Methods of treating arenaviridae and coronaviridae viral infections

Abstract

Methods for treating infections of Arenaviridae and Coronaviridae viruses by administering a nucleoside of Formula I and its prodrug, 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] This application is a divisional application of the invention patent application filed on September 16, 2016, with application number 201680066796.8 and invention title "Method for treating infections caused by viruses of the Arenaviridae and Coronaviridae families".

[0002] Cross-references to related applications

[0003] This patent application claims the benefit 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, pursuant to 35 U.S. SC §119(e). The entirety of the above applications is incorporated herein by reference. Technical Field

[0004] This invention generally relates to methods and compounds for treating infections caused by viruses of the Arenaviridae family, particularly methods and nucleosides and prodrugs for treating Lassa virus and Junin virus. This invention also generally relates to methods and compounds for treating infections caused by viruses of the Coronaviridae family, particularly methods and nucleosides and prodrugs for treating SARS virus and MERS virus. Background Technology

[0005] Lassa virus is a segmented negative-sense RNA virus belonging to the Arenaviridae family. Based on serological cross-reactivity, phylogenetic relationships, and geographic distribution, arenaviruses are further subdivided into Old World and New World virus complexes (Wulff, 1978; Bowen, 1997). The New World arenavirus complex includes viruses circulating in North America (i.e., White Water Arroyo (WWAV), Tamiami (TAMV), and Bell Canyon (BCNV) viruses) and South America (i.e., Tacaribe (TACV), Junin (JUNV), Machupo (MACV), Guanarito (GTOV), and Sabia (SABV) viruses). The Old World complex includes arenaviruses circulating in Africa, Europe, and Asia (i.e., Lymphocytic Gangliocytic Meningitis (LCMV) and Lassa (LASV) viruses). Because LCMV is associated with the globally migrating *Mus domesticus* and *M. musculus* (Salazar-Bravo, 2002), it is distributed worldwide. However, the reservoir rodent species range limits the geographic distribution of arenaviruses. LASV's reservoir hosts are rodents of the genus *Mastomys*, which are prevalent 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 prevalent in West Africa, Argentina, Bolivia, Venezuela, and Brazil, respectively. Recently, Lujo (LUJV) and Chapare (CHAPV) viruses originating in Zambia and Bolivia have also been discovered (Breise, 2009; Delgado, 2008).

[0006] Lassa virus (LASV) is endemic to West Africa, with an estimated 300,000–500,000 infections annually (McCormick, 1987). Transmission occurs through contact with infected rodents (Mastomys natalensis) or virus-contaminated rodent excrement, and human-to-human transmission has been documented, particularly in hospital settings (McCormick, 1987). Illness caused by LASV ranges from subclinical infection to mild to severe hemorrhagic fever associated with multiple organ failure. Mortality associated with LASV infection is variable, ranging from approximately 2% to 15% of hospitalized cases, and can exceed 50% in some outbreaks (McCormick, 1987; Fisher-Hoch, 1995). Despite the high incidence and associated morbidity and mortality, there is no approved treatment for human LASV infection. Maintenance therapy and early administration of ribavirin are the current standard of care.

[0007] LASV initially infects monocytes, macrophages, and dendritic cells and spreads systemically, producing primary viremia that leads to infection of internal organs. Viral replication causes elevated levels of inflammatory cytokines and the development of coagulopathy, resulting in vascular leakage, hypovolemic shock, and multiple organ failure (Hensley, 2011).

[0008] Arenavirus replication is catalyzed by the L polymerase protein, which utilizes a viral RNA template consisting of a genomic RNA encapsulated by the viral nucleocapsid protein NP and containing viral ribonucleoproteins (RNPs) (Buchmeier, 2007). Replication begins upon viral entry into the host cell, where the L polymerase, associated with viral RNPs, initiates transcription from the genomic promoter located at the 3' end of the L and S segments of each genomic RNA region. Primary transcription results in the synthesis of NP and L polymerase mRNAs encoded in the antigenomic direction from the S and L segments. Transcription terminates distal to a stem-loop (SL) structure within the intergenic region (IGR). Arenavirus utilizes a capping strategy to acquire a capped structure for its cellular mRNA to facilitate translation. Capping is mediated by the endonuclease activity of the L polymerase, which co-produces capped, non-polyadenylated mRNA through the cap-binding activity of the NP. Subsequently, the L polymerase moves through the IGR in replicase mode to produce full-length complementary antigenomic RNA (agRNA). These agRNAs serve as templates for synthesizing GPCs and Z mRNAs encoded along the genome direction from the S and L regions, respectively, as well as for synthesizing full-length genomic RNA (gRNA) (Buchmeier, 2007; Franze-Fernandez, 1987; Meyer, 1993; Qi, 2010; Lelke, 2010; Morin, 2010).

