-C8)alkyl, (C4-C8)carbocyclylalkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)alkynyl or (C2-C8)substituted alkynyl.

[0420] In one embodiment of the method of treating a Coronaviridae infection by administering a compound of Formula II, R 1 is H. In another aspect of this embodiment, R of Formula II 6 is N3, CN, halogen, (C1-C8) alkyl, (C1-C8) substituted alkyl, (C2-C8) alkenyl, (C2-C8) substituted alkenyl, (C2-C8) alkynyl or (C2-C8) substituted alkynyl. In another aspect of this embodiment, R of Formula II 6 is CN, methyl, vinyl or ethynyl. In another aspect of this embodiment, R of formula II 6 In another aspect of this embodiment, R of Formula II 6 In another aspect of this embodiment, R of Formula II 5 is H. In another aspect of this embodiment, R of Formula II 2 OR a In another aspect of this embodiment, R of Formula II 2 In another aspect of this embodiment, R2 is F. In another aspect of this embodiment, R of Formula II 3 is OR a In another aspect of this embodiment, R of Formula II 3 is OH, -OC(=O)R 11 OR-OC(=O)OR 11 In another aspect of this embodiment, R of Formula II 3 In another aspect of this embodiment, R 8 It is NR 11 R 12 In another aspect of this embodiment, R of Formula II 8 In another aspect of this embodiment, R 8 is OR 11 In another aspect of this embodiment, R of Formula II 8 In another aspect of this embodiment, R 9 is H. In another aspect of this embodiment, R of Formula II 9 NR 11 R 12 In another aspect of this embodiment, R of Formula II 9 In another aspect of this embodiment, R 7 H, -C(=O)R 11 、-C(=O)OR 11 or

[0421]

[0422] In another aspect of this embodiment, R of Formula II 7 is H. In another aspect of this embodiment, R of Formula II 7 for

[0423]

[0424] In another embodiment of the method for treating a Coronaviridae infection comprising administering a compound of Formula II, the Coronaviridae infection is caused by a Coronaviridae virus. In another aspect of this embodiment, the Coronaviridae virus is a MERS virus or a SARS virus. In another aspect of this embodiment, the Coronaviridae virus is a MERS virus. In another aspect of this embodiment, the Coronaviridae virus is a SARS virus. In another aspect of this embodiment, the Coronaviridae virus is caused by a MERS virus caused by a strain selected from known strains.

[0425] In another embodiment, a method of treating a Coronaviridae infection in a human in need thereof is provided, comprising administering a therapeutically effective amount of a compound of Formula I represented by Formula III, or a pharmaceutically acceptable salt or ester thereof:

[0426]

[0427] in,

[0428] R 6 、R 7 、R 8 and R 9 As defined above for Formula II;

[0429] Each R 2 is OR a or F; and

[0430] Each R 3 is OR a .

[0431] In one embodiment of the method of treating a Coronaviridae infection comprising administering a compound of Formula III, R 6 is N3, CN, halogen, (C1-C8) alkyl, (C1-C8) substituted alkyl, (C2-C8) alkenyl, (C2-C8) substituted alkenyl, (C2-C8) alkynyl or (C2-C8) substituted alkynyl. In another aspect of this embodiment, R of Formula III 6 is CN, methyl, vinyl or ethynyl. In another aspect of this embodiment, R of formula III 6 In another aspect of this embodiment, R of Formula III 6 In another aspect of this embodiment, R of formula III 2 is OR a In another aspect of this embodiment, R of Formula III 2 In another aspect of this embodiment, R of formula III 2 In another aspect of this embodiment, R of formula III 3 OH, -OC(=O)R 11 OR-OC(=O)OR 11 In another aspect of this embodiment, R of Formula III 3 In another aspect of this embodiment, R of formula III 8 NR 11 R 12 In another aspect of this embodiment, R of Formula III 8 In another aspect of this embodiment, R 8 is OR11 In another aspect of this embodiment, R of Formula III 8 In another aspect of this embodiment, R of formula III 9 is H. In another aspect of this embodiment, R of Formula III 9 NR 11 R 12 In another aspect of this embodiment, R of Formula III 9 In another aspect of this embodiment, R 7 H, -C(=O)R 11 、-C(=O)OR 11 or

[0432]

[0433] In another aspect of this embodiment, R of Formula III 7 is H. In another aspect of this embodiment, R of Formula III 7 for

[0434]

[0435] In another embodiment of the method of treating a Coronaviridae infection comprising administering a compound of Formula III, R 6 is N3, CN, halogen, (C1-C8)alkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)alkynyl or (C2-C8)substituted alkynyl, and R 8 In another aspect of this embodiment, R 6 is CN, methyl, vinyl or ethynyl. In another aspect of this embodiment, R of formula III 6 In another aspect of this embodiment, R of Formula III 6 In another aspect of this embodiment, R of formula III 2 is OR a In another aspect of this embodiment, R of Formula III 2 OH, -OC(=O)R 11 OR-OC(=O)OR 11 In another aspect of this embodiment, R of Formula III 2 In another aspect of this embodiment, R of formula III 2 In another aspect of this embodiment, R of formula III 3 OH, -OC(=O)R 11 OR-OC(=O)OR 11In another aspect of this embodiment, R of Formula III 3 In another aspect of this embodiment, R of formula III 9 is H. In another aspect of this embodiment, R of Formula III 9 NR 11 R 12 In another aspect of this embodiment, R of Formula III 9 In another aspect of this embodiment, R 7 H, -C(=O)R 11 、-C(=O)OR 11 or

[0436]

[0437] In another aspect of this embodiment, R of Formula III 7 is H. In another aspect of this embodiment, R of Formula III 7 for

[0438]

[0439] In another embodiment of the method of treating a Coronaviridae infection comprising administering a compound of Formula III, R 6 is CN, methyl, vinyl or ethynyl, R 8 is NH2, and R 9 is H. In another aspect of this embodiment, R of Formula III 6 In another aspect of this embodiment, R of Formula III 6 In another aspect of this embodiment, R of formula III 2 is OR a In another aspect of this embodiment, R of Formula III 2 OH, -OC(=O)R 11 OR-OC(=O)OR 11 In another aspect of this embodiment, R of Formula III 2 In another aspect of this embodiment, R of formula III 2 In another aspect of this embodiment, R of formula III 3 OH, -OC(=O)R 11 OR-OC(=O)OR 11 In another aspect of this embodiment, R of Formula III 3 In another aspect of this embodiment, R of formula III 7 H, -C(=O)R 11 、-C(=O)OR11 or

[0440]

[0441] In another aspect of this embodiment, R 7 is H. In another aspect of this embodiment, R of Formula III 7 for

[0442]

[0443] In another embodiment of the method of treating a Coronaviridae infection comprising administering a compound of Formula III, the Coronaviridae infection is caused by a Coronaviridae virus. In another aspect of this embodiment, the Coronaviridae virus is a MERS virus or a SARS virus. In another aspect of this embodiment, the Coronaviridae virus is a MERS virus. In another aspect of this embodiment, the Coronaviridae virus is a SARS virus. In another aspect of this embodiment, the Coronaviridae virus is caused by a MERS virus caused by a strain selected from known strains.

[0444] In another embodiment, a method of treating a Coronaviridae infection in a human in need thereof is provided, comprising administering a therapeutically effective amount of a compound of Formula I represented by Formula IV, or a pharmaceutically acceptable salt or ester thereof:

[0445]

[0446] where R 7 As defined above for Formula I.

[0447] In another embodiment of the method of treating a Coronaviridae infection comprising administering a compound of Formula IV, R 7 Can be H. In another embodiment of the method of treating a Coronaviridae infection, comprising administering a compound of formula IV, R 7 is selected from the group a), b) or c) as defined for formula I.

[0448] In another embodiment of the method of treating a Coronaviridae infection, comprising administering a compound of formula IV, R 7 for

[0449]

[0450] where Z 1 and Z 2 Each independently is a group having the following structure:

[0451]

[0452] And Z 3 It's Z5 .

[0453] In another embodiment of the method of treating a Coronaviridae infection comprising administering a compound of Formula IV, R 7 for

[0454]

[0455]

[0456] where Z 1 and Z 2 Each independently is a group having the following structure:

[0457]

[0458] And Z 3 It's Z 5 .

[0459] In another embodiment of the method of treating a Coronaviridae infection comprising administering a compound of Formula IV, R 7 for

[0460]

[0461] Each Q 3b is independently O or N(R). In another embodiment, each Q 3b is O and each R x Independently:

[0462]

[0463] where M12c is 1, 2, or 3 and each Q 3 is independently a bond, O, CR2 or S.

[0464] In some embodiments, R e1 and R e2 Each of R is independently H, C1-C6 alkyl or benzyl. e1 can be H, C1-C6 alkyl or benzyl, and R e2 Can be H or C1-C6 alkyl. In some embodiments, R e1 and R e2 Each of R and R is independently H or C1-C6 alkyl. e1 and R e2 R e1 can be H, methyl or benzyl, and R e2 Can be H or methyl. In some embodiments, Re1 can be H or methyl, and R e2 Can be H or methyl. In some embodiments, R e1 can be methyl, and R e2 Can be H or methyl. In some embodiments, R e1 can be H or benzyl, and R e2 It can be H or methyl.

[0465] In another embodiment of the method of treating a Coronaviridae infection comprising administering a compound of Formula IV, R 7 yes

[0466]

[0467] In another embodiment of the method of treating a Coronaviridae infection comprising administering a compound of Formula IV, R 7 for

[0468]

[0469] In another embodiment of the method of treating a Coronaviridae infection comprising administering a compound of Formula IV, R 7 for

[0470]

[0471] where R f is selected from the group consisting of H, C1-C8 alkyl, benzyl, C3-C6 cycloalkyl and -CH2-C3-C6 cycloalkyl. In another embodiment of the compound of formula IV, R f In another embodiment of the compound of formula IV, R f It is 2-ethylbutyl.

[0472] In another embodiment of the method of treating a Coronaviridae infection comprising administering a compound of Formula IV, R 7 yes

[0473]

[0474] in,

[0475] R f is selected from H, C1-C8 alkyl, benzyl, C3-C6 cycloalkyl and -CH2-C3-C6 cycloalkyl; and

[0476] R g Selected from C1-C8 alkyl, -O-C1-C8 alkyl, benzyl, -O-benzyl, -CH2-C3-C6 cycloalkyl, -O-CH2-C3-C6 cycloalkyl and CF3.

[0477] In another embodiment of the method of treating a Coronaviridae infection comprising administering a compound of Formula IV, R 7 for

[0478]

[0479] where R f is selected from the group consisting of H, C1-C8 alkyl, benzyl, C3-C6 cycloalkyl and -CH2-C3-C6 cycloalkyl. In another embodiment of the compound of formula IV, R f In another embodiment of the compound of formula IV, R f In another embodiment of the compound of formula IV, R f It is 2-ethylbutyl.

[0480] In another embodiment of the method of treating a Coronaviridae infection comprising administering a compound of Formula IV, R 7 for:

[0481]

[0482] where R g is selected from the group consisting of C1-C8 alkyl, -O-C1-C8 alkyl, benzyl, -O-benzyl, -CH2-C3-C6 cycloalkyl, -O-CH2-C3-C6 cycloalkyl and CF3. In another embodiment of the compound of formula IV, R f In another embodiment of the compound of formula IV, R f It is a C1-C6 alkyl group.

[0483] In another embodiment of the method of treating a Coronaviridae infection comprising administering a compound of Formula IV, R 7 Selected from:

[0484]

[0485] In another embodiment of the method of treating a Coronaviridae infection comprising administering a compound of Formula IV, R 7 for

[0486]

[0487] In another embodiment of the method of treating a Coronaviridae infection comprising administering a compound of Formula IV, Z 1 and Z 2 Each can be:

[0488]

[0489] In another embodiment, there is provided a method of treating a Coronaviridae infection in a human in need thereof, comprising administering a therapeutically effective amount of a compound of Formula I-IV, wherein R 11 or R 12 are independently H, (C1-C8)alkyl, (C1-C8)alkenyl, (C2-C8)alkynyl, (C4-C8)carbocyclylalkyl, optionally substituted aryl, optionally substituted heteroaryl, -C(=O)(C1-C8)alkyl, -S(O) n (C1-C8) alkyl or aryl (C1-C8) alkyl. In another embodiment, R 11 and R 12 Together with the nitrogen to which they are attached, they form a 3-7 membered heterocyclic ring, wherein any carbon atom of the heterocyclic ring may be optionally replaced by -O-, -S- or -NR a Thus, by way of example and not limitation, the group -NR 11 R 12 It can be represented by the following heterocycles:

[0490] wait.

[0491] In another embodiment, there is provided a method of treating a Coronaviridae infection in a human in need thereof, comprising administering a therapeutically effective amount of a compound of Formula I-IV, wherein each R 3 、R 4 、R 5 、R 6 、R 11 or R 12 is independently (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl or aryl(C1-C8)alkyl, wherein said (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl or aryl(C1-C8)alkyl is independently optionally substituted by one or more halogen, hydroxyl, CN, N3, N(R a )2 OR a Therefore, by way of example and not limitation, R 3 、R 4 、R 5 、R 6 、R 11 or R 12 It may represent groups such as -CH(NH2)CH3, -CH(OH)CH2CH3, -CH(NH2)CH(CH3)2, -CH2CF3, -(CH2)2CH(N3)CH3, -(CH2)6NH2, and the like.

[0492] In another embodiment, there is provided a method of treating a Coronaviridae infection in a human in need thereof, comprising administering a therapeutically effective amount of a compound of Formula I-IV, wherein R3 、R 4 、R 5 、R 6 、R 11 or R 12 is (C1-C8)alkyl, wherein one or more non-terminal carbon atoms of each of said (C1-C8)alkyl groups may be optionally replaced by -O-, -S- or -NR a - instead. Thus, by way of example and not limitation, R 3 、R 4 、R 5 、R 6 、R 11 or R 12 It may represent groups such as -CH2OCH3, -CH2OCH2CH3, -CH2OCH(CH3)2, -CH2SCH3, -(CH2)6OCH3, -(CH2)6N(CH3)2, and the like.

[0493] In another embodiment of the method of treating a Coronaviridae infection, comprising administering a compound of formula I, said compound being

[0494]

[0495]

[0496] or a pharmaceutically acceptable salt or ester thereof.

[0497] In another embodiment of the method of treating a Coronaviridae infection, comprising administering a compound of formula I, said compound being

[0498]

[0499]

[0500]

[0501] or a pharmaceutically acceptable salt or ester thereof.

[0502] In another embodiment of the method of treating a Coronaviridae infection, comprising administering a compound of formula IV, said compound being:

[0503]

[0504] or a pharmaceutically acceptable salt or ester thereof.

[0505] In another embodiment of the method of treating a Coronaviridae infection, comprising administering a compound of formula IV, said compound being:

[0506]

[0507] or a pharmaceutically acceptable salt or ester thereof.

[0508] In another embodiment of the method of treating a Coronaviridae infection, the method comprises administering a compound of Formula I-IV, which is

[0509]

[0510]

[0511] or a pharmaceutically acceptable salt or ester thereof.

[0512] In another embodiment of the method of treating a Coronaviridae infection, comprising administering a compound of Formula I-IV, said compound being

[0513]

[0514] or a pharmaceutically acceptable salt or ester thereof.

[0515] The therapeutic methods described herein include methods for treating human Coronaviridae infections, including infections caused by alphacoronavirus 229E (HCoV-229E) and NL63 (HCoV-NL63, New Haven coronavirus), betacoronavirus OC43 (HCoV-OC43), HKU1, SARS-CoV (coronavirus that causes severe acute respiratory syndrome (SARS)), and MERS-CoV (coronavirus that causes Middle East respiratory syndrome), previously known as novel coronavirus 2012 and HCoV-EMC.

[0516] The names of the compounds of the present disclosure are provided using ACD / Name software (Advanced Chemistry Development, Inc., Toronto, Canada) for naming chemical compounds. Other compounds or groups may be named using common names, systematic names, or non-systematic names. The naming and numbering of the compounds of the present invention are illustrated using representative compounds of Formula I:

[0517]

[0518] It is named (2S)--2-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propionic acid-2-ethylbutyl ester. Other compounds of the present invention include:

[0519]

[0520] It is named as (S)-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)(phenoxy)phosphoryl)amino)propionic acid-2-ethylbutyl ester, and

[0521]

[0522] It was named as 2-ethylbutyl (S)-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)(phenoxy)phosphoryl)amino)propanoate.

[0523] Any reference to a compound of the invention as described herein also includes a reference to a physiologically acceptable salt thereof. Examples of physiologically acceptable salts of compounds of the invention include those derived from a suitable base (e.g., an alkali metal or alkaline earth metal (e.g., Na + , Li + , K + , Ca +2 and Mg +2 ), ammonium and NR4 + (wherein R is as defined herein). Physiologically acceptable salts of nitrogen atoms or amino groups include: (a) acid addition salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; (b) salts formed with organic acids, such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, isethionic acid, lactobionic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, malonic acid, sulfosalicylic acid, glycolic acid, 2-hydroxy-3-naphthoate, pamoate, salicylic acid, stearic acid, phthalic acid, mandelic acid, lactic acid, ethanesulfonic acid, lysine, arginine, glutamic acid, glycine, serine, threonine, alanine, isoleucine, leucine, and the like; and (c) salts formed with elemental anions, such as chlorine, bromine, and iodine. Physiologically acceptable salts of hydroxy compounds include the anion of the compound and a suitable cation such as Na + and NR4 + combination.

[0524] Compounds of formula I-IV and pharmaceutically acceptable salts thereof can exist in different polymorphs or pseudopolymorphs. As used herein, crystalline polymorphism means the ability of a crystalline compound to exist in different crystal structures. Crystal polymorphism may be caused by differences in crystal packing (stacking polymorphism) or differences in packing between different conformers of the same molecule (conformational polymorphism). As used herein, crystalline pseudopolymorphism refers to the ability of a hydrate or solvate of a compound to exist in different crystal structures. The pseudopolymorphs of the present invention may exist due to differences in crystal packing (filling pseudopolymorphism) or due to differences in packing between different conformers of the same molecule (conformational pseudopolymorphism). The present invention comprises all polymorphs and pseudopolymorphs of compounds of formula I-III and pharmaceutically acceptable salts thereof.

[0525] The compounds of Formula I-IV and their pharmaceutically acceptable salts may also exist as amorphous solids. As used herein, an amorphous solid is a solid in which there is no long-range ordered position of atoms in the solid. This definition also applies when the crystal size is 2 nanometers or less. Additives, including solvents, may be used to produce the amorphous forms of the present invention. The present invention includes all amorphous forms of the compounds of Formula I-IV and their pharmaceutically acceptable salts.

[0526] For therapeutic use, the salts of the active ingredients of the compounds of the invention will be physiologically acceptable, i.e., they will be salts derived from physiologically acceptable acids or bases. However, salts that are not physiologically acceptable acids or bases may also be used, for example, in the preparation or purification of physiologically acceptable compounds. All salts, whether or not derived from physiologically acceptable acids or bases, are within the scope of the present invention.

[0527] Finally, it should be understood that the compositions herein comprise the unionized as well as the zwitterionic forms of the compounds of the present invention in combination with a stoichiometric amount of water as in hydrates.

[0528] It should be noted that all enantiomers, diastereomers and racemic mixtures, tautomers, polymorphs, pseudopolymorphs and pharmaceutically acceptable salts of the compounds within the scope of Formula I-IV are included in the present invention. All mixtures of these enantiomers and diastereomers are within the scope of the present invention.

[0529] The compounds of the present invention illustrated by formula I-IV may have chiral centers, such as chiral carbon or phosphorus atoms. Therefore, the compounds of the present invention include racemic mixtures of all stereoisomers, including enantiomers, diastereomers and atropisomers. In addition, the compounds of the present invention contain enriched or split optical isomers at any or all asymmetric chiral atoms. In other words, it can be seen from the description that the chiral center is a chiral isomer or a racemic mixture. Racemic and diastereomeric mixtures and separated or synthesized individual optical isomers (substantially free of their enantiomers or diastereomeric partners) are all within the scope of the present invention. Racemic mixtures are separated into their respective substantially optically pure isomers by well-known techniques, such as separation of diastereomeric salts formed with optically active auxiliary substances (such as acids or bases), and then converted back into optically active substances. In most cases, the desired optical isomers are synthesized by stereospecific reactions, starting from the appropriate stereoisomers of the desired raw materials.

[0530] Stereochemical definitions and conventions used herein generally follow those of SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active forms, meaning they have the ability to rotate the plane of plane-polarized light. When describing an optically active compound, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule about its chiral center. The prefixes d and 1, D and L, or (+) and (-) are used to indicate the sign of the compound's rotation of plane-polarized light, with S, (-) or 1 indicating that the compound is levorotatory and compounds prefixed with R, (+) or d are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Specific stereoisomers may also be referred to as enantiomers, and a mixture of these isomers is often referred to as an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur in the absence of stereoselectivity or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric substances that is optically inactive.

[0531] The compounds of the present invention may also exist as tautomers in certain circumstances. Although only one delocalized resonance structure may be depicted, all such forms are within the scope of the present invention. For example, purine, pyrimidine, imidazole, guanidine, amidine, and tetrazole systems may exist as ene-amine tautomers, and all possible tautomeric forms thereof are within the scope of the present invention.

[0532] Any formula (including compounds of Formula I) or structure given herein is also intended to represent unlabeled forms as well as isotopically labeled compounds. Isotopically labeled compounds have structures depicted by the formulas given herein except that one or more atoms are replaced by atoms having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as, but not limited to, 2H (deuterium, D), 3H (tritium), 11 C. 13 C. 14 C. 15 N. 18 F. 31 P. 32 P. 35 S. 36 Cl and 125 I. Various isotope-labeled compounds of the present disclosure, for example, compounds into which radioactive isotopes such as 3 H. 13 C and 14 Such isotopically labeled compounds are useful in metabolism studies, reaction kinetics studies, detection or imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), including drug or substrate tissue distribution assays or radiotherapy of patients.

[0533] The present disclosure also includes compounds of formula I in which 1 to n hydrogen atoms attached to the carbon atom are replaced by deuterium, where n is the number of hydrogen atoms in the molecule. Such compounds exhibit increased metabolic resistance and can therefore be used to increase the half-life of any compound of formula I when administered to a mammal, particularly a human. See, for example, Foster, "DeuteriuMIsotope Effects in Studies of Drug Metabolism", Trends Pharmacol. Sci. 5(12): 524-527 (1984). Such compounds are synthesized by methods well known in the art, for example, by using raw materials in which one or more hydrogen atoms are replaced by deuterium.

[0534] Deuterium-labeled or substituted therapeutic compounds of the present disclosure may have improved DMPK (drug metabolism and pharmacokinetics) properties, relating to distribution, metabolism and excretion (ADME). Substitution with heavier isotopes such as deuterium may provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life, reduced dosage requirements and / or improved therapeutic index. 18 F-labeled compounds can be used for PET or SPECT studies. Isotopically labeled compounds and prodrugs of the present disclosure can generally be prepared by carrying out the methods disclosed in the schemes or in the following examples and preparations by substituting readily available isotopically labeled reagents for non-isotopically labeled reagents. It should be understood that deuterium is considered a substituent in the compounds of Formula I in this context.

[0535] The concentration of such heavier isotopes, particularly deuterium, can be defined by an isotopic enrichment factor. In the compounds of the present disclosure, any atom not specifically designated as a particular isotope is intended to represent any stable isotope of that atom. Unless otherwise indicated, when a position is explicitly designated as "H" or "hydrogen," that position is understood to be hydrogen with its natural abundance isotopic composition. Thus, in the compounds of the present disclosure, any atom specifically designated as deuterium (D) is meant to represent deuterium.

[0536] Whenever a compound described herein is represented by more than one of the same designated group, such as "R" or "R 1 ", it should be understood that these groups can be the same or different, that is, each group is independently selected. Indicates a point of covalent attachment to an adjacent substructure, group, moiety, or atom.

[0537] The selected substituents comprising the compounds of Formula I-IV are present to a recursive degree. In this context, a "recursive substituent" means that a substituent can refer to another instance of itself. Due to the recursive nature of such substituents, in theory, a large number of compounds can exist in any given embodiment. For example, R x Contains R y Substituent. R y It can be R. R can be Z 3 . Z 3 Can be Z 4 , and Z 4 Can be R or contain R y Alternatively, Z 3 Can be Z 5 , which may contain R yThe number of substituents is preferably substituted with a substituent. One of ordinary skill in the art of medicinal chemistry will appreciate that the total number of such substituents is reasonably limited by the desired properties of the desired compound. By way of example and not limitation, such properties include physical properties such as molecular weight, solubility, or log P, application properties such as activity against the desired target, and practical properties such as ease of synthesis.

[0538] By way of example and not limitation, in certain embodiments, Z 3 and R y is a recursive substituent. Typically, in a given embodiment, each recursive substituent may independently occur 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 times. More typically, in a given embodiment, each recursive substituent may independently occur 12 times or less. Even more typically, in a given embodiment, each recursive substituent may independently occur 3 times or less. For example, in a given embodiment, Z 3 Will appear 0 to 8 times, R y Will occur 0 to 6 times. Even more typically, in a given embodiment, Z 3 will appear 0 to 6 times, and R y Will appear 0 to 4 times.