[0009] Human coronaviruses were first discovered in the mid-1960s; they are common viruses that infect most people at some point in their lives, typically causing mild to moderate upper respiratory and gastrointestinal illness. This novel coronavirus, known as the "MERS coronavirus" (MERS-CoV or MERS), was first reported in Saudi Arabia in 2012 and has spread to several other countries. The coronavirus responsible for Severe Acute Respiratory Syndrome (SARS), SARS-CoV, was first identified in China in 2002 and led to global outbreaks in 2002 and 2003. Summary of the Invention

[0010] Methods and compounds for treating infections caused by viruses of the Arenaviridae family are provided.

[0011] A method for treating arenaviridae infections in people of need is provided, comprising administering a therapeutically effective amount of a compound of formula I:

[0012]

[0013] Or its pharmaceutically acceptable salts or esters;

[0014] in:

[0015] Each R 1 It is H or halogen;

[0016] Each R 2 R 3 R 4 Or R 5 Independently for H, OR a 、N(R a )2, N3, CN, NO2, S(O)nR a Halogen, (C1-C8)alkyl, (C4-C8)carbocycloalkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)ynyl, (C2-C8)substituted ynyl;

[0017] Or any two R atoms on adjacent carbon atoms 2 R 3 R 4 Or R 5 Together they form -O(CO)O- or together with the ring carbon atoms they are attached to form a double bond;

[0018] R 6 Is it 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)carbocycloalkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)ynyl, (C2-C8)substituted ynyl or (C6-C 20 aryl (C1-C8) alkyl;

[0019] R7 Choose from the following groups:

[0020] 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 ,

[0021] Each R 11 Or R 12 (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)ynyl or (C6-C 20 aryl (C1-C8) alkyl groups are independently optionally bonded by one or more halogens, hydroxyl groups, CN, N3, N(R) a )2 or OR a Substitution; and wherein one or more non-terminal carbon atoms of each of the (C1-C8) alkyl groups may optionally be replaced by -O-, -S- or -NR. a -replace,

[0022]

[0023]

[0024] in:

[0025] R c Selected from phenyl, 1-naphthyl, 2-naphthyl,

[0026]

[0027] R d It is H or CH3;

[0028] R e1 and R e2 Each is independently H, (C1-C6)alkyl, or benzyl;

[0029] R f Selected from H, (C1-C8)alkyl, benzyl, (C3-C6)cycloalkyl, and -CH2-(C3-C6)cycloalkyl;

[0030] R gSelected from (C1-C8)alkyl, -O-(C1-C8)alkyl, benzyl, -O-benzyl, -CH2-(C3-C6)cycloalkyl, -O-CH2-(C3-C6)cycloalkyl and CF3; and

[0031] n' is selected from 1, 2, 3, and 4; and

[0032] d) The following groups:

[0033]

[0034] in:

[0035] Q represents O, S, NR. + N(O)(R), N(OR), + N(O)(OR) or N-NR2;

[0036] Z 1 and Z 2 Together for -Q 1 (C(R y )2)3Q 1 -;

[0037] in

[0038] Each Q 1 Independently O, S, or NR; and

[0039] Each R y 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 Rs on the same carbon atom. y Together they form a carbon ring with 3 to 7 carbon atoms;

[0040] Each Q 2 Independently for O, S, NR, + N(O)(R), N(OR), + N(O)(OR) or N-NR2; or

[0041] Z 1 and Z 2 Each group is an independent group of formula Ia:

[0042]

[0043] in:

[0044] Each Q 3 Independently for bond, O, CR2, NR, + N(O)(R), N(OR), + N(O)(OR), N-NR2, S, SS, S(O) or S(O)2;

[0045] M2 is 0, 1, or 2;

[0046] Each R x Independently for R y Or the following formula:

[0047]

[0048] in:

[0049] Each M1a, Mlc, and M1d is independently 0 or 1;