[0539] Recursive substituents are an anticipated aspect of the present invention. One of ordinary skill in the art of medicinal chemistry understands the versatility of such substituents. To the extent that recursive substituents are present in an embodiment of the present invention, the total number will be determined as described above.

[0540] The compounds of the present invention can be prepared by methods known to those skilled in the art. For example, the compounds of the present invention can be prepared according to the methods described in U.S. Patent No. 8,008,264 and U.S. Application Publication No. US2012 / 0027752.

[0541] A. Substitution forms of compounds

[0542] The compounds of formula I-IV may contain a phosphate group as R 7 , R 7 Selected from a) H, -C(=O)R 11 、-C(=O)OR 11 、-C(=O)NR 11 R 12 、-C(=O)SR 11 、-S(O)R 11 、-S(O)2R 11 、-S(O)(OR 11 )、-S(O)2(OR 11 ), -SO2NR 11 R 12, where each R 11 or R 12 are independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C4-C8)carbocyclylalkyl, optionally substituted aryl, optionally substituted heteroaryl, -C(=O)(C1-C8)alkyl, -S(O) n (C1-C8)alkyl or aryl(C1-C8)alkyl; or R 11 and R 12 Together with the nitrogen to which they are attached, they form a 3-7 membered heterocyclic ring, wherein any carbon atom of the heterocyclic ring may be optionally replaced by -O-, -S- or -NR a -replace;

[0543] Each R a are independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, aryl(C1-C8)alkyl, (C4-C8)carbocyclylalkyl, -C(=O)R, -C(=O)OR, -C(=O)NR2, -C(=O)SR, -S(O)R, -S(O)2R, -S(O)(OR), -S(O)2(OR) or -SO2NR2;

[0544] wherein each R is independently H, (C1-C8) alkyl, (C1-C8) substituted alkyl, (C2-C8) alkenyl, (C2-C8) substituted alkenyl, (C2-C8) alkynyl, (C2-C8) substituted alkynyl, C6-C 20 Aryl, C6-C 20 Substituted aryl, C2-C 20 Heterocyclic group, C2-C 20 substituted heterocyclyl, arylalkyl, or substituted arylalkyl; and

[0545] Each R 11 or R 12 Each (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl or aryl(C1-C8)alkyl group is independently optionally substituted with one or more halogen, hydroxy, CN, N3, N(R a )2 OR a and wherein each of the (C1-C8) alkyl groups has one or more non-terminal carbon atoms that may be optionally substituted with -O-, -S- or -NR a -replace,

[0546] b)

[0547]

[0548] c)

[0549]

[0550] in:

[0551] R c Selected from phenyl, 1-naphthyl, 2-naphthyl,

[0552]

[0553] R d is H or CH3;

[0554] R e1 and R e2 Each is independently H, C1-C6 alkyl or benzyl;

[0555] R f Selected from H, C1-C8 alkyl, benzyl, C3-C6 cycloalkyl and -CH2-C3-C6 cycloalkyl;

[0556] R g is selected from the group consisting of C1-C8 alkyl, -O-C1-C8 alkyl, benzyl, -O-benzyl, -CH2-C3-C6 cycloalkyl, -O-CH2-C3-C6 cycloalkyl, and CF3; and

[0557] n' is selected from 1, 2, 3 and 4; and

[0558] d) a group of the formula:

[0559]

[0560] in,

[0561] Each Q 1 are independently O, S or NR; and

[0562] Each R y are independently H, F, Cl, Br, I, OH, R, -C(=Q 2 )R、-C(=Q 2 )OR、-C(=Q 2 )N(R)2、-N(R)2、- + N(R)3, -SR, -S(O)R, -S(O)2R, -S(O)(OR), -S(O)2(OR), -OC(=Q 2 )R、-OC(=Q 2 )OR、-OC(=Q 2 )(N(R)2),-SC(=Q 2 )R、-SC(=Q 2 )OR、-SC(=Q 2 )(N(R)2),-N(R)C(=Q 2)R、-N(R)C(=Q 2 )OR、-N(R)C(=Q 2 )N(R)2, -SO2NR2, -CN, -N3, -NO2, -OR or Z 3 ; or two R on the same carbon atom y Together they form a carbocyclic ring of 3-7 carbon atoms;

[0563] Each Q 2 are independently O, S, NR, + N(O)(R), N(OR), + N(O)(OR) or N-NR2; or

[0564] Z 1 and Z 2 are each independently a group of formula Ia:

[0565]

[0566] in:

[0567] Each Q 3 are independently a bond, O, CR2, NR, + N(O)(R), N(OR), + N(O)(OR), N-NR2, S, SS, S(O) or S(O)2;

[0568] M2 is 0, 1, or 2;

[0569] Each R x Independently R y Or the following:

[0570]

[0571] in:

[0572] Each of M1a, M1c and M1d is independently 0 or 1;

[0573] M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0574] Z 3 It's Z 4 or Z 5 ;

[0575] Z 4 is R, -C(Q 2 )R y 、-C(Q 2 )Z 5 、-SO2R y or -SO2Z 5;and

[0576] Z 5 is a carbocyclic or heterocyclic ring, wherein Z 5 Independently by 0 to 3 R y Group substitution.

[0577] Z 5 Carbon ring and Z 5 The heterocyclic ring may be independently substituted by 0 to 3 R y Group substitution. Z 5 Z may be a saturated, unsaturated or aromatic ring including monocyclic or bicyclic carbocyclic or heterocyclic rings. 5 There may be 3 to 10 ring atoms, for example 3 to 7 ring atoms. When there are 3 ring atoms, Z 5 The ring is saturated, and when it contains 4 ring atoms, Z 5 The ring is saturated or monounsaturated, and when it contains 5 ring atoms, Z 5 The ring is saturated or monounsaturated or diunsaturated, and when it contains 6 ring atoms, Z 5 The rings are saturated, monounsaturated or diunsaturated, or aromatic.

[0578] Z 5 The heterocyclic ring may be a monocyclic ring having 3-7 ring members (2-6 carbon atoms and 1-3 heteroatoms selected from N, O, P and S) or a bicyclic ring having 7-10 ring members (4-9 carbon atoms and 1-3 heteroatoms selected from N, O, P and S). 5 The heterocyclic monocyclic ring can have 3-6 ring atoms (2-5 carbon atoms and 1-2 heteroatoms selected from N, O and S); or 5 or 6 ring atoms (3-5 carbon atoms and 1-2 heteroatoms selected from N and S). 5 Heterocyclic bicyclic rings have 7-10 ring atoms (6-9 carbon atoms and 1-2 heteroatoms selected from N, O and S) arranged in a bicyclic [4,5], [5,5], [5,6] or [6,6] system; or 9-10 ring atoms (8-9 carbon atoms and 1-2 heteroatoms selected from N and S) arranged in a bicyclic [5,6] or [6,6] system. 5 The heterocyclic ring can be connected to Q through a stable covalent bond via carbon, nitrogen, sulfur or other atoms. 2 bonding.

[0579] Z 5 Heterocycles include, for example, pyridyl, dihydropyridyl isomers, piperidine, pyridazinyl, pyrimidinyl, pyrazinyl, s-triazinyl, oxazolyl, imidazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, furanyl, thiofuranyl, thienyl, and pyrrolyl. 5 Examples include but are not limited to:

[0580]

[0581] As defined above, Z 5 The carbocyclic and heterocyclic rings may be independently substituted with 0 to 3 R groups. For example, substituted Z 5 Carbon rings include:

[0582]

[0583]

[0584] Examples of substituted phenyl carbocycles include:

[0585]

[0586] In another embodiment, Z of the compound of Formula I-IV 5 is a carbocyclic or heterocyclic ring, wherein Z 5 Independently by 0 to 3 R z group substituted, where each R z are independently H, F, Cl, Br, I, OH, R, -C(=Q 2 )R、-C(=Q 2 )OR、-C(=Q 2 )N(R)2、-N(R)2、- + N(R)3, -SR, -S(O)R, -S(O)2R, -S(O)(OR), -S(O)2(OR), -OC(=Q 1 )R、-OC(=Q 2 )OR、-OC(=Q 2 )(N(R)2),-SC(=Q 2 )R、-SC(=Q 2 )OR、-SC(=Q 2 )(N(R)2),-N(R)C(=Q 2 )R、-N(R)C(=Q 2 )OR、-N(R)C(=Q 2 )N(R)2, -SO2NR2, -CN, -N3, -NO2 or -OR.

[0587] Compounds of formula I-IV The implementation includes substructures such as:

[0588]

[0589] Each Q 3b is independently O or N(R). In another aspect of this embodiment, each Q 3b is O and each R xIndependently:

[0590]

[0591] where M12c is 1, 2, or 3 and each Q 3 is independently a bond, O, CR2 or S. In another aspect of this embodiment, one Q 3b -R x is NH(R), and other Q 3b -R x is OR x , where R x yes:

[0592]

[0593] wherein M12c is 2. In another aspect of this embodiment, each Q 3b is O and each R x Independently:

[0594]

[0595] wherein M12c is 2. In another aspect of this embodiment, each Q 3b is O and each R x Independently:

[0596]

[0597] Where M12c is 1 and Q 3 is a bond, O or CR2.

[0598] Compounds of formula I-IV Other embodiments include substructures such as:

[0599]

[0600] Each Q 3 is independently O or N(R). In another aspect of this embodiment, each Q 3 Is O. In another aspect of this embodiment, the substructure is:

[0601]

[0602] where R y As defined herein, R 5 .

[0603] Formula I-IV Another embodiment includes the substructure:

[0604]

[0605] Each Q 2c are independently O, N(R y ) or S.

[0606] Compounds of formula I-IV Another embodiment includes wherein Z 1 or Z 2 One of the R 3 or R 4 One of them together is -Q 3 - and Z 1 or Z 2 Another is a substructure of Formula Ia. This embodiment is represented by a compound of Formula Ib selected from the group consisting of:

[0607]

[0608]

[0609] In another aspect of the embodiment of Formula Ib, each of Q and Q 3 Is O. In another aspect of the embodiment of Formula Ib, Z 1 or Z 2 It's Q 3b -R x ; Each Q, Q 3 and Q 3b is O and R x yes:

[0610]

[0611] where M12c is 1, 2, or 3 and each Q 3 is independently a bond, O, CR2 or S. In another aspect of the embodiment of Formula Ib, Z 1 or Z 2 It's Q 3b -R x ; Each Q, Q 3 and Q 3b It is O and R x yes:

[0612]

[0613] wherein M12c is 2. In another aspect of the embodiment of Formula Ib, Z 1 or Z 2 It's Q 3b -R x ; Each Q, Q 3 and Q 3b It is O and R x yes:

[0614]

[0615] Where M12c is 1 and Q 3 is a bond, O or CR2.

[0616] Compounds of formula I-IV Another embodiment includes the substructure:

[0617]

[0618] where Z 5 is a carbocyclic ring such as phenyl or substituted phenyl. In another aspect of this embodiment, the substructure is:

[0619]

[0620] where Q 3b is O or N(R) and the phenyl carbocyclic ring is substituted with 0 to 3 R groups. In another aspect of this embodiment of the substructure, R x yes:

[0621]

[0622] where M12c is 1, 2, or 3 and each Q 3 is independently a bond, O, CR2 or S.

[0623] Formula I-IV Another embodiment includes the substructure:

[0624]

[0625] The chiral carbons of the amino acid and lactate moieties may be in the R or S configuration or as a racemic mixture.

[0626] I-IV Another embodiment of the substructure is

[0627]

[0628] Each Q 3 is independently -O- or -NH-. In another aspect of this embodiment, R y is (C1-C8)alkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)alkynyl or (C2-C8)substituted alkynyl. In another aspect of this embodiment, R yis (C1-C8)alkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)alkynyl or (C2-C8)substituted alkynyl; and R is CH3. In another aspect of this embodiment, R y is (C1-C8)alkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)alkynyl or (C2-C8)substituted alkynyl; R is CH3; and each Q 3 In another aspect of this embodiment, Z 1 and Z 2 is independently a nitrogen-linked naturally occurring amino acid or a naturally occurring amino acid ester. In another aspect of this embodiment, Z 1 and Z 2 P is independently a naturally occurring 2-hydroxycarboxylic acid or a naturally occurring 2-hydroxycarboxylic acid ester, wherein the acid or ester is linked to P via the 2-hydroxy group.

[0629] Formula I-IV Another embodiment is the substructure:

[0630]

[0631] In one aspect of this embodiment, each R x is independently (C1-C8)alkyl. In another aspect of this embodiment, each R x Independently C6-C 20 Aryl or C6-C 20 Substituted aryl.

[0632] In a preferred embodiment, Selected from

[0633]

[0634] R x Embodiments include ester, carbamate, carbonate, thioester, amide, thioamide, and urea groups:

[0635]

[0636] B. Metabolites of the Compounds of the Invention

[0637] Also within the scope of the present invention are the in vivo metabolic products of the compounds described herein, provided such products are novel and unobvious compared to the prior art. Such products may result from, for example, oxidation, reduction, hydrolysis, amidation, esterification, etc. of the administered compound, primarily due to enzymatic processes. Thus, the present invention includes novel and unobvious compounds produced by a process comprising contacting a compound of the present invention with a mammal for a period of time sufficient to produce a metabolic product thereof. Typically, the radiolabeled (e.g., 14 C or 3 H) Compounds are administered parenterally to animals such as rats, mice, guinea pigs, monkeys or humans at a detectable dose (e.g., greater than about 0.5 mg / kg), sufficient time is allowed for metabolism (usually about 30 seconds to 30 hours), and their conversion products are isolated from urine, blood or other biological samples to identify the products. These products are easily isolated because they are labeled (others are isolated by using antibodies that can bind to epitopes present in the metabolites). The metabolite structures are determined in a conventional manner, for example, by MS or NMR analysis. Generally, analysis of metabolites is performed in the same manner as conventional drug metabolism studies well known to those skilled in the art. As long as the conversion products are not present in vivo, they are useful in diagnostic assays for therapeutic doses of the compounds of the invention, even if they do not themselves have anti-Anaviridae activity.

[0638] Formulas and methods for determining the stability of compounds in surrogate gastrointestinal secretions are known. Compounds are defined herein as being stable in the gastrointestinal tract when less than about 50 mole percent of the protected groups are deprotected in surrogate intestinal or gastric fluids after incubation at 37°C for one hour. Simply because a compound is stable to the gastrointestinal tract does not mean it cannot be hydrolyzed in vivo. The prodrugs of the present invention are generally stable in the digestive system but may be substantially hydrolyzed to the parent drug in the digestive lumen, liver or other metabolic organs, or within normal cells.

[0639] III. Pharmaceutical Preparations

[0640] The compounds of the present invention are formulated with conventional carriers and excipients, which are selected according to conventional practice. Tablets will contain excipients, glidants, fillers, binders, etc. Aqueous formulations are prepared in a sterile form and are generally isotonic for delivery other than oral administration. All formulations will optionally contain excipients, such as those described in "Handbook of Pharmaceutical Excipients" (1986). Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextran, hydroxyalkyl cellulose, hydroxyalkyl methyl cellulose, stearic acid, etc. The pH range of the formulation is from about 3 to about 11, but is typically from about 7 to 10. In some embodiments, the pH range of the formulation is from about 2 to about 5, but is typically from about 3 to 4.

[0641] Although the active ingredients can be administered alone, it may be preferable to present them as a pharmaceutical formulation. The formulations of the present invention for veterinary and human use comprise at least one active ingredient as defined above together with one or more acceptable carriers and optionally other therapeutic ingredients, particularly those discussed herein. The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not physiologically harmful to the recipient thereof.

[0642] The preparation includes those preparations suitable for the above-mentioned route of administration. The preparation can be conveniently presented in unit dosage form and can be prepared by any method well-known in the pharmaceutical field. Technology and formulations can generally be found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). These methods include the step of combining the active ingredient with a carrier constituting one or more auxiliary ingredients. Generally speaking, the preparation is prepared by uniformly and closely combining the active ingredient with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product.

[0643] Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be administered as a bolus, electuary, or paste.

[0644] Tablet is prepared by compression or molding, optionally with one or more auxiliary ingredients. Compressed tablet can be prepared by compressing the active ingredient such as powder or granule of the free-flowing form optionally mixed with binding agent, lubricant, inert diluent, preservative, surfactant or dispersant in a suitable machine. Molded tablet can be prepared by molding the mixture of the powdered active ingredient moistened with an inert liquid diluent in a suitable machine. Tablet can optionally be coated or scored and optionally formulated to provide active ingredient to release slowly or controlled therefrom.

[0645] For infections of the eye or other external tissues, such as the mouth and skin, the formulation is preferably applied as a topical ointment or cream containing the active ingredient in an amount of, for example, 0.075-20% w / w (including 0.1%-20% active ingredient in increments of 0.1% w / w, such as 0.6% w / w, 0.7% w / w, etc.), preferably 0.2-15% w / w, and most preferably 0.5-10% w / w. When formulated as an ointment, the active ingredient may be used with a paraffinic or water-miscible ointment base. Alternatively, the active ingredient may be formulated as a cream with an oil-in-water cream base.

[0646] If desired, the aqueous phase of the cream base may include, for example, at least 30% w / w of a polyol, i.e., an alcohol having two or more hydroxyl groups, such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol, and polyethylene glycol (including PEG 400), and mixtures thereof. Topical formulations may desirably include compounds that enhance the absorption or penetration of the active ingredient through the skin or other affected area. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogs.

[0647] The oil phase of the emulsion of the present invention can be composed of known ingredients in a known manner. Although this phase can contain only an emulsifier (also known as an emulsifier), it ideally contains a mixture of at least one emulsifier with a fat or oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier that acts as a stabilizer. It is also preferred to include oils and fats. In summary, the emulsifier with or without a stabilizer constitutes the so-called emulsifying wax, and this wax, together with the oil, constitutes the so-called emulsifying ointment base, which forms the oily dispersed phase of the cream formulation.

[0648] Emulsifiers and emulsion stabilizers suitable for the formulations of the present invention include 60. 80, cetearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate. Other emulsifiers and emulsion stabilizers suitable for use in the formulation of the present invention include 80.

[0649] The selection of suitable oil or fat for preparation is based on realizing required cosmetic properties.Emulsifiable paste should preferably be non-greasy, non-staining and washable product, has suitable denseness to avoid leaking from pipe or other containers.Can use straight or branched monobasic or dibasic alkyl ester, for example the mixture of propylene glycol diester, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or the branched ester that is called Crodamol CAP of diisoadipate, isocetyl stearate, coconut fatty acid, three kinds of latter are preferred esters.These can be used alone or according to required character combination use.Or, use high melting point fat, for example white soft paraffin and / or liquid paraffin or other mineral oils.

[0650] Pharmaceutical formulations according to the present invention comprise a combination according to the present invention, one or more pharmaceutically acceptable carriers or excipients, and optionally, other therapeutic agents. Pharmaceutical formulations containing the active ingredient may be in any form suitable for the intended method of administration. For example, when intended for oral use, tablets, lozenges, buccal tablets, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups, or elixirs may be prepared. Compositions intended for oral use may be prepared according to any method known in the art for manufacturing pharmaceutical compositions, and such compositions may contain one or more agents, including sweeteners, flavorings, colorants, and preservatives, to provide a palatable formulation. Tablets containing the active ingredient in combination with nontoxic, pharmaceutically acceptable excipients suitable for tablet manufacture are acceptable. These excipients may include, for example, inert diluents such as calcium or sodium carbonate, lactose, calcium or sodium phosphate; granulating and disintegrants such as corn starch or alginic acid; binders such as starch, gelatin, or gum arabic; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate, alone or with a wax, may be employed.

[0651] Formulations for oral use may also be provided as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent such as calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium such as peanut oil, liquid paraffin or olive oil.

[0652] The aqueous suspensions of the present invention contain the active substance mixed with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum arabic, as well as dispersants or wetting agents such as naturally occurring phospholipids (e.g., lecithin), condensation products of ethylene oxide with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long-chain fatty alcohols (e.g., heptadecanethoxycetanol), and condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspensions may also contain one or more preservatives such as ethyl or n-propyl paraben, one or more colorants, one or more flavoring agents, and one or more sweeteners such as sucrose or saccharin. Other non-limiting examples of suspending agents include cyclodextrins and Captisol (sulfobutyl ether β-cyclodextrin; SEB-β-CD).

[0653] Oil suspensions can be prepared by suspending the active ingredient in a vegetable oil such as peanut oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. Oral suspensions can contain thickeners such as beeswax, hard paraffin or cetyl alcohol. Sweeteners (such as those mentioned above) and flavorings can be added to provide a palatable oral formulation. These compositions can be preserved by adding antioxidants such as ascorbic acid.

[0654] The dispersible powders and granules of the present invention suitable for preparing an aqueous suspension by adding water provide the active ingredient in admixture with a dispersant or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersants or wetting agents and suspending agents are exemplified by those disclosed above. Additional excipients such as sweeteners, flavorings, and coloring agents may also be present.

[0655] The pharmaceutical composition of the present invention can also be in the form of an oil-in-water emulsion. The oil phase can be a vegetable oil such as olive oil or peanut oil, a mineral oil such as liquid paraffin, or a mixture thereof. Suitable emulsifiers include naturally occurring gums such as gum arabic and gum tragacanth, naturally occurring phospholipids such as soy lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion can also contain sweeteners and flavorings. Syrups and elixirs can be formulated with sweeteners such as glycerol, sorbitol, or sucrose. Such preparations can also contain a demulcent, a preservative, a flavoring, or a coloring agent.

[0656] The pharmaceutical compositions of the present invention may be in the form of sterile injectable formulations, such as sterile injectable aqueous or oily suspensions. The suspensions may be prepared according to known techniques using suitable dispersants or wetting agents and suspending agents as mentioned above. Sterile injectable formulations may also be sterile injectable solutions or suspensions in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol or prepared as a lyophilized powder. Among acceptable carriers and solvents, water, Ringer's solution, and isotonic sodium chloride solution may be used. In addition, sterile fixed oils may generally be used as solvents or suspending media. For this purpose, any bland fixed oil may be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may also be used to prepare injections. Among acceptable carriers and solvents, water, Ringer's solution, isotonic sodium chloride solution, and hypertonic sodium chloride solution may be used.

[0657] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific mode of administration. For example, a timed-release formulation intended for oral administration to humans may contain about 1-1000 mg of active substance mixed with a suitable and convenient amount of carrier material, which may account for about 5 to about 95% (weight:weight) of the total composition. Pharmaceutical compositions can be prepared to provide an easily measured dosage. For example, an aqueous solution for intravenous infusion may contain about 3 to 500 μg of active ingredient per milliliter of solution so that a suitable volume can be infused at a rate of about 30 mL / hour.

[0658] Preparations suitable for topical administration to the eye also include eye drops in which the active ingredient is dissolved or suspended in a suitable carrier, particularly an aqueous solvent for the active ingredient. The active ingredient is preferably present in such a preparation at a concentration of 0.5-20 w / w%, advantageously 0.5-10 w / w%, particularly about 1.5 w / w%.

[0659] Formulations suitable for topical administration in the mouth include lozenges containing the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles containing the active ingredient in an inert basis such as gelatin and glycerin or sucrose and acacia; and mouthwashes containing the active ingredient in a suitable liquid carrier.

[0660] Formulations for rectal administration may be presented as a suppository with a suitable base containing for example cocoa butter or a salicylate.

[0661] Formulations suitable for intrapulmonary or intranasal administration have, for example, a particle size in the range of 0.1-500 microns, such as 0.5, 1, 30, 35 microns, etc., which are rapidly inhaled through the nasal passages or administered by oral inhalation to reach the alveoli. Suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol or dry powder administration can be prepared according to conventional methods and can be delivered with other therapeutic agents, such as compounds previously used to treat or prevent Arenaviridae infections described below.

[0662] Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

[0663] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

[0664] The preparation is presented in unit dose or multidose containers (e.g., sealed ampoules and vials) and can be stored under freeze-dried (lyophilized) conditions, requiring only the addition of a sterile liquid carrier (e.g., water for injection) immediately before use. Instant injection solutions and suspensions are prepared from sterile powders, granules, and tablets of the aforementioned types. Preferred unit dose formulations are preparations of the active ingredient containing a daily dose or unit daily subdose as described above, or an appropriate portion thereof.

[0665] It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

[0666] The present invention further provides veterinary compositions comprising at least one active ingredient as defined above and a veterinary carrier.

[0667] Veterinary carriers are materials used for the purpose of administering the composition and may be solid, liquid or gaseous materials which are inert or acceptable in the veterinary field and are compatible with the active ingredient. These veterinary compositions may be administered orally, parenterally or by any other desired route.

[0668] The compounds of the invention are useful in providing controlled-release pharmaceutical formulations ("controlled-release formulations") containing one or more compounds of the invention as active ingredients, wherein the release of the active ingredient is controlled and regulated to allow less frequent dosing or to improve the pharmacokinetic or toxicity profile of a given active ingredient.

[0669] IV. Route of Administration

[0670] One or more compounds of the present invention (referred to herein as active ingredients) are administered by any route appropriate to the condition to be treated. Suitable routes include oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), vaginal, and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural) and the like. It will be appreciated that the preferred route may vary depending on, for example, the condition of the recipient. An advantage of the compounds of the present invention is that they have oral bioavailability and can be administered orally.

[0671] In the methods of the present invention for treating Arenaviridae infections, the compounds of the present invention can be administered at any time to a human who may come into contact with a human suffering from an Arenaviridae infection or to a human who already has an Arenaviridae infection. In some embodiments, the compounds of the present invention can be administered prophylactically to a human who has come into contact with a human suffering from an Arenaviridae infection. In some embodiments, the compounds of the present invention can be administered to a human who has tested positive for an Arenaviridae infection but has not yet displayed symptoms of an Arenaviridae infection. In some embodiments, the compounds of the present invention can be administered to a human at the onset of symptoms of an Arenaviridae infection.

[0672] The effective dose of the active ingredient depends at least on the nature of the condition being treated, toxicity, whether the compound is being used prophylactically (lower doses) or against an active viral infection, the method of delivery, and the pharmaceutical formulation, and will be determined by the clinician using conventional dose escalation studies. It can be expected that about 0.0001 to about 100 mg per kilogram of body weight per day; typically about 0.01 to about 10 mg per kilogram of body weight per day; more typically about 0.01 to about 5 mg per kilogram of body weight per day; and most typically about 0.05 to about 0.5 mg per kilogram of body weight per day. For example, a candidate daily dose for an adult of about 70 kg body weight will be in the range of 1 mg to 1000 mg, preferably in the range of 5 mg to 500 mg, and can take the form of a single dose or multiple doses.

[0673] The effective dosage of the compounds of the present invention for treating Arenaviridae infections may depend on whether the dosage is used prophylactically or to treat a person already suffering from an Arenaviridae infection. Furthermore, the dosage may depend on whether the person suffering from an Arenaviridae infection is not yet symptomatic or is already showing symptoms of an Arenaviridae infection. Larger dosages may be necessary to treat a person who tests positive for an Arenaviridae infection and a person who is showing symptoms of an Arenaviridae infection, compared to a person receiving prophylactic treatment.

[0674] Any suitable time period for administering the compounds of this invention is contemplated. For example, administration can be 1 day to 100 days, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80 or 90 days. Administration can also be 1 week to 15 weeks, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 weeks. Longer administration times are also contemplated. Administration time can depend on whether the compound is administered prophylactically or the treatment is suffering from the people of Arenaviridae infection. For example, prophylactic administration can be a period of time that people and other people with Arenaviridae infection are frequently in contact with and in the appropriate time period after the last contact with the people of Arenaviridae infection. For humans who have already suffered from Arenaviridae infection, administration time can be any time length required for treating the patient, and the appropriate time period after the negative test of Arenaviridae infection to ensure that Arenaviridae infection will not recover.

[0675] V. Combination Therapy

[0676] The compositions of the present invention are also used in combination with other active ingredients. In order to treat Arenaviridae viral infections, preferably, the other active therapeutic agents are active against Arenaviridae viral infections, particularly Lassa virus and Junin virus infections. Non-limiting examples of these other active therapeutic agents are ribavirin, favipiravir (also known as T-705 or Avigan), T-705 monophosphate, T-705 diphosphate, T-705 triphosphate, ST-193, and mixtures thereof. The compounds and compositions of the present invention are also intended for use in providing general care for patients with Arenaviridae viral infections, including: parenteral fluids (including dextrose saline and lactated Ringer's solution) and nutrition, antibiotics (including metronidazole and cephalosporin antibiotics such as ceftriaxone and cefuroxime) and / or antifungal prophylaxis, fever and analgesics, antiemetics (e.g., metoclopramide) and / or antidiarrheals, vitamin and mineral supplements (including vitamin K and zinc sulfate), anti-inflammatory agents (e.g., ibuprofen), analgesics, medications for other common ailments in the affected population, such as antimalarials (including artemether and artesunate-lumefantrine combination therapy), typhoid fever (including quinolone antibiotics such as ciprofloxacin, macrolide antibiotics such as azithromycin, cephalosporin antibiotics such as ceftriaxone, or aminopenicillins such as ampicillin), or shigellosis.

[0677] Any compound of the present invention can also be combined with one or more other active therapeutic agents in a unit dosage form and administered to the patient simultaneously or sequentially. Combined therapy can be administered as a simultaneous or sequential regimen. When administered sequentially, the combination can be administered in two or more administrations.

[0678] Co-administration of a compound of the invention and one or more other active therapeutic agents generally refers to administering the compound of the invention and one or more other active therapeutic agents simultaneously or sequentially such that therapeutically effective amounts of both the compound of the invention and one or more other active therapeutic agents are present in the patient's body.

[0679] Co-administration is included in administering the compounds of this invention of unit dose before or after one or more other active therapeutic agents of a unit dose, for example, administering the compounds of this invention within a few seconds, minutes or hours of administering one or more other active therapeutic agents. For example, the compounds of this invention of a unit dose can be first administered, and then the one or more other active therapeutic agents of a unit dose are administered within a few seconds or minutes. Alternatively, one or more other therapeutic agents of a unit dose can be first administered, and then the compounds of this invention of a unit dose are administered within a few seconds or minutes. In some cases, it may be necessary to first administer the compounds of this invention of a unit dose, and then after a few hours (such as 1-12 hours), administer one or more other active therapeutic agents of a unit dose. In other cases, it may be necessary to first administer one or more other active therapeutic agents of a unit dose, and then after a few hours (such as 1-12 hours), administer the compounds of this invention of a unit dose.

[0680] Combination therapy can provide "synergy" and "synergism," i.e., the effect obtained when the active ingredients are used together is greater than the sum of the effects produced by the compounds when used alone. A synergistic effect can be obtained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combination formulation; (2) delivered as separate formulations in alternation or in parallel; or (3) by other regimens. When delivered in alternation therapy, a synergistic effect can be obtained when the compounds are administered or delivered sequentially, for example, in separate tablets, pills, or capsules, or by different injections in separate syringes. Generally, during alternation therapy, an effective dose of each active ingredient is administered sequentially, i.e., serially, whereas in combination therapy, effective doses of two or more active ingredients are administered together. A synergistic antiviral effect refers to an antiviral effect that is greater than the predicted purely additive effect of the individual compounds in the combination.

[0681] In another embodiment, the present application provides a method for inhibiting Arenaviridae polymerase in cells, comprising: contacting cells infected with Arenaviridae with an effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt, solvate and / or ester thereof, thereby inhibiting Arenaviridae polymerase.

[0682] In another embodiment, the present application provides a method for inhibiting Arenaviridae polymerase in a cell, comprising: contacting an Arenaviridae-infected cell with an effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt, solvate and / or ester thereof and at least one additional active therapeutic agent, thereby inhibiting Arenaviridae polymerase.

[0683] In another embodiment, the present application provides a method for inhibiting Arenaviridae polymerase in a cell, comprising contacting a cell infected with an Arenaviridae virus with an effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt, solvate and / or ester thereof and at least one selected additional active therapeutic agent.

[0684] In another embodiment, the present application provides a method for treating Arenaviridae virus infection in humans, comprising: administering to the patient a therapeutically effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt, solvate and / or ester thereof.

[0685] In another embodiment, the present application provides a method for treating Arenaviridae viral infection in a human, comprising: administering to the patient a therapeutically effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt, solvate and / or ester thereof and at least one additional active therapeutic agent, wherein the Arenaviridae polymerase is inhibited.

[0686] In another embodiment, the present application provides a method for treating Arenaviridae virus infection in humans, comprising administering to the patient a therapeutically effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt, solvate and / or ester thereof and at least one additional active therapeutic agent.

[0687] Also provided is a kit comprising a compound of formula I or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable ester, a stereoisomer, a mixture of stereoisomers or a tautomer. In a separate embodiment, a separate kit is provided comprising a compound selected from the group of each formula herein and each subgroup and embodiment thereof, including Formula II, Formula II, Formula IV and individual compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32 (compounds 1-32), or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable ester, a stereoisomer, a mixture of stereoisomers or a tautomer. In one aspect, the kit comprises a compound of formula I or a pharmaceutically acceptable salt thereof. Each separate kit described herein may include a label and / or instructions for using the compound to treat a disease or condition in an individual (e.g., a person) in need thereof. In some embodiments, the disease or condition is a human Arenaviridae virus infection, including Lassa virus infection or Junin virus infection. In other embodiments, each separate kit may further comprise instructions for using additional drugs in combination with the compound of Formula I for treating a disease or condition in an individual (e.g., a human) in need thereof. In some of these embodiments, the disease or condition is a human Arenaviridae virus infection, including Lassa virus infection or Junin virus infection. In each kit herein, there is another embodiment in which the kit comprises a single dose of a compound as described herein, or a pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, tautomer, polymorph, pseudopolymorph, amorphous form, hydrate, or solvate thereof. Examples of individual dosage units may include pills, tablets, capsules, prefilled syringes or syringe cartridges, intravenous bags, and the like, each containing a therapeutically effective amount of the compound or a pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, tautomer, polymorph, pseudopolymorph, amorphous form, hydrate, or solvate thereof. In some embodiments, the kit may contain a single dosage unit, and in other embodiments, a plurality of dosage units may be present, such as the number of dosage units required for a particular regimen or period.

[0688] Also provided are articles comprising a compound of Formula I or a pharmaceutically acceptable salt, pharmaceutically acceptable ester, stereoisomer, stereoisomer, or mixture of tautomers thereof; and a container. In one aspect, the article comprises a compound of Formula I, Formula II, Formula II, Formula IV and individual compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32 (Compounds 1-32) or pharmaceutically acceptable salts thereof, and a container. In a separate embodiment, the container of the article can be a vial, a can, an ampoule, a preloaded syringe, a blister pack, a tin, a can, a bottle, a box, or an intravenous bag.

[0689] Also provided as a separate embodiment is the use of a compound selected from the formulae herein, and each subgroup and embodiment thereof, in the preparation of a medicament for treating human Arenaviridae infections, including one of the specific compounds selected from Formula (I), Formula (II), Formula (III), Formula (IV), or the Examples herein, including Compounds 1-32 or pharmaceutically acceptable salts, solvates, and / or esters thereof.

[0690] VI. Methods for Inhibiting Arenaviridae Polymerases

[0691] Another aspect of the present invention relates to a method for inhibiting Arenaviridae polymerase activity, comprising the step of treating a sample suspected of containing Arenaviridae with a compound or composition of the present invention.

[0692] Arenaviruses that can be treated using the methods of the present invention are single-stranded, negative-sense RNA viruses that typically infect primates. Arenaviruses are able to reproduce in virtually all cell types.

[0693] Based on studies of nonhuman primates infected with Lassa virus, the first cells to be infected appear to be dendritic cells in lymphoid tissue. The infection progresses to Kupffer cells in the liver, parenchymal cells in the liver and adrenal glands, endothelial cells in various tissues including neural tissue, and finally to epithelial cells. Evidence of hepatitis caused by liver infection in humans has been documented (Hensley, L., 2011, Virology Journal; Yun, NE, 2012, Viruses).

[0694] A total of 30 species of arenaviruses have been identified: Allpahuayo virus (ALLV), Amapari virus (AMAV), Bear Canyon virus (BCNV), Catarina virus, Chapare virus, Cupiqui virus (CPXV), Dandenong virus, Flexal virus (FLEV), Guanarito virus (GTOV), Ippy virus (IPPYV), Junin virus (JUNV), Kodoko virus, Lassa virus (LASV; six strains - Josiah, NL, z148, Macenta, Arenaviruses are heterogeneous, ranging in diameter from 40 nm to greater than 200 nm, and consist of a nucleocapsid surrounded by a lipid envelope. Electron micrographs of the virion interior reveal a characteristic granular appearance due to the incorporation of host cell ribosomes into the viral particle during assembly. The arenavirus genome consists of two single-stranded RNA segments, small (S) and large (L). Both genomic segments have an ambiguous gene organization and encode two genes in opposite orientations. The L RNA (~7 kb) encodes the viral RNA-dependent RNA polymerase (L) and the small ring finger zinc-binding protein (Z). The S RNA (-3.4 kb) encodes the glycoprotein precursor protein (GPC) and the nucleoprotein (NP). GPC is post-translationally cleaved to produce the two envelope glycoproteins GP1 and GP2 and a stable signal peptide (SSP) (Yun, NE, 2012 Viruses).

[0695] The compositions of the present invention can be used as inhibitors of Arenaviridae polymerase, as intermediates for such inhibitors, or have other uses as described below. The inhibitor will bind to a location on the surface or within a cavity of the Arenaviridae polymerase, which has the unique geometry of the Arenaviridae polymerase. Compositions that bind to Arenaviridae polymerase can bind with varying degrees of reversibility. Compounds that bind substantially irreversibly are ideal candidates for use in the methods of the present invention. Once labeled, compositions that bind substantially irreversibly can be used as probes for detecting Arenaviridae polymerase. Thus, the present invention relates to a method for detecting Arenaviridae polymerase in a sample suspected of containing Arenaviridae polymerase, comprising the steps of: treating the sample suspected of containing Arenaviridae polymerase with a composition containing a compound of the present invention conjugated to a label; and observing the effect of the sample on the activity of the label. Suitable labels are well known in the diagnostic art and include stable free radicals, fluorophores, radioisotopes, enzymes, chemiluminescent groups, and chromophores. The compounds herein are labeled in a conventional manner using functional groups such as hydroxyl, carboxyl, sulfhydryl, or amino groups.

[0696] Within the scope of the present invention, samples suspected of containing Arenaviridae polymerases include natural or artificial materials, such as living organisms; tissue or cell cultures; biological samples, such as biological material samples (blood, serum, urine, cerebrospinal fluid, tears, sputum, saliva, tissue samples, etc.); laboratory samples; food, water, or air samples; biological product samples, such as cell extracts, particularly recombinant cells that synthesize the desired glycoprotein; and the like. Typically, the sample will be suspected of containing an organism that produces Arenaviridae polymerases, typically a pathogenic organism such as an Arenaviridae virus. The sample can be contained in any medium, including water and organic solvent / water mixtures. Samples include living organisms, such as humans, and artificial materials, such as cell cultures.

[0697] The treating step of the present invention comprises adding the composition of the present invention to the sample, or it comprises adding a precursor of the composition to the sample. The adding step comprises any of the administration methods described above.

[0698] If desired, the activity of the Arenaviridae polymerase after application of the composition can be observed by any method, including direct and indirect methods for detecting Arenaviridae polymerase activity. Quantitative, qualitative, and semi-quantitative methods for determining Arenaviridae polymerase activity are contemplated. Typically, one of the above-described screening methods is employed; however, any other method, such as observing the physiological properties of a living organism, is also suitable.

[0699] Organisms containing Arenaviridae polymerases include Arenaviridae viruses. The compounds of the present invention are useful for treating or preventing Arenaviridae infections in animals or humans.

[0700] However, when screening for compounds that inhibit human Arenaviridae, it should be remembered that results from enzyme assays may not correlate with those from cell culture assays. Therefore, cell-based assays should be the primary screening tool.

[0701] In another embodiment, the present application provides a method for treating an Arenaviridae infection in a human, comprising administering to the patient a therapeutically effective amount of a compound of Formula I-IV or a pharmaceutically acceptable salt, solvate, and / or ester thereof. In some embodiments, the Arenaviridae infection is caused by an Arenaviridae virus. In some embodiments, the Arenaviridae infection is caused by Junin virus. In some embodiments, the Arenaviridae infection is caused by Lassa virus strains Josiah, NL, z148, Macenta, AV, or CSF. In some embodiments, the Arenaviridae polymerase is inhibited.

[0702] The compounds of the present invention can be used to treat patients already infected with an Arenaviridae virus, or they can be administered prophylactically to reduce or prevent the onset of Arenaviridae infection. Physical examination of patients infected with an Arenavirus after a fever often reveals purulent pharyngitis, bilateral conjunctival hemorrhages, facial edema, and generalized abdominal tenderness. Gross pathological changes may include pleural effusions, pulmonary edema, ascites, and hemorrhagic findings in the gastrointestinal mucosa. The mortality rate for hospitalized patients varies between 5-10%.

[0703] VII. Screening for Arenaviridae Polymerase Inhibitors

[0704] The compositions of the present invention are screened for inhibitory activity against Arenaviridae polymerase by any conventional technique for evaluating enzyme activity. Within the scope of the present invention, compositions are typically first screened for inhibition of Arenaviridae polymerase in vitro, and compositions showing inhibitory activity are then screened for in vivo activity. -6 M and is preferably less than about 1×10 -7 Compositions with an in vitro Ki (inhibition constant) of M are preferred for in vivo use.

[0705] Useful in vitro screens have been described in detail and will not be described in detail here. However, these examples describe suitable in vitro assays.

[0706] VIII. Preparation of Compounds

[0707] The compounds of the present invention can be prepared by various means. For example, the protected nucleoside of formula V can be prepared by reacting the protected lactone with an iodine-substituted base under suitable coupling conditions. The compounds of the present invention can then be obtained by reacting the partially protected nucleoside with a suitable prodrug moiety, modifying the nucleoside with the prodrug moiety, and then removing the protecting group.

[0708] A. Preparation of Nucleosides by Iodine-Base

[0709] In some embodiments, the present invention provides a method for preparing a compound of formula V:

[0710]

[0711] The method for preparing the compound of formula V comprises forming a coupling agent, a halosilane, and a compound of formula VI under conditions suitable for preparing the compound of formula V:

[0712]

[0713] and a reaction mixture of a compound of formula VII:

[0714]

[0715] Wherein each PG is independently a hydroxy protecting group, or two PG groups on adjacent carbons can be combined to form -C(R 19 ) 2- Group, R 10 is H or silyl, and R 19 is H, C1-C8 alkyl, phenyl or substituted phenyl.

[0716] Any suitable coupling agent can be used in the method for preparing the compound of formula V. The coupling agent can be a lithium coupling agent, a sodium coupling agent, a magnesium coupling agent, etc. For example, the coupling agent can be a deprotonating agent such as n-butyllithium (n-BuLi), sodium hydride (NaH), lithium aluminum hydride (LAH or LiAlH4), etc. The coupling agent can also be a coupling agent based on magnesium, such as, but not limited to, MgCl2, iPrMgCl, tBuMgCl, PhMgCl, or a combination thereof. In some embodiments, the coupling agent can be a lithium coupling agent or a magnesium coupling agent. In some embodiments, the coupling agent can be n-BuLi, MgCl2, iPrMgCl, tBuMgCl, PhMgCl, or a combination thereof. In some embodiments, the coupling agent can be n-BuLi. In some embodiments, the coupling agent can be PhMgCl and iPrMgCl.

[0717] The coupling agent can be present in any suitable amount. For example, the coupling agent can be present in an amount of at least 1.0 equivalents (mol / mol) of a compound of formula V, for example, about 1.0, 2, 3, 4, 5, 6, 7, 8, 9 or about 10.0 equivalents (mol / mol). The coupling agent can also be present in an amount of about 1.0 to about 10.0 equivalents (mol / mol) of a compound of formula V, for example, about 1.0 to about 5.0 equivalents (mol / mol), or about 1.0 to about 2.0 equivalents (mol / mol). In some embodiments, the coupling agent can be present in an amount of about 1.0 to about 5.0 equivalents (mol / mol) of a compound of formula V. In some embodiments, the coupling agent can be present in an amount of about 1.0 to about 2.0 equivalents (mol / mol) of a compound of formula V.

[0718] Any suitable halosilane can be used in the method for preparing the compound of formula V. For example, the halosilane can be a fluorosilane, a chlorosilane, a bromosilane, or an iodosilane. The silane moiety can have any suitable substituent, such as an alkyl, alkenyl, alkynyl, cycloalkyl, or phenyl group. Exemplary halosilanes include, but are not limited to, Cl-Si(CH3)3 or Cl-Si(CH3)2CH2CH2Si(CH3)2-Cl. In some embodiments, the halosilane can be a chlorosilane. In some embodiments, the halosilane can be Cl-Si(CH3)3 or Cl-Si(CH3)2CH2CH2Si(CH3)2-Cl. In some embodiments, the halosilane can be TMS-Cl.

[0719] R 10 The silyl group of can be any suitable group, but can depend on the choice of halosilane. For example, when the halosilane is TMS-C1, the silyl group can be trimethylsilyl.

[0720] Halosilanes can exist in any suitable amount.For example, halosilanes can exist in the amount of at least 1.0 equivalents (mol / mol) of a compound of formula V, for example, approximately 1.0, 2, 3, 4, 5, 6, 7, 8, 9 or approximately 10.0 equivalents (mol / mol). Halosilanes can also exist in the amount of approximately 1.0 to approximately 10.0 equivalents (mol / mol) of a compound of formula V, for example, approximately 1.0 to approximately 5.0 equivalents (mol / mol), or approximately 1.0 to approximately 2.0 equivalents (mol / mol). In some embodiments, halosilanes can exist in the amount of approximately 1.0 to approximately 5.0 equivalents (mol / mol) of a compound of formula V. In some embodiments, halosilanes can exist in the amount of approximately 1.0 to approximately 2.0 equivalents (mol / mol) of a compound of formula V.

[0721] The hydroxy protecting group can be any protecting group suitable for the hydroxy functional group. Representative hydroxy protecting groups include but are not limited to silanes such as trimethylsilane (TMS), tert-butyldimethylsilane (TBDMS) or tert-butyldiphenylsilane (TBDPS); ethers such as methyl-methoxy (MOM), tetrahydropyran (THP), tert-butyl, allyl or benzyl; and esters such as acetyl, pivaloyl or benzoyl. In some embodiments, the hydroxy protecting group can be trimethylsilane (TMS), tert-butyldimethylsilane (TBDMS), tert-butyldiphenylsilane (TBDPS), methyl-methoxy (MOM), tetrahydropyran (THP), tert-butyl, allyl, benzyl, acetyl, pivaloyl or benzoyl. In some embodiments, the hydroxy protecting group can be benzyl.

[0722] The hydroxyl groups on adjacent carbons, known as 1,2-hydroxyl groups, can react with diether ketones to form cyclic protecting groups known as acetonides. Exemplary acetonides include, but are not limited to, acetonides and benzylidene acetal. In some embodiments, the hydroxyl protecting groups of the hydroxyl groups on adjacent carbons can combine to form acetonides.

[0723] When R 19 When the group is C1-C8 alkyl, R 19 It can be methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, neohexyl, heptyl or octyl. In some embodiments, R 19 The group may be methyl.

[0724] Any suitable solvent can be used in the method for the present invention.Representational solvent includes but is not limited to pentane, pentanes, hexane, hexanes, heptane, heptane, sherwood oil, cyclopentane, hexamethylene, benzene, toluene, dimethylbenzene, trifluoromethylbenzene, halogenated benzene such as chlorobenzene, fluorobenzene, dichlorobenzene and difluorobenzene, methylene dichloride, chloroform, acetone, ethyl acetate, ether, tetrahydrofuran (THF) or its combination.In some embodiments, solvent can be tetrahydrofuran (THF).Other representative solvents include but are not limited to 2-methyltetrahydrofuran, dibutyl ether, methyl tert-butyl ether, dimethoxyethane, dioxane (1.4 dioxanes), N-Methyl pyrrolidone (NMP) or its combination.

[0725] The reaction mixture of the method can be at any suitable temperature. For example, the temperature of the reaction mixture can be from about -78°C to about 100°C, or from about -50°C to about 100°C, or from about -25°C to about 50°C, or from about -10°C to about 25°C, or from about 0°C to about 20°C. In some embodiments, the temperature of the reaction mixture can be from about 0°C to about 20°C. In some embodiments, the temperature of the reaction mixture can be from about -30°C to about -10°C.

[0726] The reaction mixture of the method can be at any suitable pressure. For example, the reaction mixture can be at atmospheric pressure. The reaction mixture can also be exposed to any suitable environment, such as atmospheric atmosphere or an inert gas such as nitrogen or argon.

[0727] The methods of the invention can provide compounds of Formula V in any suitable yield. For example, compounds of Formula V can be prepared in a yield of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least about 95%.

[0728] The methods of the present invention can provide compounds of Formula V in any suitable purity. For example, compounds of Formula V can be prepared in a purity of at least about 90%, 95%, 96%, 97%, 98%, or at least about 99%. In some embodiments, compounds of Formula V can be prepared in a purity of at least 95%. In some embodiments, compounds of Formula V can be prepared in a purity of at least 98%. In some embodiments, compounds of Formula V can be prepared in a purity of at least 99%.