[0050] M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0051] Z 3 It is Z 4 or Z 5 ;

[0052] Z 4 It is R, -C(Q) 2 )R y -C(Q) 2 )Z 5 -SO2R y or -SO2Z 5 ;and

[0053] Z 5 It is a carbon ring or a hetero ring, where Z 5 Independently controlled by 0 to 3 R y Group substitution;

[0054] R 8It is 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)ynyl, (C4-C8)carbocycloalkyl, (C6-C 20 Optionally substituted aryl groups, optionally substituted heteroaryl groups, -C(=O)(C1-C8)alkyl groups, -S(O) n (C1-C8)alkyl, (C6-C 20 aryl (C1-C8) alkyl, OR 11 or SR 11 ;

[0055] Each R 9 Or R 10 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 ;

[0056] Each R 11 Or R 12Independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)ynyl, (C4-C8)carbocycloalkyl, (C6-C 20 Optionally substituted aryl groups, optionally substituted heteroaryl groups, -C(=O)(C1-C8)alkyl groups, -S(O) n (C1-C8)alkyl or (C6-C8)alkyl 20 ) aryl (C1-C8) alkyl; or R 11 and R 12 Together with nitrogen atoms that are all connected to them, they form 3-7 membered heterocycles, wherein any carbon atom of said heterocycle may optionally be -O-, -S-, or -NR. a -replace;

[0057] Each R a Independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)ynyl, (C6-C 20 )aryl (C1-C8)alkyl, (C4-C8)carbocycloalkyl, -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,

[0058] Each R is independently H, (C1-C8)alkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)ynyl, (C2-C8)substituted ynyl, (C6-C 20 )Aryl, (C6-C 20 ) substituted aryl, (C2-C 20 Heterocyclic groups, (C2-C) 20 ) substituted heterocyclic groups, (C6-C 20 aryl (C1-C8) alkyl or substituted (C6-C8) 20 aryl (C1-C8) alkyl;

[0059] Each n is independently 0, 1, or 2; and

[0060] Each R 2 R 3 R 5 R 6 R 11 Or R 12 Each (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) ynyl or (C6-C 20 aryl (C1-C8) alkyl groups are independently optionally bonded by one or more halogens, hydroxyl groups, CN, N3, N(R) a )2 or ORa Substitution; and wherein one or more non-terminal carbon atoms of each of the (C1-C8) alkyl groups may optionally be replaced by -O-, -S- or -NR. a -replace.

[0061] In another embodiment, the method comprises administering a therapeutically effective amount of a racemic, enantiomer, diastereomer, tautomer, polymorph, pseudopolymorph, amorphous form, hydrate or solvate, or a pharmaceutically acceptable salt or ester thereof of a compound of formula I to a mammal in need of it.

[0062] In another embodiment, the method includes treating a person in need of a salamander infection by administering a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt or ester thereof.

[0063] In another embodiment, the method includes treating a person in need of Lassa virus infection 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 a person in need of humpy virus infection by administering a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt or ester thereof.

[0065] In another embodiment, a method of treating a person in need of a salamander infection includes 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 with a pharmaceutically acceptable diluent or carrier.

[0066] In another embodiment, a method of treating a person in need of a salamander infection includes 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 with at least one additional therapeutic agent.

[0067] In another embodiment, the method includes administering a therapeutically effective amount of a combination of pharmaceutical agents 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 infectious arenaviruses.

[0068] In another embodiment, this application provides a method for inhibiting isnaviridae RNA-dependent RNA polymerase, comprising contacting cells infected with isnaviridae viruses with an effective amount of a compound of formula I or a pharmaceutically acceptable salt, solvate and / or ester thereof.

[0069] In another embodiment, the use of a compound of formula I or a pharmaceutically acceptable salt, solvate, and / or ester thereof for the treatment of viral infections caused by viruses of the Arenaviridae family is provided.

[0070] A method for treating coronavirus infections in people in need is provided, comprising administering a therapeutically effective amount of a compound of formula I:

[0071]

[0072] Or its pharmaceutically acceptable salts or esters;

[0073] in:

[0074] Each R 1 It is H or halogen;

[0075] Each R 2 R 3 R 4 Or R 5 Independently for H, OR a 、N(R a )2, N3, CN, NO2, S(O)nR a Halogen, (C1-C8)alkyl, (C4-C8)carbocycloalkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)ynyl, (C2-C8)substituted ynyl;

[0076] Or any two R atoms on adjacent carbon atoms 2 R 3 R 4 Or R 5 Together they form -O(CO)O- or together with the ring carbon atoms they are attached to form a double bond;

[0077] R 6 Is it 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 12Halogen, (C1-C8)alkyl, (C4-C8)carbocycloalkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)ynyl, (C2-C8)substituted ynyl or (C6-C 20 aryl (C1-C8) alkyl;

[0078] R 7 Choose from the following groups:

[0079] 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 ,

[0080] Each R 11 Or R 12 (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)ynyl or (C6-C 20 aryl (C1-C8) alkyl groups are independently optionally bonded by one or more halogens, hydroxyl groups, CN, N3, N(R) a )2 or OR a Substitution; and wherein one or more non-terminal carbon atoms of each of the (C1-C8) alkyl groups may optionally be replaced by -O-, -S- or -NR. a -replace,

[0081]

[0082] in:

[0083] R c Selected from phenyl, 1-naphthyl, 2-naphthyl,

[0084]

[0085] R d It is H or CH3;

[0086] R e1 and R e2 Each is independently H, (C1-C6)alkyl, or benzyl;

[0087] R fSelected from H, (C1-C8)alkyl, benzyl, (C3-C6)cycloalkyl, and -CH2-(C3-C6)cycloalkyl;

[0088] 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

[0089] n' is selected from 1, 2, 3, and 4; and

[0090] d) The following groups:

[0091]

[0092] in:

[0093] Q represents O, S, NR. + N(O)(R), N(OR), + N(O)(OR) or N-NR 2 ;

[0094] Z 1 and Z 2 Together for -Q 1 (C(R y )2)3Q 1 -;

[0095] in

[0096] Each Q 1 Independently O, S, or NR; and

[0097] Each R y 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 Rs on the same carbon atom. y Together they form a carbon ring with 3 to 7 carbon atoms;

[0098] Each Q 2 Independently for O, S, NR, + N(O)(R), N(OR), + N(O)(OR) or N-NR2; or

[0099] Z 1 and Z 2 Each group is an independent group of formula Ia:

[0100]

[0101] in:

[0102] Each Q 3 Independently for bond, O, CR2, NR, + N(O)(R), N(OR), + N(O)(OR), N-NR2, S, SS, S(O) or S(O)2;

[0103] M2 is 0, 1, or 2;

[0104] Each R x Independently for R y Or the following formula:

[0105]

[0106] in:

[0107] Each M1a, Mlc, and M1d is independently 0 or 1;

[0108] M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0109] Z 3 It is Z 4 or Z 5 ;

[0110] Z 4 It is R, -C(Q) 2 )R y -C(Q) 2 )Z 5 -SO2R y or -SO2Z 5 ;and

[0111] Z 5It is a carbon ring or a hetero ring, where Z 5 Independently controlled by 0 to 3 R y Group substitution;

[0112] R 8 It is 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)ynyl, (C4-C8)carbocycloalkyl, (C6-C 20 Optionally substituted aryl groups, optionally substituted heteroaryl groups, -C(=O)(C1-C8)alkyl groups, -S(O) n (C1-C8)alkyl, (C6-C 20 aryl (C1-C8) alkyl, OR 11 or SR 11 ;

[0113] Each R 9 Or R 10 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 SR11 ;

[0114] Each R 11 Or R 12 Independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)ynyl, (C4-C8)carbocycloalkyl, (C6-C 20 Optionally substituted aryl groups, optionally substituted heteroaryl groups, -C(=O)(C1-C8)alkyl groups, -S(O) n (C1-C8)alkyl or (C6-C8)alkyl 20 ) aryl (C1-C8) alkyl; or R 11 and R 12 Together with nitrogen atoms that are all connected to them, they form 3-7 membered heterocycles, wherein any carbon atom of said heterocycle may optionally be -O-, -S-, or -NR. a -replace;

[0115] Each R a Independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)ynyl, (C6-C 20 )aryl (C1-C8)alkyl, (C4-C8)carbocycloalkyl, -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,

[0116] Each R is independently H, (C1-C8)alkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)ynyl, (C2-C8)substituted ynyl, (C6-C 20 )Aryl, (C6-C 20 ) substituted aryl, (C2-C 20 Heterocyclic groups, (C2-C) 20 ) substituted heterocyclic groups, (C6-C 20 aryl (C1-C8) alkyl or substituted (C6-C8) 20 aryl (C1-C8) alkyl;