[0729] In some embodiments, the method comprises preparing a compound of formula V:

[0730]

[0731] The method comprises forming a compound of formula VI with TMS-Cl, PhMgCl, iPrMgCl under conditions suitable for preparing a compound of formula V:

[0732]

[0733] and a reaction mixture of a compound of formula VII:

[0734]

[0735] In some embodiments, the present invention provides compounds

[0736]

[0737] B. Addition of Prodrug Moieties

[0738] The present invention also provides methods of coupling a prodrug moiety to a nucleoside to provide a compound of the present invention. In some embodiments, the present invention provides a method of preparing a compound of formula VIII:

[0739]

[0740] The method comprises forming a reaction mixture comprising a coupling agent, a non-nucleophilic base, and a compound of formula IX under conditions suitable for forming a compound of formula VIII:

[0741]

[0742] and a reaction mixture of a compound of formula X:

[0743]

[0744] Each R a is H or PG, each PG group is a hydroxyl protecting group, or two PG groups are combined to form C(R 19 )2-,R e1 and R e2 Each independently represents H, C1-C6 alkyl or benzyl, R f is H, C1-C8 alkyl, benzyl, C3-C6 cycloalkyl or -CH2-C3-C6 cycloalkyl, R 19 is H, C1-C8 alkyl, phenyl or substituted phenyl, and LG is a leaving group.

[0745] As described above for the method of preparing the compound of formula V, any suitable coupling agent can be used in the method of preparing the compound of formula VIII. In some embodiments, the coupling agent can be a magnesium coupling agent. In some embodiments, the coupling agent can be MgCl2, iPrMgCl, tBuMgCl, PhMgCl or a combination thereof. In some embodiments, the coupling agent can be MgCl2.

[0746] Any suitable non-nucleophilic base can be used in the method for preparing the compound of formula VIII. Representative non-nucleophilic bases include, but are not limited to, triethylamine, diisopropylethylamine, N,N-diethylaniline, pyridine, 2,6-lutidine, 2,4,6-collidine, 4-dimethylaminopyridine, and quinuclidine. In some embodiments, the non-nucleophilic base can be diisopropylethylamine (DIPEA).

[0747] As described above with respect to the method for preparing the compound of formula V, the protecting group PG can be any suitable hydroxy protecting group. An exemplary protecting group PG can be benzyl, or the PG groups can be combined to form an acetonide. Exemplary acetonides include, but are not limited to, acetonides and benzylidene acetal. In some embodiments, the hydroxy protecting groups of hydroxy groups on adjacent carbons can be combined to form an acetonide. In some embodiments, the PG groups are combined to form -C(R 19 ) 2- In some embodiments, each R a is a protecting group PG in which the PG group is bonded to form -C(Me)2.

[0748] When R e When the group is a C1-C8 alkyl group, each R eR may be methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, neohexyl, heptyl, or octyl. e The group may be methyl.

[0749] When R f When the group is a C1-C8 alkyl group, R f Can be methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, neohexyl, heptyl or octyl. In some embodiments, R f The group may be methyl, ethyl, isopropyl, tert-butyl or isohexyl. f When the group is a C3-C6 cycloalkyl group, R f Can be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, R f It may be cyclobutyl, cyclopentyl or cyclohexyl.

[0750] When R 19 When the group is a C1-C8 alkyl group, R 19 Can be methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, isohexyl, neohexyl, heptyl or octyl. In some embodiments, R 19 The group may be methyl.

[0751] The leaving group can be any suitable leaving group. Suitable leaving groups LG include, but are not limited to, chloride, bromide, mesylate, tosylate, triflate, 4-nitrobenzenesulfonate, 4-chlorobenzenesulfonate, 4-nitrophenoxy, pentafluorophenoxy, and the like. In some embodiments, the leaving group LG can be 4-nitrophenoxy or pentafluorophenoxy. In some embodiments, the leaving group LG can be 4-nitrophenoxy.

[0752] In some embodiments, each R a is where the PG groups combine to form C(R 19 )2-PG, R f is a C1-C8 alkyl group, R 19 is a C1-C8 alkyl group, and the leaving group LG is a 4-nitrophenoxy group or a pentafluorophenoxy group.

[0753] In some embodiments, the coupling agent is MgCl 2 and the non-nucleophilic base is diisopropylethylamine.

[0754] In some embodiments, the compound of formula VIII can be

[0755]

[0756] In some embodiments, the compound of formula VIII can be

[0757]

[0758] In some embodiments, the compound of formula VIII can be

[0759]

[0760] In some embodiments, the method of preparing a compound of Formula VIII comprises forming a reaction mixture comprising MgCl, DIPEA, a compound of Formula IX, and a compound of Formula X under conditions suitable for the formation of a compound of Formula VIII:

[0761]

[0762] When R a When the group is a hydroxy protecting group PG, the method may comprise removing the protecting group to form a a is H. In some embodiments, the method of preparing the compound of Formula VIII comprises the step of forming a compound of Formula VIII wherein each R a is H under the conditions of the compound of formula VIII to form a compound comprising a deprotecting agent and a compound of formula VIII (wherein each R a The second reaction mixture wherein the protecting group is a protecting group PG. The deprotecting agent can be any suitable reagent for removing the protecting group PG such as hydrogen and a hydrogenation catalyst or an acid. For example, if the protecting group PG is a benzyl group, the deprotecting agent can be hydrogen and platinum on carbon. Alternatively, when the protecting group PG is an acetonide, the deprotecting agent can be an acid. Representative acids include but are not limited to acetic acid, glacial acetic acid, trifluoroacetic acid (TFA), hydrochloric acid, concentrated hydrochloric acid, and the like. In some embodiments, the method for preparing a compound of formula VIII comprises preparing a compound suitable for forming a compound wherein each R a is H under conditions to form a compound of formula VIII comprising an acid and a compound of formula VIII (wherein R a The groups combine to form C(R 19 ) 2- In some embodiments, the acid can be hydrochloric acid.

[0763] Any suitable solvent can be used in the method of the present invention. Representative solvents include but are not limited to pentane, pentanes, hexane, hexanes, heptane, heptanes, petroleum ether, cyclopentane, cyclohexane, benzene, toluene, xylene, trifluorotoluene, halogenated benzenes such as chlorobenzene, fluorobenzene, dichlorobenzene and difluorobenzene, methylene chloride, chloroform, acetone, ethyl acetate, ether, tetrahydrofuran, acetonitrile or a combination thereof. In some embodiments, the solvent can be acetonitrile.

[0764] The reaction mixture of the method can be at any suitable temperature. For example, the temperature of the reaction mixture can be from about -78°C to about 100°C, or from about -50°C to about 100°C, or from about -25°C to about 50°C, or from about -10°C to about 25°C, or from about 0°C to 20°C. In some embodiments, the temperature of the reaction mixture can be from about 0°C to about 20°C.

[0765] The reaction mixture of the method can be at any suitable pressure. For example, the reaction mixture can be at atmospheric pressure. The reaction mixture can also be exposed to any suitable environment, such as atmospheric atmosphere or an inert gas such as nitrogen or argon.

[0766] The methods of the present invention can provide the compound of formula VIII in any suitable yield. For example, the compound of formula VIII can be prepared in a yield of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least about 95%.

[0767] The methods of the present invention can provide compounds of Formula VIII in any suitable purity. For example, compounds of Formula VIII can be prepared in a purity of at least about 90%, 95%, 96%, 97%, 98%, or at least about 99%. In some embodiments, compounds of Formula VIII can be prepared in a purity of at least 95%. In some embodiments, compounds of Formula VIII can be prepared in a purity of at least 98%. In some embodiments, compounds of Formula VIII can be prepared in a purity of at least 99%.

[0768] In some embodiments, the present invention provides compounds

[0769] Example

[0770] Certain abbreviations and acronyms are used to describe the experimental details. Although most of them will be understood by those skilled in the art, Table 1 contains a list of many of these abbreviations and acronyms.

[0771] Table 1. List of abbreviations and acronyms

[0772] Abbreviations and meanings

[0773] Ac2O acetic anhydride

[0774] AIBN 2,2'-azobis(2-methylpropionitrile)

[0775] Bn benzyl

[0776] BnBr benzyl bromide

[0777] BSA bis(trimethylsilyl)acetamide

[0778] BzCl Benzoyl chloride

[0779] CDI Carbonyldiimidazole

[0780] DABCO 1,4-diazabicyclo[2.2.2]octane

[0781] DBN 1,5-diazabicyclo[4.3.0]non-5-ene

[0782] DDQ 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone

[0783] DBU 1,5-diazabicyclo[5.4.0]undec-5-ene

[0784] DCA dichloroacetamide

[0785] DCC Dicyclohexylcarbodiimide

[0786] DCM dichloromethane

[0787] DMAP 4-dimethylaminopyridine

[0788] DME 1,2-dimethoxyethane

[0789] DMTC1 Dimethoxytrityl chloride

[0790] DMSO dimethyl sulfoxide

[0791] DMTR 4,4'-dimethoxytrityl

[0792] DMF dimethylformamide

[0793] EtOAc

[0794] ESI electrospray ionization

[0795] HMDS Hexamethyldisilazane

[0796] HPLC High-pressure liquid chromatography

[0797] LDA lithium diisopropylamide

[0798] LRMS low-resolution mass spectrometry

[0799] MCPBA m-Chloroperbenzoic acid

[0800] MeCN Acetonitrile

[0801] MeOH methanol

[0802] MMTC Monomethoxytrityl chloride

[0803] m / z or m / e mass-to-charge ratio

[0804] MH + Mass plus 1

[0805] MH - Mass minus 1

[0806] MsOH methanesulfonic acid

[0807] MS or MS mass spectrometry

[0808] NBS N-bromosuccinimide

[0809] Ph Phenyl

[0810] rt or rt room temperature

[0811] TBAF Tetrabutylammonium fluoride

[0812] TMSCl trimethylsilyl chloride

[0813] TMSBr trimethylsilyl bromide

[0814] TMSI trimethylsilyl iodide

[0815] TMSOTf (trimethylsilyl) trifluoromethanesulfonate

[0816] TEA triethylamine

[0817] TBA Tributylamine

[0818] TBAP Tributylammonium pyrophosphate

[0819] TBSCl tert-Butyldimethylsilyl chloride

[0820] TEAB triethylammonium bicarbonate

[0821] TFA trifluoroacetic acid

[0822] TLC or TLC thin layer chromatography

[0823] Tr trityl

[0824] Tol 4-methylbenzoyl

[0825] Turbo Grignard 1:1 mixture of isopropylmagnesium chloride and lithium chloride

[0826] δ parts per million downfield from tetramethylsilane

[0827] A. Preparation of Compounds

[0828] Example 1. Ethyl (2S)-2-(chloro(phenoxy)phosphorylamino)propionate (chloride A)

[0829]

[0830] Alanine ethyl ester hydrochloride (1.69 g, 11 mmol) was dissolved in anhydrous CH2Cl2 (10 mL) and the mixture was cooled to 0°C under N2(g) and stirred. Phenyl dichlorophosphate (1.49 mL, 10 mmol) was added, and Et3N was then added dropwise over 10 min. The reaction mixture was then slowly warmed to room temperature and stirred for 12 hours. Anhydrous Et2O (50 mL) was added and the mixture was stirred for 30 minutes. The solid formed was removed by filtration and the filtrate was concentrated under reduced pressure. The residue was subjected to silica gel chromatography and eluted with a hexane solution of 0-50% EtOAc to obtain intermediate A (1.13 g, 39%). 1 H NMR (300MHz, CDCl3) δ7.39-7.27(m,5H),4.27(m,3H),1.52(m,3H),1.32(m,3H). 31 P NMR (121.4MHz, CDCl3) δ8.2,7.8.

[0831] Example 2. 2-Ethylbutyl (2S)-2-(chloro(phenoxy)phosphorylamino)propanoate (Chloride B)

[0832]

[0833] Alanine chlorophosphoramido-2-ethylbutyl ester B was prepared using the same procedure as for chloride A, except that alanine ethyl ester was replaced with alanine 2-ethylbutyl ester. This material was used crude in the next reaction. Treatment with methanol or ethanol formed a displacement product with the desired LCMS signal.

[0834] Example 3. Isopropyl (2S)-2-(Chloro(phenoxy)phosphorylamino)propionate (Chloride C)

[0835]

[0836] Alanine chlorophosphoramido isopropyl ester C was prepared using the same procedure as for chloride A, except that alanine isopropyl ester was used instead of alanine ethyl ester. This material was used crude in the next reaction. Treatment with methanol or ethanol formed a displacement product with the desired LCMS signal.

[0837] Example 4. (2R,3R,4S,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile (Compound 1)

[0838]

[0839] The preparation of (2R,3R,4S,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile is as follows.

[0840]

[0841] Under N2(g), commercial lactose (10 g, 23.8 mmol) was dissolved in anhydrous DMSO (30 mL). AC2O (20 mL) was added and the resulting reaction mixture was stirred at room temperature for 48 hours. The reaction mixture was poured into ice water (500 mL) and the mixture was stirred for 20 minutes. The mixture was extracted with EtOAc (3 × 200 mL), and then the combined organic extracts were washed with H2O (3 × 200 mL). The organic extracts were dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The residue was dissolved in CH2Cl2 and subjected to silica gel chromatography, eluted with a hexane solution of 25% EtOAc to obtain a lactone (9.55 g, 96%). 1 H NMR (400 MHz, DMSO) δ 7.30-7.34 (m, 13H), 7.19-7.21 (m, 2H), 4.55-4.72 (m, 6H), 4.47 (s, 2H), 4.28 (d, J = 3.9 Hz, 1H), 3.66 (m, 2H). LCMS m / z 436.1 [M+H2O], 435.2 [M+OH] - T = 2.82 min. HPLC T = 4.59 [2-98% ACN in H2] in 5 minutes at a flow rate of 2 ml / min.

[0842]

[0843] Under N2(g), bromopyrazole (prepared according to WO2009 / 132135) (0.5 g, 2.4 mmol) was suspended in anhydrous THF (10 mL). The suspension was stirred and TMSCl (0.67 mL, 5.28 mmol) was added. The mixture was stirred at room temperature for 20 minutes, then cooled to -78°C, after which a solution of nBuLi (6 mL, 1.6 N in hexane, 9.6 mmol) was slowly added. The reaction mixture was stirred at -78°C for 10 minutes, and then lactone (1 g, 2.4 mmol) was added via a syringe. When the reaction was complete as measured by LCMS, AcOH was added to quench the reaction. The mixture was concentrated under reduced pressure and the residue was dissolved in a mixture of CH2Cl2 and H2O (100 mL, 1:1). The organic layer was separated and washed with H2O (50 mL). The organic layer was then dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel eluting with 0-50% EtOAc in hexanes to afford the product as a 1:1 mixture of anomers (345 mg, 26% yield). LCMS m / z 553 [M+H].

[0844]

[0845] Hydroxy nucleoside (1.1g, 2.0mmol) is dissolved in anhydrous CH2Cl2 (40mL) and the solution is cooled to 0 ℃ under stirring under N2(g). TMSCN (0.931mL, 7mmol) is added and the mixture is stirred for another 10 minutes. TMSOTf (1.63mL, 9.0mmol) is slowly added to the reaction and the mixture is stirred for 1 hour. The reaction mixture is then diluted with CH2Cl2 (120mL) and a NaHCO3 aqueous solution (120mL) is added to quench the reaction. The reaction mixture is stirred for another 10 minutes and the organic layer is separated. The aqueous layer is extracted with dichloromethane (150 milliliters), and the combined organic extracts are dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue is dissolved in a minimum amount of CH2Cl2 and subjected to silica gel chromatography, eluted with a gradient of 0-75% EtOAc and hexane to obtain tribenzyl cyanide nucleoside, a mixture of end isomers (0.9g, 80%). 1H NMR(300MHz,CD3CN)δ7.94(s,0.5H),7.88(s,0.5H),7.29-7.43(m,13H),7.11-7.19(m,1H),6.82-6.88(m,1H),6.70-6.76(m,1H) LCMS m / z 562[M+H].

[0846]

[0847] Tribenzyl cyanide nucleoside (70 mg, 0.124 mmol) was dissolved in anhydrous CH2Cl2 (2 mL) and cooled to -78 ° C under N2 (g). A solution of BCl3 (1N in CH2Cl2, 0.506 mL, 0.506 mmol) was added and the reaction mixture was stirred at -78 ° C for 1 hour. When the reaction was completed by LC / MS, MeOH was added to quench the reaction. The reaction mixture was allowed to warm to room temperature and the solvent was removed under reduced pressure. The residue was subjected to C18 reverse phase HPLC and eluted with H2O (0.1% TFA) for 5 minutes, and then eluted with 0-70% MeCN in H2O solution (0.1% TFA) in a gradient of 35 minutes to elute the α-anomer (20 mg, 37%) and β-anomer 1 (20 mg, 37%). (α-anomer) 1 H NMR (300 MHz, D2O) δ 7.96 (s, 1H), 7.20 (d, J = 4.8 Hz, 1H), 6.91 (d, J = 4.8 Hz, 1H), 4.97 (d, J = 4.4 Hz, 1H), 4.56-4.62 (m, 1H), 4.08-4.14 (m, 1H), 3.90 (dd, J = 12.9, 2.4 Hz, 1H), 3.70 (dd, J = 13.2, 4.5 Hz, 1H). (β-anomer) 1H NMR (400MHz, DMSO) δ7.91 (s, 1H), 7.80-8.00 (br s, 2H), 6.85-6.89 (m, 2H), 6.07 (d, J = 6.0Hz, 1H), 5.17 (br s, 1H), 4.90 (br s,1H),4.63(t,J=3.9Hz,1H),4.02-4.06(m,1H),3.94(br s,1H),3.48-3.64(m,2H).LCMS m / z 292.2[M+H],290.0[MH].Tr=0.35min.13C NMR(400MHZ,DMSO),156.0,148.3,124.3,117.8,117.0,111.2,101.3,85.8,79.0,74.7,70.5,61.4.HPLCTr=1.32min.

[0848] Example 5. (2R,3R,4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile (Compound 2)

[0849]

[0850] The preparation of (2R,3R,4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile is as follows.

[0851]

[0852] 2-Deoxy-2-fluoro-4,5-O,O-dibenzyl-D-arabinose. 1'-Methoxy-2-deoxy-2-fluoro-4,5-O,O-dibenzyl-D-arabinose (1.0 g, 2.88 mmol) in TFA (13.5 mL) was treated with H2O (1.5 mL) and the resulting mixture was stirred for 5 hours. The mixture was then diluted with EtOAc (100 mL) and treated with saturated NaHCO3 (50 mL). The organic layer was separated and washed with NaCl (50 mL), dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The residue was subjected to silica gel chromatography (80 g SiO2 Combiflash HP Gold Column) and eluted with 0-100% EtOAc in hexane to give 2-deoxy-2-fluoro-4,5-O,O-dibenzyl-D-arabinose (695 mg, 72%) as a white solid: R f = 0.52 (25% EtOAc in hexane). 1H NMR (300MHz, CDCl3) δ7.30(m,10H),5.35(m,1H),4.68–4.29(m,7H),3.70(d,J=10.5Hz,1H),3.50(d,J=10.5Hz,2H). 19 F NMR (282.2MHz, CDCl3) δ-207(m), -211(m).LCMSm / z 350[M+H2O].

[0853]

[0854] (3R,4R,5R)-4-(Benzyloxy)-5-(Benzyloxymethyl)-3-fluorodihydrofuran-2(3H)-one. 2-Deoxy-2-fluoro-4,5-O,O-dibenzyl-D-arabinose (4.3 g, 12.8 mmol) was dissolved in CH2Cl2 (85 mL) and MS (10 g) and pyridinium dichromate (14.4 g, 38.3 mmol) were treated. The resulting mixture was stirred for 24 hours and then filtered through a pad of celite. The eluent was concentrated under reduced pressure and the residue was subjected to silica gel chromatography (120 g SiO2 HP Gold Combiflash Column) eluted with 0-100% EtOAc in hexane to give (3R, 4R, 5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorodihydrofuran-2(3H)-one (3.5 g, 83%) as a clear oil: R f = 0.25 (25% EtOAc in hexane). 1 HNMR (300MHz, CDCl3) δ7.37 (m, 10H), 5.45 (dd, J=49, 5.7, Hz, 1H), 4.85 (d, J=1 1.7Hz,1H),4.52(m,4H),4.29(d,J=5.4Hz,1H),2.08(dd,J=15.3,10.2Hz,2H). 19 F NMR (282.2 MHz, CDCl3) δ-216. LCMS m / z 348 [M+H2O]. HPLC (6–98% MeCN–H2O gradient, 0.05% TFA modifier) ​​t R = 5.29 min. Phenomenex Synergi 4M Hydro-RP 80A, 50 x 4.60 mm, 4 microns; flow rate 2 ml / min.

[0855]

[0856] (3R,4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-ol. A solution of 7-bromopyrrolo[1,2-f][1,2,4]-triazin-4-amine (68 mg, 0.319 mmol) in THF (1.4 mL) was treated with TMSCl (89 μL, 0.703 mmol) and the mixture was stirred for 2 hours. The mixture was then cooled to -78°C and treated with nBuLi (1.0 M in hexanes, 1.09 mL, 1.09 mmol). The solution was stirred for 30 minutes and then treated dropwise with a THF (1.4 mL) solution of (3R, 4R, 5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorodihydrofuran-2(3H)-one (106 mg, 0.319 mmol). The resulting mixture was stirred for 30 minutes and then added with AcOH (83 μL, 1.44 mmol) in THF (1.0 mL) to quench the reaction. The mixture was warmed to room temperature and then concentrated under reduced pressure. The residue was diluted with EtOAc (100 mL) and washed with saturated NaCl solution (50 mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel (40 g SiO2 HP Gold Combiflash Column) eluting with 0-100% EtOAc in hexanes followed by a 0-100% gradient (20% MeOH in EtOAc) in EtOAc to afford (3R,4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-ol as a white solid (68 mg, 44%, 60 / 40 α / β isomer mixture). f =0.32 (EtOAc). 1 H NMR(300MHz, CDCl3)δ8.05(s,1H),7.86(s,1H),7.81(s,1H),7.64(s,1H),7.26(m,1 0H),6.95(m,1H),6.71(m,1H),6.08(m,1H),5.34(m,1H),4.65(m,6H),4.71(m,2H). 19 FNMR (282.2 MHz, CDCl3) δ-211 (m). LCMS m / z 465 [M+H]. HPLC (6-98% MeCN-H2O gradient, 0.05% TFA modifier) ​​t R = 4.37 min (α-isomer), 4.54 min (β-isomer).

[0857]

[0858] (3R,4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-carbonitrile: A solution of (3R,4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-ol (195 mg, 0.42 mmol) in MeCN (1.4 mL) was treated with TMSCN (336 μL, 2.52 mmol) and In(OTf)3 (708 mg, 1.26 mmol). The solution was stirred at 70°C for 18 hours and then cooled to 0°C. The mixture was treated with saturated NaHCO3 solution (20 drops), then warmed to room temperature and diluted with EtOAc (100 mL) and H2O (50 mL). The organic layer was separated and washed with saturated NaCl solution (50 mL), dried over MgSO4, filtered and concentrated under reduced pressure. The residue was subjected to silica gel chromatography (40 g SiO2HP Gold Combiflash Column) eluting with 0-100% EtOAc in hexanes to give (3R,4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-carbonitrile as a white solid (110 mg, 55%, 60 / 40 α / β isomers). Data for both isomers: Rf = 0.53 (EtOAc). R f =0.53 (EtOAc). 1 H NMR (300MHz, CDCl3) δ8.01(s,1H),7.94(s,1H),7.30(m,10H),7.00(d,J=4.5Hz,1H),6.93(d,J=4.8Hz,1H),6.87(d,J=5.4H z,1H),6.70(d,J=4.8Hz,1H),5.85(dd,J=52,3.3Hz,1H),5.55(dd,J=53,4.5Hz,1H),4.71(m,7H),3.87(m,2H),3.72(m,2H). 19 F NMR (282.2 MHz, CDCl3) δ -196 (m), -203 (m). LCMS m / z 474 [M+H]. HPLC (6-98% MeCN-H2O gradient, 0.05% TFA modifier) ​​t R =4.98min.

[0859]

[0860] (2R,3R,4R,5R)-2-(4-Aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile (2) (3R,4R,5R)-2-(4-Aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-carbonitrile (110 mg, 0.23 mmol) was dissolved in CHCl (1.5 mL) and cooled to 0° C. The reaction mixture was treated with BCl (1.0 M in CHCl, 766 μL, 0.77 mmol) and stirred for 2 h. The mixture was then cooled to -78°C and treated with EtN (340 μL, 2.44 mmol), then treated with MeOH (2 mL), and then warmed to RT. The reaction was concentrated under reduced pressure and then co-evaporated with MeOH (3 × 5 mL). The residue was then suspended in H O (5 mL) and treated with NaHCO (1 g). The solution was stirred for 10 minutes and then concentrated under reduced pressure. The residue was filtered and washed with MeOH (3 × 10 mL) on a fritted glass funnel (thick), and the eluent was concentrated under reduced pressure. The residue was subjected to reverse phase HPLC (6-98% MeCN in H2O gradient with 0.05% TFA modifier) ​​to afford (2R,3R,4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile 2 as a white solid (16.8 mg, 25%) and the α-isomer. Data for the β-isomer: R f =0.13 (10% MeOH in EtOAc). 1 HNMR(300MHz,CD3OD)δ8.09(s,1H),7.28(d,J=5.1Hz,1H),7.17(d,J=5.1Hz,1H),5. 42(dd,J=53,3.3Hz,1H),4.20(m,2H),3.99(d,J=3.6Hz,1H),3.77(d,J=3.6Hz,1H). 19 F NMR (282.2 MHz, CDCl3) δ-197 (m). LCMS m / z 294 [M+H]. HPLC (2–98% MeCN–H2O gradient, 0.05% TFA modifier) ​​t R =1.49min.

[0861] Example 6. (2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-fluoro-2- (Hydroxymethyl)-5-methyltetrahydrofuran-3-ol (Compound 3)

[0862]

[0863] The preparation of (2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-fluoro-2-(hydroxymethyl)-5-methyltetrahydrofuran-3-ol is as follows.

[0864]

[0865] The starting nucleoside (prepared as described in the synthesis of compound 2) (0.355 g, 0.765 mmol) was dissolved in anhydrous THF (35 mL) and stirred under N2(g) and cooled to 0 ° C. A solution of methylmagnesium chloride (2 mL, 6 mmol) (3N in THF) was added and the resulting mixture was stirred overnight. Acetic acid (7 mmol) was added to quench the reaction and the solvent was then removed by rotary evaporation under reduced pressure. The residue was redissolved in CH2Cl2 and the solution was subjected to a silica gel plug to isolate the product (0.355 g) as a crude mixture. LC / MS (m / z: 480, M +1 ). The crude material was dissolved in anhydrous CH2Cl2 (20 mL) and placed under N2(g). The solution was stirred and treated with methanesulfonic acid (0.2 mL, 2.74 mmol). The reaction mixture was stirred at room temperature for 12 hours and then quenched by the addition of Et3N (3.5 mmol). The mixture was concentrated under reduced pressure and the residue was subjected to silica gel chromatography to obtain the methyl-substituted nucleoside (0.174 g, 0.377 mmol, 44% yield) as a 4:1 mixture of β- and α-anomers, respectively. 1 H NMR (300 MHz, CD3CN) major anomer δ 7.87 (s, 1H), 7.27-7.40 (m, 10H), 6.77 (d, J = 4.5 Hz, 1H), 6.70 (d, J = 4.5 Hz, 1H), 6.23 (br s, 2H), 5.53 (dd, J = 55, 3.3 Hz, 1H), 4.42-4.75 (m, 4H), 4.19-4.26 (m, 1H), 3.65-4.00 (m, 3H), 1.74 (d, J = 3.9 Hz, 3H). 19 F NMR (282.2 MHz, CD3CN) major anomer δ-207 (m, 1F). LCMS m / z 463 [M+H].

[0866]

[0867] Benzylated nucleoside substance (0.134 g, 0.290 mmol), Degussa catalyst (0.268 g) and AcOH (30 mL) were mixed together. H (g) was added to the reaction atmosphere and the reaction was stirred for 2 hours. The catalyst was removed by filtration and the mixture was concentrated under reduced pressure. Residue was dissolved in a minimum of H O and reversed-phase HPLC (C18hydrORP column) was performed to separate β-anomer 3 (0.086 g, 0.217 mmol, 57% yield). 1 H NMR(300MHz,D2O)δ7.87(s,1H),7.22(d,J=4.8Hz,1H),6.87(d,J=4.8Hz,1H),5.35(dd,J=54,3.6Hz,1H ),3.97-4.10(m,2H),3.81(dd,J=12.6,2.1Hz,1H),3.64(dd,J=12.6,4.8Hz,1H),1.65(d,J=4.2Hz,3H). 19 F NMR(282.2MHz,CD3CN)δ-207(m,1F).

[0868] The minor α anomer was characterized as follows. 1 H NMR(300MHz,D2O)δ7.86(s,1H),7.26(d,J=4.8Hz,1H),6.85(d,J=4.8Hz,1H),5.31(dd,J=54,3.9Hz,1H),4.39(ddd ,J=26.1,9.9,3.6Hz,2H),4.00-4.05(m,1H),3.90(dd,J=12.3,2.1Hz,1H),3.66(dd,J=12.6,4.8,1H),1.56(s,3H). 19 F NMR (282.2MHz, CD3CN) δ-198 (dd, J=54, 26Hz, 1F).

[0869] Example 7. (2R)-2-((((2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7- (4-fluoro-3-hydroxy-5-methyltetrahydrofuran-2-yl)-(methoxy)-(phenoxy)phosphorylamino)propionic acid isopropyl ester ( Compound 4)

[0870]

[0871] Nucleoside 3 (0.011g, 0.04mmol) is dissolved in trimethyl phosphate (2mL) and is cooled to 0 ℃.Mixture is stirred under N2(g) atmosphere and 1-methylimidazole (0.320mL, 5mmol) is added, then alanine monoisopropyl monophenol phosphoryl chloride C (0.240mL, 4.4mmol) is added.Reaction mixture is stirred at 0 ℃ for 2 hours, then it is slowly warmed to room temperature.Monitored by LC / MS simultaneously.When completing by LCMS, reaction mixture H2O (5mL) is processed, then concentrated under reduced pressure.Residue is dissolved in CH2Cl2 and carries out silica gel chromatography, eluted with 0-100%EtOAc in hexane.Collect product fractions and concentrate.Residue is carried out preparation HPLC, obtain alanine isopropyl monoamide prodrug 4, is a mixture of isomers (4.7mg, 0.003mmol, 6%). 1 H NMR(300MHz,CD3CN)δ7.87(s,1H),7.17-7.44(m,5H),6.71-6.83(m,2H),6.14(br,s,2H),5.38(dd,J=5 6,3.3Hz,1H),4.92-5.01(m,1H),3.86-4.46(m,6H),3.58(m,1H),1.73(m,3H),1.18-1.34(m,9H).LCMS m / z 552[M+H].

[0872] Example 8. (2R)-2-((((2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7- 4-Fluoro-3-hydroxy-5-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propionic acid ethyl ester (compound 5)

[0873]

[0874] Nucleoside 3 (0.026 g, 0.092 mmol) was dissolved in trimethyl phosphate (2 mL) and cooled to 0 ° C. The mixture was stirred under N2 (g) and 1-methylimidazole (0.062 mL, 0.763 mmol) was added, followed by chloride A (0.160 g, 0.552 mmol). The reaction mixture was stirred at 0 ° C for 2 hours and then slowly warmed to room temperature. H2O (5 mL) was added to quench the reaction, and the mixture was then concentrated under reduced pressure. The residue was dissolved in CH2Cl2 and subjected to silica gel chromatography, eluting with a 0-100% EtOAc hexane solution. The product fractions were collected and concentrated. The crude product was eluted with a 0-100% EtOAc hexane solution. The crude product was collected and concentrated under reduced pressure. The residue was subjected to preparative HPLC to obtain 5 (2.0 mg, 4% yield). LCMS m / z 538 [M+H].

[0875] Example 9. ((2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-fluoro- 3-Hydroxy-5-methyltetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate (Compound 6)

[0876]

[0877] Nucleoside 3 (0.022 g, 0.056 mmol) was dissolved in trimethyl phosphate (1 mL) and stirred under N2(g). Phosphorus oxychloride (0.067 mL, 0.73 mmol) was added and the mixture was stirred for 2 hours. The time when >80% of the monophosphate was formed was determined by monitoring on an analytical ion exchange column. A solution of tributylamine (0.44 mL, 1.85 mmol) and triethylammonium pyrophosphate (0.327 g, 0.72 mmol) dissolved in anhydrous DMF (1 mL) was added. The reaction mixture was stirred for 20 minutes and then quenched by adding a 1N aqueous solution of triethylammonium bicarbonate (5 mL). The mixture was concentrated under reduced pressure and the residue was redissolved in H2O. The solution was subjected to ion exchange chromatography to give the title product 6 (1.7 mg, 6% yield). LCMS m / z 521 [MH]. Tr = 0.41. HPLC ion exchange TR = 9.40 minutes.

[0878] Example 10. (2R,3R,5S)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3-hydroxy-5- (Hydroxymethyl)-tetrahydrofuran-2-carbonitrile (Compound 7)

[0879]

[0880] The preparation of (2R,3R,5S)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-carbonitrile is as follows.

[0881]

[0882] ((3αR,5S,6αR)-2,2-dimethyl-tetrahydrofuro[2,3-d][1,3]dioxolan-5-yl)methanol. The acetate starting material (1.2 g, 5.5 mmol) (J.Org.Chem.1985,50,3547, De Bernardo et al.) was dissolved in a 1:1 mixture of MeOH and THF (10 mL). 1N NaOH (aqueous solution) (10 mL) solution was added until the pH was 13. The reaction mixture was stirred for 2 hours and then neutralized to pH 8-9 by adding AcOH. The mixture was extracted with EtOAc (10×30 mL), and the combined organic extracts were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was subjected to silica gel chromatography, eluting with 0-70% EtOAc in hexane to give the desired product (866 mg, 90%). 1H NMR (300MHz, CDCl3) δ5.84(d,J=3.6Hz,1H),4.78(t,J=4.5Hz,1H),4.38(m,1H),3.93-3.54(m,2H),2.04-1.84(m,2H),1.52(s,3H),1.33(s,3H).

[0883]

[0884] (3αR,5S,6αR)-5-(benzyloxymethyl)-2,2-dimethyl-tetrahydrofuran [2,3-d] [1,3] dioxolane. Sodium hydride (188 mg, 7.46 mmol) was dissolved in anhydrous THF (5 mL) and stirred at room temperature under N2 (g). Alcohol (866 mg, 4.97 mmol) was dissolved in anhydrous THF (3 mL) and then added to the sodium hydride mixture in batches over 5 min. The resulting mixture was stirred for 20 minutes. Benzyl bromide (892 μL, 7.46 mmol) was then added. The reaction mixture was stirred for 2 hours and then poured into a mixture of ice-cold NaHCO3 aqueous solution and EtOAc (30 mL). The organic layer was separated and the aqueous layer was re-extracted with EtOAc (30 mL). The combined organic extracts were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel eluting with 0-40% EtOAc in hexanes to give the benzyl ether product (912 mg, 69%). 1 H NMR (300MHz, CDCl3) δ7.35-7.27(m,5H),5.86(d,J=3.6Hz,1H),4.74(t,J=4.2Hz,1H),4.60(s,2H) ,4.42(m,1H),3.69-3.53(m,2H),2.10-2.04(m,1H),1.83-1.77(m,1H),1.52(s,3H),1.33(s,3H).

[0885]

[0886] (3R,5S)-5-(Benzyloxymethyl)-tetrahydrofuran-2,3-diol. The benzyl ether (910 mg, 3.44 mmol) was dissolved in a 1:1 mixture of AcOH and H2O (20 mL) and stirred at 60°C for 7 hours. The mixture was concentrated under reduced pressure and the residue was chromatographed on silica gel, eluting with 0-70% EtOAc in hexanes to give the diol product (705 mg, 91%). 1H NMR (300MHz, CDCl3) δ7.36-7.27(m,5H),5.40(d,J=3.9Hz,0.5H),5.17(s,0.5H),4.67-4.56(m,3H),4.33(m, 0.5H), 4.24 (d, J = 4.8Hz, 0.5H), 3.71-3.67 (m, 1H), 3.56-3.42 (m, 2H), 2.31-2.22 (m, 1H), 2.08-1.89 (m, 2H).

[0887]

[0888] (3R,5S)-5-(benzyloxymethyl)-3-hydroxy-dihydrofuran-2(3H)-one. The diol (705 mg, 3.14 mmol) was dissolved in benzene (30 mL) and treated with a mixture of silver carbonate and celite (3.46 g, 6.28 mmol). The resulting mixture was stirred at 80 ° C for 2 h under N2 (g). The mixture was then cooled to room temperature, filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel, eluting with 0-70% EtOAc in hexane to give the lactone product (600 mg, 86%). 1 H NMR (300MHz, CDCl3) δ7.39-7.27(m,5H),4.75-4.68(m,1H),4.60-4.49(m,2H),3.74-3.54(m,2H),2.61-2.35(m,2H),2.38-2.28(m,1H).

[0889]

[0890] (3R, 5S) -3- (benzyloxy) -5- (benzyloxymethyl) - dihydrofuran -2 (3H) -one. The lactone (600 mg, 2.7 mmol) was dissolved in EtOAc (30 mL) and treated with silver oxide (626 mg, 2.7 mmol), followed by benzyl bromide (387 μL, 3.24 mmol). The reaction mixture was then stirred at 50 ° C for 8 hours under N2 (g). Additional silver oxide (300 mg) was then added, and the resulting mixture was stirred at 50 ° C for 16 hours. Benzyl bromide (50 μL) and silver oxide (150 mg) were additionally added and the mixture was stirred for another 8 hours. The reaction mixture was cooled, filtered, and then concentrated under reduced pressure. The residue was subjected to silica gel chromatography and eluted with 0-20% EtOAc in hexane to give the title product (742 mg, 88%). 1H NMR (300MHz, CDCl3) δ7.39-7.27(m,10H),4.99(d,J=11.4Hz,1H),4.72(m,2H ),4.56(m,2H),4.39(t,J=8.1Hz,1H),3.72-3.51(m,2H),2.42-2.25(m,2H).

[0891]

[0892] (3R,5S)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3-(benzyloxy)-5-(benzyloxymethyl)-tetrahydrofuran-2-ol. 7-Bromopyrrolo[1,2-f][1,2,4]triazin-4-amine (607 mg, 2.85 mmol) was dissolved in anhydrous THF (10 mL) and stirred at room temperature under Ar(g). TMSCl (1.1 mL, 8.55 mmol) was added dropwise and the mixture was stirred for 2 h. The reaction was concentrated under reduced pressure and then dried under high vacuum. The residue was suspended in THF (20 mL) and stirred at -78 °C under Ar(g). 2.5 M n-butyllithium in hexane (2.28 mL, 5.7 mmol) was added dropwise over 10 minutes. The resulting mixture was stirred for 60 minutes. The lactone (742 mg, 2.37 mmol) dissolved in anhydrous THF (7 mL) was added to the above mixture over 20 minutes. The reaction mixture was stirred for 2 hours. It was then quenched with AcOH until the pH was 5-6. The mixture was allowed to warm to room temperature and then diluted with EtOAc. The solution was washed with saturated NaHCO₃ solution, saturated NaCl, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was chromatographed on silica gel, eluting with 0-80% EtOAc in hexane, to give the title product (250 mg, 24%). LCMS m / z 447.2 [M+H], 445.1 [M–H].

[0893]

[0894] (3R,5S)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-3-(benzyloxy)-5-(benzyloxymethyl)-tetrahydrofuran-2-carbonitrile. The alcohol (250 mg, 0.56 mmol) was dissolved in anhydrous CHCl (10 mL) and stirred at -15 °C under Ar(g). TMSCN (448 μL, 3.36 mmol) was added dropwise and the mixture was stirred for 10 minutes. TMSOTf (466 μL, 2.58 mmol) was added dropwise over 10 minutes, and the resulting mixture was stirred at -15 °C for 90 minutes. Additional TMSCN (224 μL, 3 equiv.) and TMSOTf (202 μL, 2 equiv.) were added and stirring was continued for 5 hours. Saturated aqueous NaHCO3 was added to quench the reaction, and the mixture was stirred for 10 minutes. The organic layer was separated and washed with saturated aqueous NaHCO₃ and saturated NaCl solution, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was chromatographed on silica gel, eluting with 0-70% EtOAc in hexanes to yield the title product (150 mg, 59%). LCMS m / z 456.3 [M+H], 454.1 [M–H].

[0895]

[0896] (2R,3R,5S)2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-carbonitrile (7). Benzyl ether (150 mg, 0.329 mmol) was dissolved in anhydrous CHCl (2 mL) and the mixture was stirred at -20°C under Ar(g). A 1 M solution of BCl in CHCl (724 μL, 0.724 mmol) was added dropwise and the resulting mixture was stirred for 2 hours. An additional 1 M solution of BCl in CHCl (724 μL, 0.724 mmol) was added and stirring continued for 2 hours. The mixture was then cooled to -78°C and slowly treated with a 2:1 mixture of EtN and MeOH (3 mL). The mixture was stirred for 10 minutes and then treated with MeOH (10 mL). The reaction was allowed to warm to room temperature and then concentrated under reduced pressure. The residue was dissolved in MeOH and concentrated under reduced pressure. The residue was dissolved in MeOH again and treated with solid NaHCO 3 . The mixture was stirred for 5 minutes and then filtered to remove the solid. The solution was concentrated under reduced pressure and subjected to preparative HPLC to provide the desired product 7 (10 mg, 11%). 1H NMR(300MHz,D2O)δ7.71(s,1H),6.75(d,J=4.5Hz,1H),6.65(d,J=4.8Hz,1H),4.91(t,J=6.3Hz,1H),4.57(m,1H),3.67-3.47(m,2H),2.18(m,2H).LCMS m / z 276.1[M+H],274.0[M–H].

[0897] Example 11. (2S)-2-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7- Isopropyl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)-phosphorylamino)propionate (compound Object 8)

[0898]

[0899] Nucleoside 1 (45mg, 0.15mmol) is dissolved in anhydrous trimethyl phosphate (0.5mL) and solution is stirred under N (g) at 0 ℃.Methylimidazole (36 μ L, 0.45mmol) is added in the solution.Chlorophosphoramidite C (69mg, 0.225mmol) is dissolved in anhydrous THF (0.25mL) and dropwise added in the nucleoside mixture.When completing by LCMS reaction, the reaction mixture is diluted with EtOAc and washed with saturated NaHCO The aqueous solution, saturated NaCl are washed, through anhydrous Na SO Drying is filtered and concentrated under reduced pressure.Resistates is carried out silica gel chromatography, with the CH of 0-5%MeOH Cl Solution wash-out, then by preparation HPLC, obtain product (20.9mg, 25%). 1 H NMR (300MHz, CD3OD) δ7.95(m,1H),7.31-6.97(m,7H),4.94(m,1H),4.78(m,1H),4.43(m,3H),4.20(m,1H),3.80(d,1H),1.30-1.18(m,9H). 31 P NMR (121.4MHz, CD3OD) δ3.8.LCMS m / z561.0[M+H],559.0[MH].

[0900] Example 12. (2S)-2-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7- 2-Ethylbutyl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propanoate Ester (Compound 9)

[0901] Compound 9 can be prepared by the following methods.

[0902] Program 1

[0903]

[0904] Prepared from compound 1 and chloride B according to the same preparation method used for compound 8.

[0905] 1 H NMR (300MHz, CD3OD) δ7.87(m,1H),7.31-7.16(m,5H),6.92-6.89(m,2H),4.78(m,1H),4.50-3.80(m,7H),1.45-1.24(m,8H),0.95-0.84(m,6H). 31 P NMR(121.4MHz,CD3OD)δ3.7.LCMS m / z603.1[M+H],601.0[MH].

[0906] Program 2

[0907]

[0908] 2-Ethylbutyl (2S)-2-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate. 2-Ethylbutyl (2S)-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (1.08 g, 2.4 mmol) was dissolved in anhydrous DMF (9 mL) and stirred at room temperature under a nitrogen atmosphere. (2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile (350 mg, 1.2 mmol) was added to the reaction mixture in one portion. A solution of tert-butylmagnesium chloride in THF (1 M, 1.8 mL, 1.8 mmol) was then added dropwise to the reaction over 10 minutes. The reaction was stirred for 2 hours, at which time the reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated aqueous sodium bicarbonate (3 x 15 mL), followed by saturated aqueous sodium chloride (15 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting oil was purified by silica gel column chromatography (0-10% MeOH in DCM) to afford 2-ethylbutyl (2S)-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (311 mg, 43%, 1:0.4 mixture of diastereomers at phosphorus) as a white solid. 1H NMR (400MHz, CD3OD) δ7.85(m,1H),7.34-7.23(m,2H),7.21-7.09(m,3H),6.94-6.84(m,2H),4.78(d,J=5.4Hz,1H),4.4 6-4.33(m,2H),4.33-4.24(m,1H),4.18(m,1H),4.05-3.80(m,3H),1.52-1.39(m,1H),1.38-1.20(m,7H),0.85(m,6H). 31 P NMR (162 MHz, CD3OD) δ 3.71, 3.65. LCMS m / z 603.1 [M+H], 600.9 [MH]. HPLC (2-98% MeCN-H2O gradient in 8.5 minutes, containing 0.1% TFA modifier, 1.5 mL / min, column: Phenomenex Kinetex C18, 2.6 μm, 4.6x 100mm)t R =5.544min,5.601min.

[0909] Separation of (S) and (R) diastereomers

[0910] 2-Ethylbutyl (2S)-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate was dissolved in acetonitrile. The resulting solution was loaded onto a Lux Cellulose-2 chiral column equilibrated in acetonitrile and eluted with isocratic acetonitrile / methanol (95:5 v / v). The retention time of the first eluting diastereomer was 17.4 minutes, and the retention time of the second eluting diastereomer was 25.0 minutes.

[0911] The first eluting diastereomer is (S)-2-ethylbutyl 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)(phenoxy)phosphoryl)amino)propanoate:

[0912]

[0913] 1HNMR (400MHz, CD3OD) δ8.05 (s, 1H), 7.36 (d, J = 4.8Hz, 1H), 7.29 (br t,J=7.8Hz,2H),7.19-7.13(m,3H),7.11(d,J=4.8Hz,1H),4.73(d,J=5.2Hz,1H),4.48-4.38(m,2H),4.37-4.28(m,1H),4.17(t,J=5.6Hz,1H ),4.08-3.94(m,2H),3.94-3.80(m,1H),1.48(sep,J=12.0,6.1Hz,1H),1.34(p,J=7.3Hz,4H),1.29(d,J=7.2Hz,3H),0.87(t,J=7.4Hz,6H). 31 P NMR (162 MHz, CD3OD) δ 3.71 (s). HPLC (2–98% MeCN–H2O gradient in 8.5 min, containing 0.1% TFA modifier, 1.5 mL / min, column: Phenomenex Kinetex C18, 2.6 μm, 4.6x 100mm)t R =5.585min.

[0914] The second eluting diastereomer is (S)-2-ethylbutyl 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)(phenoxy)phosphoryl)amino)propanoate:

[0915]

[0916] 1 HNMR(400MHz,CD3OD)δ8.08(s,1H),7.36-7.28(m,3H),7.23-7.14(m,3H),7.08(d,J=4.8Hz,1H),4.71(d,J=5.3Hz,1H),4.45-4.34(m,2H),4.3 2-4.24(m,1H),4.14(t,J=5.8Hz,1H),4.08-3.94(m,2H),3.93-3.85(m, 1H), 1.47 (sep, J=6.2Hz, 1H), 1.38-1.26 (m, 7H), 0.87 (t, J=7.5Hz, 6H). 31P NMR (162 MHz, CD3OD) δ 3.73 (s). HPLC (2–98% MeCN–H2O gradient in 8.5 min, containing 0.1% TFA modifier, 1.5 mL / min, column: Phenomenex Kinetex C18, 2.6 μm, 4.6x 100mm)t R =5.629min.

[0917] Example 13. (2S)-2-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7- 5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propionic acid ethyl ester (compound 10)

[0918]

[0919] The preparation of ethyl (2S)-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate is described below.

[0920] Procedure 1. Preparation via Chloride A

[0921]

[0922] Prepared from compound 1 and chloride A using the same preparation method used for compound 8.

[0923] 1 H NMR (300MHz, CD3OD) δ7.95(m,1H),7.32-6.97(m,7H),4.78(m,1H),4.43-4.08(m,6H),3.83(m,1H),1.31-1.18(m,6H). 31 P NMR (121.4MHz, CD3OD) δ3.7.LCMS m / z 547.0[M+H],545.0[MH].

[0924] Procedure 2. Preparation via Nitrobenzene Compound L

[0925]

[0926] Compound 1 (50 mg, 0.17 mmol) was dissolved in NMP-THF (1: 1 mL) and cooled with an ice bath. tBuMgCl (0.257 mL, 0.257 mmol) was then added over 5 minutes. The resulting mixture was allowed to warm to room temperature and stirred for 30 minutes. A solution of compound L (prepared according to US20120009147, 74.6 mg, 0.189 mmol) in THF (2 mL) was then added. After 30 minutes, the reaction mixture was purified by HPLC (aqueous solution of 10-80% acetonitrile) to give compound 29 as a yellow solid. The solid was further purified by silica gel chromatography (MeOH 0-20% DCM) to give compound 29 (23 mg, 24%, a diastereoisomer mixture of 2.5: 1). 1 H NMR(400MHz,CD3OD)δ7.76(d,J=6.0Hz,1H),7.25-7.14(m,2H),7.11-6.99(m,3 H),6.87-6.72(m,2H),4.70(d,J=5.4Hz,1H),4.39-4.24(m,2H),4.20(dddd,J= 9.7,7.9,5.1,2.8Hz,1H),4.10(dt,J=12.8,5.5Hz,1H),4.06-3.91(m,2H),3.7 2(ddq,J=14.3,9.3,7.1Hz,1H),1.17(dd,J=7.1,1.0Hz,1H),1.14-1.06(m,5H). 31 P NMR(162MHz,CD3OD)δ3.73,3.68.MS m / z=547(M+1) + .