[0117] Each n is independently 0, 1, or 2; and

[0118] Each R 2 R 3 R 5 R 6 R 11 Or R 12 Each (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) ynyl or (C6-C20 aryl (C1-C8) alkyl groups are independently optionally bonded by one or more halogens, hydroxyl groups, CN, N3, N(R) a )2 or OR a Substitution; and wherein one or more non-terminal carbon atoms of each of the (C1-C8) alkyl groups may optionally be replaced by -O-, -S- or -NR. a -replace.

[0119] In another embodiment, the method comprises administering a therapeutically effective amount of a racemic, enantiomer, diastereomer, tautomer, polymorph, pseudopolymorph, amorphous form, hydrate or solvate, or a pharmaceutically acceptable salt or ester thereof of a compound of formula I to a mammal in need of it.

[0120] In another embodiment, the method comprises treating a person in need of a coronavirus infection by administering a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt or ester thereof.

[0121] In another embodiment, the method comprises treating a person in need of MERS virus infection by administering a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt or ester thereof.

[0122] In another embodiment, the method comprises treating a person in need of SARS virus infection by administering a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt or ester thereof.

[0123] In another embodiment, a method of treating a person in need of a coronavirus infection includes 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 with a pharmaceutically acceptable diluent or carrier.

[0124] In another embodiment, a method of treating coronavirus infection in a person in need includes 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 with at least one additional therapeutic agent.

[0125] In another embodiment, the method includes administering a therapeutically effective amount of a combination of pharmaceutical agents 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 infectious coronaviruses.

[0126] In another embodiment, this application provides a method for inhibiting coronavirus RNA-dependent RNA polymerase, comprising contacting cells infected with coronaviruses with an effective amount of a compound of formula I or a pharmaceutically acceptable salt, solvate and / or ester thereof.

[0127] In another embodiment, the use of a compound of formula I or a pharmaceutically acceptable salt, solvate, and / or ester thereof for the treatment of viral infections caused by coronaviruses is provided. Attached Figure Description

[0128] Figure 1 Changes in body weight of mice treated with the vector and compound 32 after infection;

[0129] Figure 2A and 2B Viral load in lung tissue of mice treated with vector and compound 32 on days 2 and 5 post-infection;

[0130] Figure 3A -F: Whole-body volume plethysmography of mice infected with SARS-CoV;

[0131] Figure 4A Changes in body weight in monkeys treated with the vector and compound 32 after infection;

[0132] Figure 4B Changes in body temperature in monkeys treated with the vector and compound 32 after infection;

[0133] Figure 4C Changes in respiratory rate in monkeys treated with the vector and compound 32 after infection;

[0134] Figure 5 : Viral RNA concentration in the tissues of the treatment group. Viral load was measured by qRT-PCR. Invention Details

[0135] I. Definition

[0136] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings:

[0137] When the product name is used in this document, the applicant intends to independently include the product under the product name and the active pharmaceutical ingredient of that product.

[0138] As used herein, “compound of the present invention” or “compound of formula I” means a compound of formula I or a pharmaceutically acceptable salt thereof. Similarly, with respect to separable intermediates, the phrase “compound of formula (number)” means a compound of that formula or a pharmaceutically acceptable salt thereof.

[0139] "Alkyl" is a hydrocarbon containing a positive carbon atom, a secondary carbon atom, a tertiary carbon atom, or a cyclic carbon atom. For example, alkyl groups can have 1-20 carbon atoms (i.e., C1-C2). 20 Alkyl groups, having 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), and 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).

[0140] "Alkoxy" refers to a group having the formula -O-alkyl, wherein the alkyl group as defined above is attached to the parent molecule via an oxygen atom. The alkyl portion of an alkoxy group can have 1-20 carbon atoms (i.e., C1-C2). 20 alkoxy group), 1-12 carbon atoms (i.e., C1-C12) 12Alkoxy groups are alkoxy groups with 1-6 carbon atoms (i.e., C1-C6 alkoxy groups). Examples of suitable alkoxy groups include, but are not limited to, methoxy (-O-CH3 or -OMe), ethoxy (-OCH2CH3 or -OEt), tert-butoxy (-OC(CH3)3 or -OtBu), etc.