[0927] Example 14. (2S)-2-(((2R,3R,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7- 5-cyano-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propionic acid ethyl ester (compound 11)

[0928]

[0929] Compound 11 was prepared from Compound 2 and Chloride A using the same preparation method used for Compound 8. 1 H NMR (300MHz, CD3OD) δ7.91(m,1H),7.33-7.16(m,5H),6.98-6.90(m,2H),5.59(m,1H),4.50-4.15(m,4H),4.12-3.90(m,3H),1.33-1.18(m,6H). 31 P NMR(121.4MHz,CD3OD)δ3.8.LCMS m / z549.0[M + H],547.1[MH].

[0930] Example 15. (2S,2'S)-2,2'-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4] triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)dipropionic acid di Ethyl ester (Compound 12)

[0931]

[0932] Nucleoside 1 (14.6 mg, 0.05 mmol) was dissolved in anhydrous trimethyl phosphate (0.5 mL) and stirred at room temperature under N2 (g). POCl3 (9.2 μL, 0.1 mmol) was added and the mixture was stirred for 60 minutes. Alanine ethyl ester hydrochloride (61 mg, 0.4 mmol) was added, followed by Et3N (70 μL, 0.5 mmol). The resulting mixture was stirred for 15 minutes. Et3N (70 μl, 0.5 mmol) was then added to obtain a solution of pH 9-10. The mixture was stirred for 2 hours. It was then diluted with EtOAc, washed with a saturated NaHCO3 aqueous solution, and then washed with a saturated NaCl aqueous solution. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was subjected to preparative HPLC (C18 column) to obtain product 12 (5.5 mg, 16%). 1 H NMR (400MHz, CD3OD) δ8.13(s,1H),7.41(d,J=4.8Hz,1H),7.18(d,J=4.8Hz,1H),4.7 8(d,J=5.6Hz,1H),4.36(m,1H),4.25-4.08(m,7H),3.83(m,2H),1.33-1.23(m,12H). 31 P NMR(121.4MHz,CD3OD)δ13.8.LCMS m / z570.0[M+H],568.0[MH].

[0933] Example 16. (2S,3R,4S,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-2-ethynyl 5-(Hydroxymethyl)tetrahydrofuran-3,4-diol (Compound 13)

[0934]

[0935] The preparation of (2S,3R,4S,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-2-ethynyl-5-(hydroxymethyl)tetrahydrofuran-3,4-diol is as follows.

[0936]

[0937] The nucleoside alcohol (0.6 g, 1.08 mmol) (prepared as described in the synthesis of compound 1) was dissolved in anhydrous THF (8 mL) and placed under N2(g). The reaction mixture was stirred and cooled to 0°C and then treated with 0.5N ethynylmagnesium bromide in THF (17.2 mL, 17.2 mmol). The reaction mixture was stirred at room temperature overnight. AcOH (1.5 mL) was added to quench the reaction. The mixture was concentrated under reduced pressure and the residue was redissolved in CHCl2. The solution was passed through a plug of silica gel and eluted with 0-80% EtOAc in hexanes to give the title product as a crude mixture. LCMS m / z 579 [M+H].

[0938]

[0939] The crude acetylene alcohol (0.624 g, 1.08 mmol) was dissolved in anhydrous CH2Cl2 (10 mL) and placed under N2(g). The mixture was stirred and sulfonic acid (0.2 mL, 2.74 mmol) was added. The reaction mixture was stirred at room temperature for 12 hours. When complete by LCMS, Et3N (0.56 mL) was added to quench the reaction. The reactants were concentrated under reduced pressure and the residue was subjected to silica gel chromatography, eluting with 0-75% EtOAc in hexane to give the acetylene nucleoside as a mixture of anomers (0.200 g, 33% over 2 steps). LCMSm / z 561[M+H].

[0940]

[0941] Tribenzyl nucleoside (0.650 g, 1.16 mmol) was dissolved in anhydrous CH2Cl2 (30 mL) and cooled to -78 ° C under N2 (g). Boron tribromide (1N in CH2Cl2, 5.5 mL) solution was added and the reaction mixture was stirred at -78 ° C for 1 hour. A solution of MeOH (10 mL) and pyridine (2 mL) was added to quench the reaction and the mixture was allowed to warm to room temperature. The mixture was concentrated under reduced pressure and subjected to preparative HPLC to provide α-anomer (20 mg) and β-anomer 13 (110 mg). (β-anomer) 1HNMR (300 MHz, DMSO) δ 7.81 (s, 1H), 7.76 (br s, 2H), 6.80-6.85 (m, 2H), 5.11 (d, J = 7.2 Hz, 1H), 4.90 (d, J = 6.0 Hz, 1H), 4.82 (dd, J = 7.2, 4.8 Hz, 1H), 4.62 (t, J = 6.3 Hz, 1H), 3.95-3.99 (m, 1H), 3.85-3.91 (dd, J = 11.4, 5.7 Hz, 1H), 3.61-3.67 (m, 1H), 3.47-3.55 (m, 1H), 3.52 (d, J = 0.9 Hz, 1H). (α-anomer) 1 H NMR (300MHz, DMSO) δ7.80(s,1H),7.59(bs,2H),6.80(d,J=4.5Hz,1H),6.54(d,J=4.2Hz,1H),5.00(d,J=7.2Hz,1H),4.89(d,J=4.8Hz,1H) ,4.74(t,J=5.7Hz,1H),4.58(t,J=4.5Hz,1H),4.27(m,1H),3.88(m,1H),3.64-3.72(m,1H),3.51-3.59(m,1H),3.48(d,J=0.6Hz,1H).LCMS m / z 291[M+H].

[0942] Example 17. (2R,3R,4R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-1,3,4-triazine (Benzyloxy)hexane-2,5-diol (Compound 14)

[0943]

[0944] The preparation of (2R,3R,4R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-1,3,4-tris(benzyloxy)hexane-2,5-diol is described below.

[0945]

[0946] Tribenzyl alcohol (0.250 g, 0.453 mmol) from the synthesis of compound 1 was dissolved in anhydrous THF (25 mL) and stirred under N2(g). The reaction mixture was cooled to 0°C, and then a 3.0 N solution of methylmagnesium chloride in THF (1.2 mL, 3.62 mmol) was added. The reaction mixture was stirred at room temperature overnight. Acetic acid (1.5 mL) was added to quench the reaction, and the mixture was then concentrated under reduced pressure. The residue was redissolved in CH2Cl2 and passed through a silica gel plug, eluting with 0-80% EtOAc in hexane. The crude product (0.452 g) was then used in the next reaction without further purification. LCMS m / z 569 [M+H].

[0947]

[0948] The crude methyl nucleoside (0.452 g, 0.796 mmol) was dissolved in anhydrous CH2Cl2 (20 mL) and stirred under N2(g). Methanesulfonic acid (0.2 mL, 2.78 mmol) was added and the reaction was stirred at room temperature for 12 hours. Et3N (0.56 mL) was added to quench the reaction, and the mixture was then concentrated under reduced pressure. The residue was chromatographed on silica gel, eluting with 0-75% EtOAc in hexanes to give the product as a mixture of anomers (0.20 g, 46% over 2 steps). LCMS m / z 551 [M+H].

[0949]

[0950] Tribenzyl nucleoside (0.20 g, 0.364 mmol) was dissolved in AcOH (30 mL). Pd / C (Degussa) (400 mg) was added. The stirred mixture was flushed with N2(g) three times, and then H2(g) was introduced. The reaction was stirred under H2(g) for 2 hours. The catalyst was then removed by filtration. The solution was concentrated under reduced pressure and the residue was redissolved in H2O. The solution was subjected to preparative HPLC under neutral conditions to provide 81% yield of α-anomer and β-anomer 14. (α-anomer) 1 HNMR (300 MHz, D2O) δ7.81 (s, 1H), 7.22 (d, 1H), 6.75 (d, 1H), 4.47 (d, 1H), 4.25-4.31 (m, 1H), 3.88-4.95 (m, 1H), 3.58-3.86 (dd, 2H), 1.50 (s, 3H). (β-anomer) 1 H NMR(300MHz,D2O)δ7.91(s,1H),7.26(d,1H),6.90(d,1H),4.61(d,1H),4.00-4.09(m,2H),3.63-3.82(dd,2H),1.67(s,3H).LCMS m / z 281[M+H].

[0951] Example 18. S,S'-2,2'-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazol-2-yl)-1,2-dextrin (oxazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl) (methoxy)phosphoryl)bis(oxy)bis(ethane-2,1-diyl) Bis(2,2-dimethylpropionic acid thioester) (Compound 15)

[0952]

[0953] Nucleoside 1 (0.028 g, 0.096 mmol) was dissolved in trimethyl phosphate (1 mL). The reaction was stirred under N2 (g) and then treated with 1H-tetrazole (0.021 g, 0.29 mmol). The reaction mixture was cooled to 0 ° C and phosphane (NucleosideNucleotides, Nucleic acids; 14; 3-5; 1995; 763-766. Lefebvre, Isabelle; Pompon, Alain; Perigaud, Christian; Girardet, Jean-Luc; Gosselin, Gilles; etc.) (87 mg, 0.192 mmol) was added. The reaction was stirred for 2 hours. It was then quenched with 30% hydrogen peroxide (0.120 mL). The mixture was stirred at room temperature for 30 minutes and then treated with saturated sodium thiosulfate aqueous solution (1 mL). The mixture was stirred for 10 minutes. It was then concentrated under reduced pressure. The residue was subjected to preparative HPLC to separate the title product 15. 1 H NMR(300MHz,CD3CN)δ7.98(s,1H),6.92(d,1H),6.81(d,1H),6.44(bs,2H),4.82(m, 2H),4.47(m,1H),4.24(m,2H),4.00(m,4H),3.80(bs,1H),3.11(m,4H),1.24(s,9H). 31 P NMR(121.4MHz,CD3CN)δ-1.85(s).LCMS m / z 661[M+H].

[0954] Example 19. S,S'-2,2'-((((2R,3S,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazol-2-yl)-1,2-diol (oxazin-7-yl)-5-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)phosphoryl)bis(oxy)bis(ethane-2,1-diol 1-Methyl)bis(2,2-dimethylpropionic acid thioester) (Compound 16)

[0955]

[0956] Compound 16 was prepared using the same method as compound 15, except that compound 13 was used as the starting nucleoside. 1 HNMR(300MHz,CD3CN)δ7.91(s,1H),6.86(d,J=4,8Hz,1H),6.76(d,J=4.5Hz,1H),6.29(bs,2H),4.69(t,J=2.7Hz,1H),4.5 8(d,J=5.7Hz,1H),4.14-4.33(m,5H),3.99-4.07(m,4H),3.53(d,J=5.4Hz,1H),3.11(q,J=5.7Hz,4H),1.22(s,18H).LCMS m / z 658.9[M+].Tr=2.31

[0957] Example 20. ((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-cyano 3,4-dihydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate (Compound 17)

[0958]

[0959] Compound 17 was prepared from compound 1 using a procedure similar to that used to prepare compound 6. The product was isolated as a sodium salt. 1 H NMR(400MHz,D2O)δ7.76(s,1H),6.88(d,J=4.8Hz,1H),6.73(d,J=4.4Hz,1H) ,4.86(d,J=5.2Hz,1H),4.43(m,1H),4.39(m,1H),4.05(m,1H),3.94(m,1H). 31 P NMR (121.4 MHz, D2O) δ -5.4 (d, 1P), -10.8 (d, 1P), -21.1 (t, 1P). LCMS m / z 530 [MH], 531.9 [M+H] Tr = 0.22 min. HPLC ion exchange Tr = 9.95 min.

[0960] Example 21. ((2R,3S,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-ethyl Alkynyl-3,4-dihydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate (Compound 18)

[0961]

[0962] Compound 18 was prepared from compound 13 using a procedure similar to that used to prepare compound 6. The product was isolated as a TEA salt. 1 H NMR(300MHz,D2O)δ7.85(s,1H),7.09(d,J=4.6Hz,1H),6.95(d,J=4.7Hz,1H ), 4.23 (m, 2H), 4.08 (m, 2H), 3.06 (q, J = 7.4Hz, 20H), 1.14 (t, J = 7.3Hz, 30H). 31 P NMR (121.4 MHz, D2O) δ -10.8 (d, 1P), -11.2 (d, 1P), -23.2 (t, 1P). LCMS m / z 530.8 [M+H], Tr = 0.46. HPLC ion exchange Tr = 9.40 min.

[0963] Example 22. ((2R,3S,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3,4- Dihydroxy-5-methyltetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate (Compound 19)

[0964]

[0965] Compound 19 was prepared from Compound 14 using a procedure similar to that used to prepare Compound 6. 1H NMR(400MHz,D2O)δ7.78(s,1H),6.98(m,1H),6.84(m,1H),4.45(m,1H),4.04(m,4H),1.54(s,3H). 31 P NMR(161MHz,D2O)δ-10.6(m),-23.0(m).LCMS m / z 521.0[M+H].

[0966] Example 23. ((2R,3R,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-cyano 4-(4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate (Compound 20)

[0967]

[0968] Compound 20 was prepared from Compound 2 using a procedure similar to that used to prepare Compound 6. 1 H NMR (400MHz, D2O) δ7.78 (s, 1H), 6.93 (d, J = 4.4Hz, 1H), 6.78 (d, J = 4.8Hz, 1H), 5.45 (dd, J = 53, 4.4Hz, 1H), 4.38-4.50 (m, 2H), 4.13-4.20 (m, 2H). 31 P NMR (161 MHz, D2O) δ -5.7 (d, 1P), -11.0 (d, 1P), -21.5 (t, 1P). LCMS m / z 533.9.0 [M+H], 532.0 [MH] Tr = 1.25 min. HPLC ion exchange TR = 11.0 min.

[0969] Example 24. (2S)-2-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine- 7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropionic acid ethyl Esters (21)

[0970]

[0971] The preparation of ethyl (2S)-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate is as described below.

[0972] Preparation of (S)-2-amino-3-phenylpropionic acid ethyl ester hydrochloride.

[0973]

[0974] L-phenylalanine (5 g, 30 mmol) was dissolved in EtOH (30 mL). TMSCl (6.915 mL, 54 mmol) was added to the reaction at room temperature. The reaction vessel was equipped with a reflux condenser and the reaction was placed in an 80°C bath. The reaction was stirred overnight. The next day, the reaction was cooled to room temperature, concentrated under reduced pressure and the resulting residue was dissolved in Et2O. The resulting slurry was filtered and the isolated solid was further washed with Et2O. The washed solid was placed under high vacuum to give Example (S)-ethyl 2-amino-3-phenylpropionate hydrochloride (6.86 g, 99%). 1 H NMR(400MHz,DMSO-d6)δ8.52(s,3H),7.30(m,5H),4.24(ABX,J AX =7.8Hz,J BX =6.2Hz,1H),4.11(m,2H),3.17,3.05(ABX,J AB =-14Hz,J BX =5.8Hz,J AX =7.6Hz,2H),1.09(t,J=6.8Hz,3H).

[0975] Ethyl (2S)-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)-3-phenylpropionate (Compound D) preparation

[0976]

[0977] (S)-2-amino-3-phenylpropionic acid ethyl ester hydrochloride (1.01g, 4.41mmol) is dissolved in DCM (50mL). The solution is cooled to 0 ℃, PhOP(O)Cl (0.656mL, 4.41mmol) is added, and then EtN (1.62mL, 11.5mmol) is slowly added over 5 minutes. The cooling bath is removed, and the reaction is allowed to warm to room temperature and stirred for 80 minutes. p-NOPhOH (0.583g, 4.19mmol) is added, and then EtN (0.3mL, 2.1mmol) is added. The reaction progress is monitored by LC / MS. After the reaction is completed, it is diluted with EtO and the obtained solid is removed by filtration. The filtrate was concentrated and compound D (1.25 g, 60%, as a mixture of diastereomers) was isolated by silica gel column chromatography (25 g dry load, 120 g column; eluent: 100% hexanes rising to 55% EtOAc in hexanes). 1 H NMR (400MHz, CD3OD) δ8.17(m,2H),7.33(m,2H),7.09-7.25(m,10H),4.17(m,1H),4.07(m,2H),3.08(m,1H),2.84(m,1H),1.14(m,3H).31 P NMR (162MHz, DMSO-d6)δ-1.479(s),-1.719(s).MS m / z=471.01[M+1].

[0978] (2S)-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano 3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropionic acid ethyl ester (compound 21) Preparation

[0979]

[0980] Compound 1 (0.030g, 0.103mmol) is dissolved in DMF (1mL), then THF (0.5mL) is added. Under vigorous stirring, t-BuMgCl (1M / THF, 154.5 μ L, 0.154 μ mol) is added dropwise to the reaction. The gained white slurries are stirred at room temperature for 30 minutes. A solution of compound D (0.058g, 0.124mmol) in THF (1mL) is added dropwise to the reactant at room temperature. The reaction process is monitored by LC / MS. When the reaction proceeds to 50% conversion, the reactant is cooled in an ice bath and quenched with glacial acetic acid (70 μ L). The reaction is concentrated and separated from residue by reversed-phase HPLC. Compound 21 (22mg, 34% is a 2.6: 1 mixture of diastereomers). 1 H NMR (400MHz, DMSO-d6) δ7.91(d,J=4Hz,1H),7.90(brs,2H),7.09-7.30(m,8H),7.01,(t,J=8.2Hz,2H),6.89(d,J=4.4Hz,1H),6.82(t,J=4.4H z,1H),6.27(m,1H),6.14(m,1H),5.34(m,1H),4.62(t,J=5.6Hz,1H),4 .15(m,1H),3.78-4.01(m,6H),2.92(m,1H),2.78(m,1H),1.04(m,3H). 31 P NMR (162MHz, DMSO-d6) δ3.69 (s), 3.34 (s).MS m / z=623.0[M+H].

[0981] Example 25. (2S)-2-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine- 7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-methylbutanoic acid ethyl Esters (22)

[0982]

[0983] The preparation of ethyl (2S)-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-methylbutanoate is as described below.

[0984] Ethyl (2S)-3-methyl-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)butanoate (Compound E) preparation

[0985]

[0986] (S)-2-amino-3-methylbutanoic acid ethyl ester (0.351 g, 1.932 mmol) was dissolved in DCM (17 mL). The solution was cooled in an ice bath and PhOP(O)Cl2 (0.287 mL, 1.932 mmol) was added, followed by the slow addition of Et3N (1.62 mL, 11.4 mmol) over 5 minutes. The cooling bath was removed and the reaction allowed to warm to room temperature and stirred for 1 hour. p-NO2PhOH (0.255 g, 1.836 mmol) was added and the reaction progress was monitored by LC / MS. After completion of the reaction, the mixture was diluted with Et2O and the resulting solid was removed by filtration. The filtrate was concentrated and the compound E (0.642 g, 79%, as a mixture of diastereomers) was isolated by silica gel column chromatography (12 g dry carrier column, 80 g column; eluent: 100% hexane to 55% EtOAc in hexane). 1 H NMR(400MHz, DMSO-d6)δ8.30(d,J=9.2Hz,2H),7.48(t,J=9.6Hz,2H),7.40(t,J=7.8Hz,2H),7.20-7.27 (m,3H),6.60(quart,J=11.6Hz,1H),4.01(m,2H),3.61(m,1H),1.93(m,1H),1.11(m,3H),0.79(m,6H). 31 P NMR (162MHz, DMSO-d6)δ-0.342(s),-0.578(s).MS m / z=422.9[M+H].

[0987] (2S)-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano 3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-methylbutanoic acid ethyl ester (compound 22) Preparation

[0988]

[0989] Compound 1 (0.040 g, 0.137 mmol) was dissolved in NMP (1.5 mL) and THF (0.25 mL) was added. The solution was cooled in an ice bath and t-BuMgCl (1 M / THF, 425.7 μ L, 0.426 μ mol) was added dropwise under vigorous stirring. The ice bath was removed and the resulting white slurry was stirred at room temperature for 15 minutes. A solution of THF (0.5 mL) of compound E (0.081 g, 0.192 mmol) was added dropwise to the reaction at room temperature. The reaction process was monitored by LC / MS. When the reaction was carried out to 50% conversion, the reactant was cooled in an ice bath and quenched with glacial acetic acid (70 μ L). The reaction was concentrated and semi-purified compound 22 (22 mg, 34%) was obtained from residue by reversed-phase HPLC. The semi-pure material was further purified by silica gel column chromatography (12 g dry load, 40 g column; eluent: 100% EtOAc rising to 10% MeOH in EtOAc) to afford compound 22 (0.034 g, 43% as a 1.8:1 mixture of diastereomers). 1 H NMR(400MHz,DMSO-d6)δ7.91(d,J=1.6Hz,1H),7.88(brs,2H),7.32(m,2H),7.1 5(m,3H),6.90(t,J=4.2Hz,1H),6.84(d,J=4.8Hz,1H),6.26(dd,J=13.4,6.2Hz ,1H),5.87(quart.J=11.2Hz,1H),5.35(m,1H),4.64(m,1H),4.25(m,2H),3.93 -4.15(m,4H),3.45(m,1H),1.87(m,1H),1.09-1.16(m,3H),0.70-0.83(m,6H). 31 P NMR(162MHz, DMSO-d6)δ4.59(s),4.47(s).MS m / z=575.02[M+H].

[0990] Example 26. (S)-2-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7- Isobutyl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (23)

[0991]

[0992] The preparation of isobutyl (S)-2-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate is as described below.

[0993]

[0994] Compound 1 (60.0 mg, 206 μmol) was dissolved in NMP (0.28 mL). THF (0.2 mL) was added, followed by tert-butylmagnesium chloride (1.0 M tetrahydrofuran solution, 0.309 mL) at room temperature under an argon atmosphere. After 20 minutes, a solution of compound F (prepared according to Cho, A. et al J. Med. Chem. 2014, 57, 1812-1825., 81 mg, 206 μmol) in THF (0.2 mL) was added, and the resulting mixture was warmed to 50° C. After 3 hours, the reaction mixture was cooled to room temperature and purified directly by preparative HPLC (Phenominex Synergi 4u Hydro-RR80 150×30 mm column, 5-100% acetonitrile / water gradient) to give compound 23 (44 mg, 38%, as a single diastereomer). 1 H NMR(400MHz,CD3OD)δ7.86(s,1H),7.34-7.26(m,2H),7.21-7.12(m,3H),6.91 (d,J=4.6Hz,1H),6.87(d,J=4.6Hz,1H),4.92(sept,J=6.3Hz,1H),4.80(d,J=5 .4Hz,1H),4.43-4.34(m,1H),4.33-4.24(m,1H),4.18(t,J=5.6Hz,1H),3.82(d q,J=9.7,7.1Hz,2H),1.27(dd,J=7.1,1.0Hz,3H),1.18(dd,J=6.3,4.8Hz,6H). 31 P NMR(162MHz,CD3OD)δ3.72(s).LC / MS:t R =1.39 min, MS m / z=561.11 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μXB-C18100A, 50×4.6 mm; Solvent: ACN containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% ACN, 2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN, 3.2 min-3.5 min 2% ACN, 2 μl / min. HPLC: t R= 2.523 min; HPLC system: Agilent 1100 Series. Column: Gemini 5μ C18110A, 50×4.6 mm; Solvents: ACN containing 0.1% TFA, water containing 0.1% TFA; Gradient: 2-98% ACN from 0 min to 5.0 min, 98% ACN from 5.0 min to 6.0 min, 2 mL / min.

[0995] Example 27. (2S)-2-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine- 7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propionic acid cyclobutyl ester (24)

[0996]

[0997] The preparation of cyclobutyl (2S)-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate is as described below.

[0998] (2S)-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propionic acid cyclobutyl ester (Compound G)

[0999]

[1000] Phenyl dichlorophosphate (1.49mL, 10mmol) is dissolved in 10mL anhydrous DCM and stirred in an ice bath under a nitrogen atmosphere. Disposable addition of L-alanine isobutyl ester hydrochloride (0.9g, 5mmol). Then triethylamine (765 μL, 5.5mmol) is added dropwise. The reaction is stirred for 1 hour. More triethylamine (765 μL, 5.5mmol) is added dropwise and the reaction is stirred for 45 minutes. Disposable addition of p-nitrophenol (1.25g, 9mmol) is added and stirred for 30 minutes. Triethylamine (765 μL, 5.5mmol) is added and the reaction mixture is stirred for 2 hours. Additional p-nitrophenol (1.25g, 9mmol) and triethylamine (765 μL, 5.5mmol) are then added, and the reaction is stirred for another 2 hours. The reaction mixture is concentrated under reduced pressure. The gained crude product is diluted with EtOAc and washed twice with 5% citric acid aqueous solution, then washed with saturated sodium chloride aqueous solution. Then the organic layer is dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified on a silica gel column (0-20-50% EtOAc in hexanes) to afford compound G (1.48 g, 70% yield as a mixture of diastereomers). 1H NMR (400MHz, CD3OD) δ8.33-8.23(m,2H),7.52-7.33(m,4H),7.33-7.17(m,3H),4.96-4.85(m,1H),4. 07-3.96(m,1H),2.27(m,2H),2.07-1.91(m,2H),1.83-1.70(m,1H),1.70-1.55(m,1H),1.32(m,3H). 31 P NMR (162MHz, CD3OD) δ-1.36,-1.59.MS m / z=420.9[M+H].

[1001] (2S)-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano Cyclobutyl (3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (Compound 24)

[1002]

[1003] Compound 1 (58 mg, 0.2 mmol) was mixed with compound G (101 mg, 0.24 mmol) in 2 mL of anhydrous DMF. Magnesium chloride (42 mg, 0.44 mmol) was added at one time. The reaction mixture was heated to 50 ° C. DIPEA (87 μL, 0.5 mmol) was added and the reaction was stirred at 50 ° C for 2 hours. The reaction mixture was cooled to room temperature, diluted with EtOAc and washed with 5% citric acid aqueous solution, then washed with saturated sodium chloride aqueous solution. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by silica gel column (0-2-5% MeOH in DCM solution) to obtain compound 24 (42 mg, 37% yield, a mixture of diastereomers). 1 H NMR (400 MHz, methanol-d4) δ 7.85 (m, 1H), 7.34-7.22 (m, 2H), 7.22-7.08 (m, 3H), 6.94-6.84 (m, 2H), 4.95-4.85 (m, 1H), 4.79 (m, 1H), 4.46-4.34 (m, 2H), 4.34-4.24 (m, 1H), 4.19 (m, 1H), 3.81 (m, 1H), 2.27 (m, 2H), 2.01 (m, 2H), 1.84-1.68 (m, 1H), 1.62 (m, 1H), 1.30-1.16 (m, 3H). 31 P NMR(162MHz,cd3od)δ3.70,3.65.MS m / z=573.0[M+H].

[1004] Example 28. (2S)-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7- Isopropyl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate Esters (25)

[1005]

[1006] The preparation of isopropyl (2S)-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate is as described below.

[1007] Isopropyl (2S)-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate (Compound H) Preparation

[1008]

[1009] Phenyl dichlorophosphate (718 μL, 4.8 mmol) was dissolved in 10 mL of anhydrous DCM and stirred in an ice bath under a nitrogen atmosphere. L-phenylalanine isopropyl ester hydrochloride (1 g, 4.1 mmol) was added once. 10 mL of anhydrous DCM was added. Triethylamine (736 μL, 5.3 mmol) was added dropwise and the reaction mixture was stirred for 30 minutes. More triethylamine (736 μL, 5.3 mmol) was then added dropwise and the reaction mixture was stirred for 30 minutes. Another triethylamine (736 μL, 5.3 mmol) was then added dropwise and the reaction mixture was stirred for 15 minutes. p-Nitrophenol (600 mg, 4.32 mmol) was then added. The ice bath was then removed, the reaction mixture was warmed to room temperature and stirred for 2 hours. More p-Nitrophenol (50 mg) and triethylamine (736 μL, 5.3 mmol) were added and the reaction mixture was stirred for 1 hour.

[1010] The reaction mixture was then concentrated under reduced pressure, diluted with EtOAc and washed twice with 5% aqueous citric acid solution, followed by washing with saturated aqueous sodium chloride solution. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column (0-15% EtOAc in hexane) to give compound H (1.57 g, 68% yield, diastereomeric mixture). 1 H NMR (400MHz, CDCl3) δ8.17(m,2H),7.38-7.13(m,10H),7.13-7.02(m,2H),4.95( m,1H),4.31(m,1H),3.69(m,1H),3.02(dd,J=6.1,1.8Hz,2H),1.21-1.08(m,6H). 31 P NMR (162MHz, cdcl3)δ-2.96,-2.98.MS m / z=485.0[M+H].

[1011] (2S)-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano Isopropyl 3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate (compound Preparation of Compound 25

[1012]

[1013] Compound 1 (58mg, 0.2mmol) and compound H (116mg, 0.24mmol) are mixed and 2mL anhydrous DMF is added.Under nitrogen atmosphere, the reaction mixture is stirred at room temperature.With 3 minutes, 1M tBuMgCl THF solution (300 μ L, 0.3mmol) is added dropwise, and then the reaction mixture is stirred for 16 hours.The reaction mixture is diluted with EtOAc and washed with 5% citric acid aqueous solution, saturated sodium bicarbonate aqueous solution, and then with saturated sodium chloride aqueous solution.The organic layer is dried over anhydrous sodium sulfate and concentrated under reduced pressure.The thick residue is purified with silica gel column (0-5%MeOH DCM solution) to obtain compound 25 (40mg, 32%, diastereomeric mixture). 1 H NMR (400MHz, CD3OD) δ7.84(m,1H),7.27-7.08(m,8H),7.08-6.97(m,2H),6.88(m,2H),4.91-4.84(m,1H),4.74( m,1H),4.26(m,1H),4.19-4.04(m,2H),4.04-3.91(m,2H),2.97(m,1H),2.82(m,1H),1.14(m,3H),1.06(m,3H). 31 P NMR (162MHz, CD3OD) δ3.63,3.25.MS m / z=637.0[M+H].

[1014] Example 29. (S)-2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazol-2-yl)-1,2-diol (oxazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propionic acid methyl ester (26)

[1015]

[1016] The preparation of methyl (S)-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)(phenoxy)phosphoryl)amino)propanoate is as described below.

[1017]

[1018] Compound 1 (100mg, 0.34mmol) is dissolved in THF (2mL) and cooled with an ice-water bath. Then 1M t-BuMgCl (0.52mL, 0.77mmol) is slowly added dropwise. The resulting mixture is stirred at room temperature for 30 minutes. Then a solution of compound 1 (prepared according to WO2012142085, 29mg, 0.52mmol) in THF (2mL) is added over 5 minutes, and the resulting mixture is stirred at room temperature for 24 hours. The reaction mixture is then diluted with EtOAc, cooled in an ice-water bath, washed with a NaHCO3 aqueous solution (2mL), washed with brine, dried over sodium sulfate and concentrated in vacuo. The resulting mixture is purified by silica gel column chromatography (a solution of MeOH 0-20% in DCM) and preparative HPLC (aqueous solution of acetonitrile 10-80%) to obtain compound 26 (12mg, 6.6%, a single diastereomer). 1H NMR (400MHz, CD3OD) δ7.86 (s, 1H), 7.29 (dd, J=8.6, 7.2Hz, 2H), 7.21-7.09 (m, 3H), 6.94-6.81 (m, 2H), 4.79 (d, J=5.4Hz, 1H), 4.38 (ddq, J= 10.8,5.3,2.7Hz,2H),4.33-4.23(m,1H),4.18(t,J=5.5Hz,1H),3.86(dq,J=9.9,7.1Hz,1H),3.62(s,3H),1.27(dd,J=7.2,1.1Hz,3H).MS m / z=533(M+1) + .

[1019] Example 30. (S)-2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]]triazolidine (oxazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propionic acid neopentyl ester (27)

[1020]

[1021] The preparation of (S)-neopentyl 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)(phenoxy)phosphoryl)amino)propanoate is as described below.

[1022]

[1023] Compound 1 (100mg, 0.34mmol) is dissolved in THF (2mL) and cooled in an ice-water bath.Then 1M t-BuMgCl (0.52mL, 0.77mmol) is slowly added dropwise. The gained mixture is stirred at room temperature for 30 minutes.Then compound J (prepared according to WO2012075140, 248mg, 0.52mmol) is added over 5 minutes, and the gained mixture is stirred at room temperature for 24 hours, diluted with EtOAc, cooled in an ice-water bath, and NaHCO is used The aqueous solution (2mL) is processed, washed with salt water, dried over sodium sulfate, and concentrated in vacuo. By silica gel column chromatography (MeOH 0-20% DCM solution) and preparative HPLC (acetonitrile 10-80% aqueous solution) purified gained mixture, compound 27 (12mg, 10% is a single diastereomer) is obtained. 1 H NMR(400MHz,CD3OD)δ7.86(s,1H),7.36-7.24(m,2H),7.23-7.10(m,3H),6.96 -6.85(m,2H),4.78(d,J=5.4Hz,1H),4.38(tdd,J=10.0,4.9,2.5Hz,2H),4.32- 4.24(m,1H),4.17(t,J=5.6Hz,1H),3.91(dq,J=9.8,7.1Hz,1H),3.81(d,J=10 .5Hz,1H),3.69(d,J=10.5Hz,1H),1.31(dd,J=7.2,1.1Hz,3H),0.89(s,9H).MS m / z=589(M+1) + .

[1024] Example 31. (2S)-2-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7- (28)

[1025]

[1026] The preparation of cyclopentyl (2S)-2-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate is as described below.

[1027]

[1028] Compound 1 (100mg, 0.34mmol) is dissolved in THF (2mL) and cooled in an ice-water bath.Then 1M t-BuMgCl (0.52mL, 0.77mmol) is slowly added dropwise. The resulting mixture is stirred at room temperature for 30 minutes.Then a solution of compound K (prepared according to WO2012075140, 247mg, 0.52mmol) in THF (2mL) is added over 5 minutes, and the resulting mixture is stirred at room temperature for 24 hours, diluted with EtOAc, cooled in an ice-water bath, and treated with NaHCO3aqueous solution (2mL), washed with salt water, dried over sodium sulfate and concentrated in vacuo. The resulting mixture is purified by silica gel column chromatography (aqueous solution of MeOH 0-20%) and preparative HPLC (aqueous solution of acetonitrile 10-80%) to obtain Example 28 (47mg, 23%, 27:1 mixture of diastereomers). 1H NMR (400MHz, CD3OD) δ7.85 (s, 1H), 7.33-7.22 (m, 2H), 7.14 (tdd, J = 7.6, 2.1, 1.1Hz, 3 H),6.95-6.87(m,2H),5.13-5.00(m,1H),4.78(d,J=5.4Hz,1H),4.48-4.35(m,2H),4. 30(ddd,J=10.6,5.7,3.6Hz,1H),4.19(t,J=5.4Hz,1H),3.78(dq,J=9.2,7.1Hz,1H), 1.81(dtd,J=12.5,5.9,2.4Hz,2H),1.74-1.49(m,6H),1.21(dd,J=7.1,1.2Hz,3H).MS m / z=587(M+1) + .

[1029] Example 32. (2S)-2-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine- (7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propionic acid cyclohexyl ester (29)

[1030]

[1031] The preparation of cyclohexyl (2S)-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate is as described below.

[1032]

[1033] To a mixture of compound 1 (50 mg, 0.343 mmol), compound M (prepared according to US20130143835, 93 mg, 0.209 mmol) and MgCl2 (24.5 mg, 0.257 mmol) in DMF (1 mL) was added diisopropylethylamine (0.075 mL, 0.43 mmol) at 0 ° C over 5 minutes. The resulting mixture was stirred at 50 ° C for 1 h. The reaction mixture was then cooled with an ice-water bath, treated with 1 M citric acid (0.5 mL), and purified directly by preparative HPLC (ACN 0-70% in water) to give compound 29 (20 mg, 19% as a mixture of diastereomers). 1 H NMR(400MHz,CD3OD)δ7.84(s,1H),7.32-7.23(m,2H),7.18-7.10(m,3H),6.93 -6.87(m,2H),4.78(d,J=5.4Hz,1H),4.67(td,J=8.7,4.2Hz,1H),4.48-4.35( m,2H),4.30(ddd,J=10.8,5.7,3.7Hz,1H),4.20(t,J=5.4Hz,1H),3.88-3.71( m,1H),1.83-1.63(m,4H),1.58-1.46(m,1H),1.46-1.24(m,5H),1.24(s,3H). 31 P NMR(162MHz,CD3OD)δ3.75.MS m / z=601(M+1) + .

[1034] Example 33. 2-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)- Ethyl 5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-2-methylpropanoate (30)

[1035]

[1036] The preparation of ethyl 2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-2-methylpropanoate is as described below.

[1037] Preparation of ethyl 2-((tert-butoxycarbonyl)amino)-2-methylpropionate

[1038]

[1039] Triphenylphosphine (6.18g, 25.00mmol) is dissolved in THF (30mL). Next, DIAD (4.92mL, 25.00mmol) is loaded and stirred at room temperature for 10 minutes. 2-((tert-butoxycarbonyl) amino)-2-methylpropionic acid (5.08g, 25.00mmol) is dissolved in THF (20mL) and added to the reaction mixture, then ethanol (2.19mL, 37.49mmol) is added. The reaction is stirred at room temperature for 1 hour. The solvent is removed under reduced pressure, and the crude product is dissolved in 1:1 Et2O: hexane (120mL). Solid triphenylphosphine oxide is filtered out and the solvent is removed under reduced pressure. The crude product is dissolved in a minimum amount of CH2Cl2 and purified by silica gel chromatography 0-50%EtOAc / Hex to obtain ethyl 2-((tert-butoxycarbonyl) amino)-2-methylpropanoate (2.71g, 47%). 1 H NMR (400 MHz, chloroform-d) δ 4.18 (q, J = 7.1 Hz, 2H), 1.49 (s, 6H), 1.43 (s, 9H), 1.27 (t, J = 7.1 Hz, 3H).

[1040] Preparation of ethyl 2-amino-2-methylpropionate hydrochloride

[1041]

[1042] Ethyl 2-((tert-butoxycarbonyl)amino)-2-methylpropanoate (2.71 g, 11.72 mmol) was dissolved in CH Cl (25 mL) and a 4N HCl solution in dioxane (25 mmol) was slowly added and stirred at room temperature. After 1 hour, TLC confirmed the reaction was complete. The solvent was removed under reduced pressure and the crude product was co-evaporated with Et O twice and then placed under high vacuum to give ethyl 2-amino-2-methylpropanoate hydrochloride (2.02 g, 102%). 1 H NMR (400MHz, DMSO-d6) δ8.70 (s, 3H), 4.18 (q, J = 7.1Hz, 2H), 1.46 (s, 6H), 1.21 (t, J = 7.1Hz, 3H).

[1043] Preparation of ethyl 2-methyl-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propionate (Compound N)

[1044]

[1045] Phenyl dichlorophosphate (0.97 mL, 6.50 mmol) and ethyl 2-amino-2-methylpropanoate hydrochloride (1.09 g, 6.50 mmol) were dissolved in CH2Cl2 (50 mL). The reaction mixture was cooled to 0°C and TEA (1.75 mL, 12.45 mmol) was slowly added. The cooling bath was removed and the reaction mixture was stirred at room temperature. After 2 hours, the mixture was dried over a slurry of water and the mixture was dried over a slurry of water. The slurry ...31 P NMR confirmed that the addition of amino acid was complete. p-Nitrophenol (0.860 g, 6.17 mmol) was loaded, followed by the addition of TEA (0.87, 7.69 mmol). The reaction was stirred at room temperature. After 2 hours, LCMS confirmed that the reaction was complete. The reaction was diluted with Et2O and the TEA*HCl salt was filtered off. The crude product was concentrated and purified by silica gel chromatography (0-50% EtOAc / Hex) to give compound N (1.79 g, 68%). 1 H NMR(400MHz,DMSO-d6)δ8.37-8.21(m,2H),7.55-7.44(m,2H),7.43-7.33(m,2H),7.30-7.0 9(m,3H),6.57(d,J=10.1Hz,1H),3.99(q,J=7.1Hz,2H),1.39(s,6H),1.08(t,J=7.1Hz,3H). 31 P NMR(162MHz,DMSO-d6)δ-2.87.LC / MS:t R =1.65 min, MS m / z=408.97 [M+1]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQFleet; column: Kinetex 2.6μXB-C18 100A, 50×3.00 mm; solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; gradient: 0 min-2.4 min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN, 1.8 mL / min.

[1046] 2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3, 4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-2-methylpropionic acid ethyl ester (Compound 30) preparation

[1047]

[1048] Compound 1 (66 mg, 0.23 mmol) was dissolved in NMP (2.0 mL). The mixture was cooled to 0 ° C and tBuMgCl (1.0 M THF solution, 0.34 mL, 0.34 mmol) was slowly added. The reaction was stirred at 0 ° C for 30 minutes, and then a solution of compound N (139 mg, 0.34 mmol) dissolved in THF (1.0 mL) was added. The cold bath was removed and the reaction solution was placed in a 50 ° C preheated oil bath. After 2 hours, the reaction was cooled to room temperature and quenched with acetic acid and methanol. The crude product was concentrated and purified by reverse phase HPLC without modifier to obtain compound 30 (32 mg, 25% as a mixture of diastereomers). 1 H NMR (400MHz, DMSO-d6) δ7.89 (m, 3H), 7.31 (q, J = 8.1Hz, 2H), 7.22-7.05 (m, 3H),6.87(d,J=4.5,1H),6.80(d,J=4.5Hz,1H),6.27(d,J=11.7,1H),5.81( d,J=9.7,1H),5.35(d,J=5.6Hz,1H),4.64(dt,J=9.0,5.6Hz,1H),4.24(m,2 H),4.11(m,1H),4.04-3.90(m,3H),1.39-1.23(m,6H),1.10(t,J=7.1,3H). 31 P NMR(162MHz,DMSO-d6)δ2.45,2.41.LC / MS:t R =1.03 min, MS m / z=561.03 [M+1]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQFleet; column: Kinetex 2.6μ XB-C18100A, 50×3.00 mm; solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; gradient: 0 min-2.4 min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN, 1.8 mL / min.

[1049] Example 34. 2-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)- Isopropyl 5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-2-methylpropanoate (31)

[1050]

[1051] The preparation of isopropyl 2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-2-methylpropanoate is as described below.

[1052] Preparation of Isopropyl 2-((tert-Butoxycarbonyl)amino)-2-methylpropionate

[1053]

[1054] Triphenylphosphine (6.17g, 25.00mmol) is dissolved in THF (30mL). Next, DIAD (4.92mL, 25.00mmol) is loaded and at room temperature stirred 10 minutes. 2-((tert-butoxycarbonyl) amino)-2-methylpropionic acid (5.07g, 25.00mmol) is dissolved in THF (20mL) and added in the reaction mixture, then add isopropyl alcohol (1.91mL, 25.00mmol). The reaction was stirred at room temperature for 1 hour. Removal of solvent under reduced pressure, the crude product is dissolved in 1: 1 Et o: in hexane (120mL). Filter out solid triphenylphosphine oxide, removal of solvent under reduced pressure. The crude product is dissolved in minimum CH cl in and by silica gel chromatography (0-50% EtOAc / Hex) purification to provide 2-((tert-butoxycarbonyl) amino)-2-methylpropionic acid isopropyl ester (4.09g, 67%). 1 H NMR (400 MHz, chloroform-d) δ 5.03 (p, J = 6.2 Hz, 1H), 1.48 (s, 6H), 1.40 (d, J = 6.2 Hz, 9H), 1.24 (d, J = 6.3 Hz, 6H).

[1055] Preparation of 2-amino-2-methylpropionic acid isopropyl ester hydrochloride

[1056]

[1057] Isopropyl 2-((tert-butoxycarbonyl)amino)-2-methylpropanoate (4.09 g, 16.67 mmol) was dissolved in CH2Cl2 (50 mL) and 4N HCl in dioxane (50 mmol) was slowly added and stirred at room temperature. After 1 hour, the reaction was determined to be complete by TLC. The solvent was removed under reduced pressure and the crude product was co-evaporated twice with Et2O and then placed under high vacuum to give isopropyl 2-amino-2-methylpropanoate hydrochloride (3.06 g, 101%). 1 H NMR (400MHz, DMSO-d6) δ8.61 (s, 3H), 4.96 (p, J = 6.2Hz, 1H), 1.44 (s, 6H), 1.22 (d, J = 6.2Hz, 6H).

[1058] Preparation of isopropyl 2-methyl-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propionate (Compound O) Preparation

[1059]

[1060] Phenyl dichlorophosphate (0.83 mL, 5.58 mmol) and isopropyl 2-amino-2-methylpropanoate hydrochloride (1.01 g, 5.58 mmol) were dissolved in CH2Cl2 (50 mL). The reaction mixture was cooled to 0°C and TEA (1.61 mL, 11.45 mmol) was slowly added. The cooling bath was removed and the reaction mixture was stirred at room temperature. After 2 hours, the mixture was dried over a mixture of 4% ethanol and 1% ethanol. 31 P NMR confirmed that the addition of amino acid was complete. p-Nitrophenol (0.74 g, 5.30 mmol) was loaded, followed by TEA (0.81, 5.84 mmol). The reaction was stirred at room temperature. After 2 hours, LCMS confirmed that the reaction was complete. The reaction was diluted with Et2O and the TEA*HCl salt was filtered off. The crude product was concentrated and purified by silica gel chromatography (0-50% EtOAc / Hex) to give compound O (1.45 g, 62%). 1 H NMR (400MHz, DMSO-d6) δ8.42-8.19(m,2H),7.55-7.43(m,2H),7.39(dd,J=8.6,7.2Hz,2H),7.30- 7.12(m,3H),6.53(d,J=10.1Hz,1H),4.82(hept,J=6.3Hz,1H),1.38(s,6H),1.09(d,J=6.3,6H). 31 P NMR(162MHz,DMSO-d6)δ-2.84.LC / MS:t R =1.73 min, MS m / z=422.92 [M+1]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQFleet; column: Kinetex 2.6μXB-C18100A, 50×3.00 mm; solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; gradient: 0 min-2.4 min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN, 1.8 mL / min.

[1061] 2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3, Isopropyl (4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-2-methylpropanoate (Compound 31) Preparation

[1062]

[1063] Compound 1 (66 mg, 0.23 mmol) was dissolved in NMP (2.0 mL). The mixture was cooled to 0 ° C and tBuMgCl (1.0 M THF solution, 0.57 mL, 0.57 mmol) was slowly added. The reaction was stirred at 0 ° C for 30 minutes, and then a solution of compound O (143 mg, 0.34 mmol) dissolved in THF (1.0 mL) was added. The cold bath was removed and the reaction solution was placed in a 50 ° C preheated oil bath. After 2 hours, the reactant was cooled to room temperature and quenched with acetic acid and methanol. The crude product was concentrated and purified by reverse phase HPLC without modifier to obtain compound 31 (48 mg, 37%, a mixture of diastereomers). 1 H NMR(400MHz,DMSO-d6)δ7.88(m,3H),7.30(td,J=8.5,7.0Hz,2H),7.20-7.04(m ,3H),6.87(d,J=4.5,1H),6.80(d,J=4.5Hz,1H),6.27(d,6.1Hz,1H),5.75(t,J= 9.1Hz,1H),5.34(d,J=5.7Hz,1H),4.81(p,J=6.3Hz,1H),4.71-4.50(m,1H),4.2 3(m,2H),4.11(m,1H),4.03-3.83(m,1H),1.37-1.23(m,6H),1.18-1.04(m,6H). 31 P NMR(162MHz,dmso)δ2.47,2.43.LC / MS:t R =1.08 min, MS m / z=575.06 [M+1]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Kinetex 2.6μXB-C18100A, 50×3.00 mm; solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid; gradient: 0 min-2.4 min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN, 1.8 mL / min.

[1064] Example 35. (S)-2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazol-2-yl)-1,2-diol (oxazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy) (phenoxy)phosphoryl)amino)propanoic acid-2-ethyl Butyl ester (32)

[1065]

[1066] The preparation of (S)-ethylbutyl 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)(phenoxy)phosphoryl)amino)propanoate is as described below.

[1067] Preparation of (3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)dihydrofuran-2(3H)-one.

[1068]

[1069] (3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol (15.0 g) was combined with MTBE (60.0 mL), KBr (424.5 mg), aqueous KHPO (2.5 M, 14.3 mL), and TEMPO (56 mg). The mixture was cooled to approximately 1°C. Aqueous bleach solution (7.9 wt %) was slowly added portionwise until the starting material was completely consumed as indicated by the starch / iodide test. The layers were separated, and the aqueous layer was extracted with MTBE. The combined organic phases were dried over MgSO and concentrated under reduced pressure to yield a solid product.

[1070] Preparation of (4-amino-7-iodopyrrolo[2,1-f][1,2,4]triazine)

[1071]

[1072] To a cold solution of 4-aminopyrrolo[2,1-f][1,2,4]-triazine (10.03 g; 74.8 mmol) in N,N-dimethylformamide (70.27 g) was added N-iodosuccinimide (17.01 g; 75.6 mmol) portionwise while maintaining the contents at approximately 0°C. After the reaction was complete (approximately 3 hours at approximately 0°C), the reaction mixture was transferred to a 1 M aqueous sodium hydroxide solution (11 g NaOH and 276 mL water) while maintaining the contents at approximately 20-30°C. The resulting slurry was stirred at approximately 22°C for 1.5 hours and then filtered. The solid was rinsed with water (50 mL) and dried under vacuum at approximately 50°C to yield 4-amino-7-iodopyrrolo[2,1-f][1,2,4]triazine as a solid. 1 H NMR (400MHz, DMSO-d6) δ7.90 (s, 1H), 7.78 (br s, 2H), 6.98 (d, J = 4.4Hz, 1H), 6.82 (d, J = 4.4Hz, 1H). 13 C NMR (101MHz, DMSO-d6) δ155.7,149.1,118.8,118.1,104.4,71.9.MS m / z=260.97[M+H].

[1073] Preparation of (3R,4R,5R)-2-(4-aminopyrrole) by (4-amino-7-iodopyrrolo[2,1-f][1,2,4]triazine (2,1-f][1,2,4]triazin-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol

[1074]

[1075] In the reactor under nitrogen atmosphere, iodine base 2 (81g) and THF (1.6LV) are added. The gained solution is cooled to about 5 ℃, and TMSCl (68g) is added. Then PhMgCl (345mL, 1.8M THF solution) is slowly added while keeping internal temperature at about≤5 ℃. The reaction mixture is stirred at about 0 ℃ for 30 minutes, then cooled to about-15 ℃. iPrMgCl-LiCl (311mL, 1.1M THF solution) is slowly added while keeping internal temperature below about-12 ℃. After stirring at about-15 ℃ for about 10 minutes, the reaction mixture is cooled to about-20 ℃, THF (400mL) solution of lactone 1 (130g) is added. Then the reaction mixture is stirred at about-20 ℃ for about 1 hour and quenched with AcOH (57mL). The reaction mixture is warming up to about 0 ℃ and is adjusted to pH 7-8 with NaHCO The aqueous solution (5 weight %, 1300mL) is adjusted to pH 7-8. The reaction mixture was then diluted with EtOAc (1300 mL), and the organic layer and aqueous layer were separated. The organic layer was washed with 1N HCl (1300 mL), NaHCO3 aqueous solution (5 wt%, 1300 mL) and salt solution (1300 mL), then anhydrous Na2SO4 was used and concentrated to dryness. Purification by silica gel column chromatography was performed using a gradient consisting of a mixture of MeOH and EtOAc to obtain the product.

[1076] ((2S)-2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propionic acid-ethylbutyl ester) (Sp and Rp Preparation of mixture):

[1077]

[1078] L-alanine 2-ethyl butyl ester hydrochloride (5.0g, 23.84mmol) is combined with dichloromethane (40mL), cooled to about -78 ℃, and dichlorophenyl phosphate (3.65mL, 23.84mmol) is added. Triethylamine (6.6mL, 47.68mmol) is added at about -78 ℃ for about 60 minutes, and the resulting mixture is stirred for 3h at ambient temperature. The reaction mixture is cooled to about 0 ℃, pentafluorophenol (4.4g, 23.84mmol) is added. Triethylamine (3.3mL, 23.84mmol) is added over about 60 minutes. The mixture is stirred for about 3 hours at ambient temperature and concentrated under reduced pressure. The residue is dissolved in EtOAc, washed several times with aqueous sodium carbonate solution, and concentrated under reduced pressure. The residue is purified by silica gel column chromatography using a gradient of EtOAc and hexane (0-30%). The product-containing fractions were concentrated under reduced pressure to give 2-ethylbutyl (2S)-2(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate as a solid. 1 H NMR (400 MHz, chloroform-d) δ 7.41-7.32 (m, 4H), 7.30-7.17 (m, 6H), 4.24-4.16 (m, 1H), 4.13-4.03 (m, 4H), 4.01-3.89 (m, 1H), 1.59-1.42 (m, 8H), 1.40-1.31 (m, 8H), 0.88 (t, J = 7.5 Hz, 12H). 31 P NMR (162 MHz, chloroform-d) δ-1.52. 19 F NMR (377MHz, chloroform-d) δ-153.63,-153.93(m),-160.05(td,J=21.9,3.6Hz),-162.65(qd,J=22.4,20.5,4.5Hz).MS m / z=496[M+H].

[1079] Preparation of the title compound (mixture of Sp and Rp):

[1080]

[1081] At ambient temperature, nucleosides (29 mg, 0.1 mmol), phosphamide (60 mg, 0.12 mmol) and N, N-dimethylformamide (2 mL) were combined. Tert-butylmagnesium chloride (1 M in THF, 0.15 mL) was slowly added. After about 1 hour, the reaction was diluted with ethyl acetate and washed with citric acid aqueous solution (5 wt %), saturated NaHCO aqueous solution and saturated brine solution. The organic phase was dried over Na SO and concentrated under reduced pressure. Residue was purified by silica gel column chromatography using a gradient of methanol and CH Cl (0-5%). The fraction containing the product was concentrated under reduced pressure to provide the product.

[1082] (3aR,4R,6R,6aR)-4-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-6-(hydroxymethyl)-2, 2-Dimethyltetrahydrofuro[3,4-d][1,3]dioxolane-4-carbonitrile:

[1083]

[1084] To a mixture of (2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile (5.8 g, 0.02 mol), 2,2-dimethoxypropane (11.59 mL, 0.09 mol), and acetone (145 mL) was added sulfuric acid (18 M, 1.44 mL) at ambient temperature. The mixture was warmed to approximately 45°C. After approximately 30 minutes, the mixture was cooled to ambient temperature, and sodium bicarbonate (5.8 g) and water (5.8 mL) were added. After 15 minutes, the mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (150 mL) and water (50 mL). The aqueous layer was extracted with ethyl acetate (2 x 50 mL). The combined organic phases were dried over sodium sulfate and concentrated under reduced pressure to give crude (2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile. 1 H NMR (400MHz, CD3OD) δ7.84(s,1H),6.93(d,J=4.6Hz,1H),6.89(d,J=4.6Hz,1H),5.40(d,J=6.7Hz,1 H),5.00(dd,J=6.7,3.3Hz,1H),4.48-4.40(m,1H),3.81-3.72(m,2H),1.71(s,3H),1.40(s,3H).MS m / z=332.23[M+1].

[1085] (2S)-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano Preparation of 2-ethylbutyl methyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate Preparation:

[1086]

[1087] Acetonitrile (100 mL) was combined with 2-ethylbutyl (2S)-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (9.6 g, 21.31 mmol), the substrate alcohol (6.6 g, 0.02 mol), and magnesium chloride (1.9 g, 19.91 mmol) at ambient temperature. The mixture was stirred for approximately 15 minutes, and N,N-diisopropylethylamine (8.67 mL, 49.78 mmol) was added. After approximately 4 hours, the reaction was diluted with ethyl acetate (100 mL), cooled to approximately 0°C, and combined with aqueous citric acid (5 wt%, 100 mL). The organic phase was washed with aqueous citric acid (5 wt%, 100 mL), saturated aqueous ammonium chloride (40 mL), aqueous potassium carbonate (10 wt%, 2 x 100 mL), and saturated brine (100 mL). The organic phase was dried over sodium sulfate and concentrated under reduced pressure to provide the crude product. 1 H NMR(400MHz,CD3OD)δ7.86(s,1H),7.31-7.22(m,2H),7.17-7.09(m,3H),6.93-6.84(m,2H ),5.34(d,J=6.7Hz,1H),4.98(dd,J=6.6,3.5Hz,1H),4.59-4.50(m,1H),4.36-4.22(m,2H ),4.02(dd,J=10.9,5.7Hz,1H),3.91(dd,J=10.9,5.7Hz,1H),3.83(dq,J=9.7,7.1Hz,1H) ,1.70(s,3H),1.50-1.41(m,1H),1.39(s,3H),1.36-1.21(m,7H),0.86(t,J=7.4Hz,6H).MS m / z=643.21[M+1].

[1088] (S)-2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)- 2-Ethylbutyl 5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate Preparation of (Compound 32)

[1089]

[1090] Crude acetone reduction (12.85g) is merged with tetrahydrofuran (50mL) and concentrated under reduced pressure. The residue is dissolved in tetrahydrofuran (100mL), cooled to about 0 ℃, and slowly added concentrated HCl (20mL). The mixture is warmed to ambient temperature. After the initial acetone reduction indicated by HPLC analysis is consumed, water (100mL) is added, followed by saturated sodium bicarbonate aqueous solution (200mL). The mixture is extracted with ethyl acetate (100mL), the organic phase is washed with saturated saline solution (50mL), dried over sodium sulfate and concentrated under reduced pressure. The gradient (0 to 20%) of methanol and ethyl acetate is used to purify the residue by silica gel column chromatography. The fraction containing the product is concentrated under reduced pressure to provide the product.

[1091] B. Antiviral activity

[1092] Another aspect of the present invention relates to a method of inhibiting a viral infection comprising the step of treating a sample or individual suspected of being in need of such inhibition with a composition of the present invention.

[1093] Within the scope of the present invention, samples suspected of containing viruses include natural or artificial materials, such as living organisms; tissue or cell cultures; biological samples such as biological material samples (blood, serum, urine, cerebrospinal fluid, tears, sputum, saliva, tissue samples, etc.); laboratory samples; food, water or air samples; biological product samples such as cell extracts, especially recombinant cells that synthesize the desired glycoprotein; etc. Typically, the sample will be suspected of containing an organism that induces viral infection, usually a pathogen such as a tumor virus. The sample can be contained in any medium, including water and organic solvent / water mixtures. The sample includes organisms such as humans, as well as artificial materials such as cell cultures.

[1094] If desired, the antiviral activity of the compounds of the invention can be observed after administration of the composition by any method, including direct and indirect methods for detecting such activity. Quantitative, qualitative, and semi-quantitative methods for determining such activity are contemplated. Typically, one of the above-described screening methods is employed; however, any other method, such as observing the physiological properties of living organisms, is also suitable.

[1095] The antiviral activity of the compounds of the present invention can be measured using known standard screening protocols. For example, the antiviral activity of the compounds can be measured using the following general protocol:

[1096]

[1097] HCS: High Content Imaging

[1098] HeLa: HeLa epithelial cells (cervical cancer)

[1099] Example 36. Antiviral activity and cytotoxicity assays for Lassa virus and Junin virus

[1100] The antiviral activity of compounds 1, 9, and 32 against Lassa virus (LASV) and Junin virus (JUNV) was measured. All studies using wild-type viruses were conducted in a Biosafety Level 4 (BSL-4) facility at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). Antiviral studies using an attenuated strain of JUNV were conducted in a BSL-2 laboratory at Utah State University. Lassa virus antiviral assays were performed using HeLa cells. Junin virus antiviral assays were performed in Vero and HeLa cells.

[1101] Antiviral assays were performed in 384 or 96-well plates in BSL-4 containment using a high-content imaging system to quantify viral antigen production as a measure of viral replication. A "no virus" control (column 2) and a "1% DMSO" control (column 3) were included on each plate to determine 0% and 100% viral replication signals, respectively. The primary antibodies used to detect viral antigens were mm L52-161-6 anti-GP; LASV and mm Y-GQC03_BF11 anti-GP; JUNV and DyLight 488 anti-mouse IgG were used as secondary detection antibodies. The primary antibody was diluted 1000-fold in blocking buffer (1×PBS containing 3% BSA) and added to each well of the assay plate. The assay plate was incubated at room temperature for 60 minutes. The primary antibody was removed and the cells were washed 3 times with 1×PBS. The secondary antibody was diluted 1000-fold in blocking buffer and added to each well of the assay plate. The assay plate was incubated at room temperature for 60 minutes. Cell nuclei were stained using Draq5 (Biostatus, Shepshed Leicestershire, UK, catalog number DR05500) diluted in 1× PBS. Cell images were acquired using a Perkin Elmer Opera confocal microscope (Perkin Elmer, Waltham, MA) using a 10× air objective to collect 5 images per well. Virus-specific antigens were quantified by measuring fluorescence emission at a wavelength of 488 nm, and nuclei were quantified by measuring fluorescence emission at a wavelength of 640 nm. Z' values ​​for all antiviral assays were >0.3.

[1102] The percentage inhibition of each tested concentration relative to the 0% and 100% inhibition controls was calculated, and the EC of each compound was determined by nonlinear regression. 50 The value was taken as the effective concentration of the compound that inhibited viral replication by 50%.

[1103] Example 37. Junin Virus Assay - Vero

[1104] Vero or Vero E6 cells are seeded in 96-well plates at 20,000 cells / well in 100 μL MEM+2% FBS. The compound diluted in DMSO is mixed with 120 μL of MEM+2% FBS. 100 μL of each test compound is transferred to 2 wells of a 96-well plate. 20 μL of virus solution in MEM+20% FBS is added so that the final test concentration is 47, 4.7, 0.47, 0.047 μM and the multiplicity of infection is 0.003 pfu / cell. The test plate is incubated until the untreated virus control approaches the maximum cytopathic effect (CPE) (5-7 days). The plate is then stained with neutral red dye for 2 hours, then eluted in citrate / ethanol buffer and read on a spectrophotometer at 540 nm. EC is calculated by regression analysis 50 The values ​​were expressed as the concentration of test compound required to reduce virus-induced CPE by 50% as measured by neutral red staining.

[1105] Example 38. Junin virus assay-HeLa

[1106] HeLa cells were seeded at 2,000 cells per well in 384-well plates, and compounds were added to the assay plates as described in Section 3.2.1. The assay plates were transferred to a BSL-4 facility and infected with 0.3 pfu / cell of JUNV, resulting in ~50% cell expression of viral antigen within 48 hours. The assay plates were incubated for 48 hours, and viral replication was quantified by immunostaining with antibodies that recognize viral glycoproteins.

[1107] Example 39. Lassa virus assay

[1108] HeLa cells were seeded at 2,000 cells per well in 384-well plates, and compounds were added to the assay plates as described in Section 3.2.1. The assay plates were transferred to a BSL-4 facility and infected with 0.1 pfu / cell of LASV, resulting in >60% cell expression of viral antigen within 48 hours. The assay plates were incubated for 48 hours, and viral replication was quantified by immunostaining with antibodies that recognize viral glycoproteins.

[1109] Table 2: Lassa virus and Junin virus antiviral assays

[1110] Table 2: In vitro antiviral activity of compounds 1, 9 and 32 against arenaviruses

[1111]

[1112] ND not detected

[1113] JUNV = Junin virus, LASV = Lassa virus

[1114] Example 40. MERS-CoV and SARS-CoV antiviral activity and cytotoxicity assays

[1115] The antiviral activities of compound 9 and compound 32 against MERS virus (MERS-CoV) and SARS virus (SARS-CoV) were measured.

[1116] Antiviral assays were performed at USAMRIID and the University of North Carolina at Chapel Hill.

[1117] Example 41. MERS-CoV antiviral assay (USAMRIID)

[1118] Vero E6 cells were seeded in 384-well plates, and serial dilutions of compound 32 or compound 9 were added to the assay plate by direct titration using an HP D300 digital dispenser (Hewlett-Packard, Palo Alto, CA). The plates were transferred to a BSL-4 kit and infected with MERS-CoV (strain Jordan N3) at a multiplicity of infection of 0.5 plaque-forming units (pfu) per cell. The infected cultures were incubated for 48 hours. The level of viral replication in compound-treated and control vehicle-treated cultures was determined by quantifying the level of virus-specific antigens after immunostaining with antibodies against MERS-CoV Spike (S) protein. The primary antibody (40069-RP02rb-HCoV-EMC / 2012 Spike (S) protein) was diluted 1000-fold in blocking buffer (1× phosphate-buffered saline (PBS) containing 3% BSA) and added to each well of the assay plate. The assay plate was incubated at room temperature for 60 minutes. The primary antibody was removed and the cells were washed 3 times with 1× PBS. The secondary detection antibody was an anti-rabbit IgG conjugated to Dylight488 (Thermo Fisher Scientific, Waltham, MA, catalog number 405310). The secondary antibody was diluted 1000-fold in blocking buffer and added to each well of the assay plate. The assay plate was incubated at room temperature for 60 minutes. Cell nuclei were stained using Draq5 (Biostatus, Shepshed Leicestershire, UK, Cat#DR05500) diluted in 1× PBS. Cells were counterstained with CellMask Deep Red (Thermo Fisher Scientific, Waltham, MA, catalog number C10046) to enhance detection of the cytoplasmic compartment. Cell images were acquired using a Perkin Elmer Opera confocal microscope (Perkin Elmer, Waltham, MA) using a 10× air objective to collect 5 images per well. Virus-specific antigens were quantified by measuring fluorescence emission at a wavelength of 488 nm, and nuclei were quantified by measuring fluorescence emission at a wavelength of 640 nm. High-content image analysis was performed to quantify the percentage of infected cells and cell viability. Dose-response analysis to determine EC50 values ​​was performed using GeneData Screener software using the Levenberg-Marquardt algorithm for a curve-fitting strategy.

[1119] Example 42. MERS-CoV and SARS-CoV antiviral assays

[1120] HAE cell cultures isolated from lung tissue were cultured at the air-liquid interface for 6 weeks to promote differentiation. The apical surface of the HAE culture was washed 24 hours and 1 hour before infection with 1×PBS, and then infected with 1×PBS at 37°C for>1 hour. Recombinant MERS-CoV expressing red fluorescent protein (MERS-CoV RFP) and recombinant SARS-CoV expressing green fluorescent protein (SARS-CoVGFP) were used to infect differentiated HAE cultures at a multiplicity of infection of 0.1pfu / cell. In order to infect HAE cultures, the top washing solution was removed, the viral inoculum was added, and the inoculated culture was incubated at 37°C for 2.5 hours. The inoculum was removed and the apical surface of the HAE culture was washed 3 times with 500μL 1×PBS to remove residual viruses. 5 3-fold serial dilutions of compound 9 were prepared starting at 10μM in triplicate and added to the HAELI culture medium on the basolateral side of the culture approximately 30 minutes before infection. Viral replication was assessed by fluorescence imaging of cell culture after 48 hours of incubation. In addition, viral replication was quantified by measuring infectious virus yield in HAE apical washes by plaque assay on Vero cell monolayers and by quantifying viral RNA yield from total cellular RNA by real-time PCR assay.

[1121] Table 3: MERS antiviral assay

[1122] Table 3: In vitro antiviral activity of compound 32 against coronavirus

[1123] <![CDATA[EC 50 (μM)]]> Determination Virus MERS-CoV cell lines Vero Compound 9 0.46 Compound 32 0.58

[1124] MERS = Middle East Respiratory Syndrome

[1125] Example 43. Real-time PCR assay for MERS-CoV and SARS-CoV

[1126] At 48 hours post-infection, primary HAE cultures from the antiviral assay described above were harvested in 500 μL TRIzol. RNA was purified using the Direct-zol RNA MiniPrep Kit (Zymo Research Corporation, Irvine, CA, USA). First-strand cDNA was generated for each sample using SuperScript III (Life Technologies, Grand Island, NY, USA) incubated at 55°C. After first-strand cDNA generation, ORF1 (genomic RNA) and ORF8 or ORF9 (MERS-CoV and SARS-CoV subgenomic RNA, respectively) were quantified by real-time PCR using the following primers: MERS-CoV: leader sequence forward (5′-GAA TAG CTT GGC TAT CTC AC-3′), ORF1 reverse (5′-CAC AAT CCC ACC AGA CAA-3′), ORF8 reverse (5′-TTG TTA TCG GCA AAG GAA AC-3′); and SARS-CoV: leader sequence forward (5′-AGC CAACCA ACC TCG ATC TCT TGT-3′), ORF1 reverse (5′-TGA CAC CAA GAA CAA GGC TCT CCA-3′), ORF9 reverse (5′-ATT GGT GTT GAT TGG AAC GCC CTG-3′). Reads were normalized to GAPDH using the following primers: GAPDH forward (5'-TGC ACC ACC AAC TGC TTA GC-3') and GAPDH reverse (5'-GGC ATG GACTGT GGT CAT GAG-3'). Results are expressed as the log10-fold change in viral ORF1 and ORF8 encoding RNA (MERS-CoV) and ORF9 encoding RNA (SARS-CoV) copy number in treated versus untreated cells using the ΔΔCt method {10431}.

[1127] Example 44. In vitro efficacy in Calu-32B4 cells

[1128] 48 hours before infection, Calu-3 2B4 cells were plated at 5 × 10 4Cells / well were seeded in 96-well black-walled, clear-bottom plates. 24 hours before infection, the medium was changed. A 20 mM stock solution of compound 32 was serially diluted in 100% DMSO in 3-fold increments to obtain a ten-point dilution series. MERS-nLUC was diluted in DMEM 10% FBS and 1% antibiotic / antimycin to obtain a multiplicity of infection (MOI) of 0.08. Each drug dilution was infected in triplicate for 1 hour, after which the virus was aspirated, the culture was rinsed once, and fresh medium containing drug or vehicle was added. At 48 hours after infection, viral replication was quantified by nanoluciferase assay (Promega) on a Spectramax (Molecular Devices) plate reader according to the manufacturer's protocol. For our 100% inhibition control, diluted MERS-nLUC was exposed to shortwave ultraviolet light (LLC, Upland, CA) for 6 minutes to inhibit the ability of the virus to replicate. For our 0% inhibition control, cells were infected in the presence of vehicle. DMSO was kept constant at 0.05% by volume (v / v) in all conditions. Values ​​from triplicate wells for each condition were averaged and compared to controls to generate a percent inhibition value for each drug dilution. 50 The value was defined as the concentration at which viral replication was reduced by 50%. Data were analyzed using GraphPad PrisM 6.0 (La Jolla, CA). EC values ​​were calculated by nonlinear regression analysis using a dose-response (variable slope) equation (four-parameter logistic equation). 50 and CC 50 Value: Y = bottom + (top - bottom) / (1 + 10^((LogEC 50 = -X)*Hill Slope). The "bottom" and "top" values ​​are defined by the minimum and maximum Y values. The Hill Slope is a parameter used to define the steepness of the dose-response curve. Calculate EC 50 and CC 50 Values ​​are presented as the mean of 2–4 independent experiments.

[1129] Table 4: Antiviral activity and cytotoxicity of compound 1 and compound 32 against MERS-CoV and SARS-CoV.

[1130]

[1131] 1 All values ​​are the mean of >3 independent experiments. HAE = human airway epithelial cells. Calu-3 = human lung epithelial cell line Calu-3 (Calu3-2B4). HAE studies were performed by three donors.

[1132] Example 45. Evaluation of Subcutaneous Compound 32 Against Severe Acute Respiratory Syndrome in Esterase-Deficient (Ces1C- / -) Mice Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)

[1133] Male and female mice (25-28 weeks) were genetically deficient in carboxylesterase 1C (Ces1C- / -) (Jackson Laboratories stock 014096). (Ces1C- / -) mice were used because rodents express high levels of carboxylesterase activity in plasma relative to other animal species, thereby reducing the plasma half-life of compound 32. Genetic deletion of carboxylesterase 1C improved the plasma stability of compound 32, resulting in a pharmacokinetic profile similar to that observed in humans and other animal species.

[1134] The study design is documented in Table 4. The efficacy study was conducted in an animal biosafety level 3 (ABSL3) facility. All work was performed according to protocols approved by the UNC Chapel Hill Institutional Animal Care and Use Committee, in accordance with guidelines established by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and the United States Department of Agriculture (USDA).

[1135] Table 4: Experimental design (subcutaneous injection)

[1136]

[1137]

[1138] Ketamine / xylazine were exposed to 4 Group 1 (vehicle), Group 2 (Compound 32BID 25 mg / kg) and Group 3 (Compound 32QD 50 mg / kg) were anesthetized with pfu of SARS-CoV / 50ul. Group 4 (vehicle) and Group 5 (Compound 32BID 25 mg / kg) remained uninfected and used as controls for whole-body plethysmography evaluation. The vehicle contained a 12% aqueous solution of sulfobutyl ether-β-cyclodextrin (containing HCl / NaOH, pH 5.0). On Day 0, the animals were exposed to the virus. On Day 2 and Day 5 after infection, the animal groups were euthanized by an overdose of isoflurane, and the left lobe of the lung was placed in a 2 mL screw-cap tube with 1 mL DPBS and glass beads and frozen at -80°C until analysis by plaque assay. The right lower lobe was placed in 10% formalin buffer and stored at 4°C until histological analysis.

[1139] Changes in lung function were determined by whole body plethysmography (WBP, Buxco pulmonary function testing system, Data Sciences International). After acclimatization for 30 minutes in the plethysmograph chamber, 11 respiratory responses and several quality control indicators were measured continuously, every 2 seconds for 5 minutes, for a total of 150 data points. The mean value of each parameter was determined in DSI Finepoint software.

[1140] Histological analysis was per...

Claims

1. A therapeutically effective amount of a compound having the following structure: Or the use of a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a coronavirus infection in a person in need thereof, wherein the coronavirus infection is caused by a SARS virus or a MERS virus, wherein the compound or a pharmaceutically acceptable salt thereof inhibits a coronavirus RNA-dependent RNA polymerase.

2. The use according to claim 1, wherein a compound having the following structure is used:

3. The use according to claim 1 or 2, wherein the medicament further comprises a pharmaceutically acceptable carrier or excipient.

4. The use according to claim 1 or 2, wherein the medicament further comprises a therapeutically effective amount of at least one other therapeutic agent selected from corticosteroids, anti-inflammatory signal transduction regulators, β2-adrenergic receptor agonists bronchodilators, anticholinergics, mucolytics, hypertonic saline and other drugs for treating coronavirus virus infections; or mixtures thereof.

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