[0141] "Haloalkyl" is an alkyl group as defined above, wherein one or more hydrogen atoms of the alkyl group are replaced by halogen atoms. The alkyl portion of a haloalkyl group can have 1-20 carbon atoms (i.e., C1-C2). 20 Halogenated alkyl groups), 1-12 carbon atoms (i.e., C1-C2) 12 Alkyl halogen (or alkyl halogroup) or 1-6 carbon atoms (i.e., C1-C6 alkyl). Examples of suitable alkyl halogroups include, but are not limited to, -CF3, -CHF2, -CFH2, -CH2CF3, etc.

[0142] "Alkenyl" refers to a group containing at least one unsaturated site (i.e., carbon-carbon sp). 2 Hydrocarbons with double bonds (ortho-, secondary, tertiary, or cyclic carbon atoms). For example, alkenyl groups can have 2 to 20 carbon atoms (i.e., C2-C2). 20 Alkenyl (2 to 8 carbon atoms, i.e., C2-C8 alkenyl) or 2 to 6 carbon atoms (i.e., C2-C6 alkenyl). Examples of suitable alkenyl groups include, but are not limited to, ethylene or vinyl (-CH=CH2), allyl (-CH2CH=CH2), cyclopentenyl (-C5H7), and 5-hexenyl (-CH2CH2CH2CH2CH=CH2).

[0143] "Alynyl" is a hydrocarbon containing a normal, secondary, tertiary, or cyclic carbon atom with at least one unsaturated site (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 Alkyne groups consist of 2 to 8 carbon atoms (i.e., C2-C8 alkynes) or 2 to 6 carbon atoms (i.e., C2-C6 alkynes). Examples of suitable alkyne groups include, but are not limited to, ethynyl (-C≡CH), propynyl (-CH2C≡CH), etc.

[0144] "Alkylene" refers to a saturated branched, straight, or cyclic hydrocarbon group having two monovalent groups at its center derived by removing two hydrogen atoms from the same or two different carbon atoms of a parent alkane. For example, alkylenes can have 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. Typical alkylenes 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-), and 1,4-butyl (-CH2CH2CH2CH2-).

[0145] "Alkenyl" refers to an unsaturated branched, straight, or cyclic hydrocarbon group having two monovalent groups at its center derived by removing two hydrogen atoms from the same or two different carbon atoms of the parent olefin. For example, alkenyl groups can have 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. Typical alkenyl groups include, but are not limited to, 1,2-ethylene (-CH=CH-).

[0146] "Imyynyl" refers to an unsaturated branched, straight, or cyclic hydrocarbon group having two monovalent groups at its center, obtained by removing two hydrogen atoms from the same or two different carbon atoms of the parent alkyne. For example, an ynylyl group can have 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. Typical ynylyl groups include, but are not limited to, acetylenyne (-C≡C-), propynyne (-CH2C≡C-), and 4-pentynyne (-CH2CH2CH2C≡C-).

[0147] "Amino" usually refers to a nitrogen group, which can be considered a derivative of ammonia and has the formula -N(X)2, where each "X" is independently H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted carbocyclic group, a substituted or unsubstituted heterocyclic group, etc. The hybridization of nitrogen is approximately sp... 3 Non-limiting types of amino groups include -NH2, -N(alkyl)2, -NH(alkyl), -N(carbocyclic)2, -NH(carbocyclic), -N(heterocyclic)2, -NH(heterocyclic), -N(aryl)2, -NH(aryl), -N(alkyl)(aryl), -N(alkyl)(heterocyclic), -N(carbocyclic)(heterocyclic), -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)2, -NH(benzyl), -N(benzyl)2, etc. Substituted alkylamino groups generally refer to alkylamino groups as defined above, wherein at least one substituted alkyl group as defined herein is attached to an 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, etc.

[0148] "Aryl" refers to an aromatic hydrocarbon group derived by removing a hydrogen atom from a single carbon atom in a parent aromatic ring system. For example, aryl groups can have 6-20 carbon atoms, 6-14 carbon atoms, or 6-10 carbon atoms. Typical aryl groups include, but are not limited to, groups derived from benzene (e.g., phenyl), substituted benzenes, naphthalenes, anthracene, biphenyls, etc.

[0149] "Arylalkyl" refers to an acyclic alkyl group in which the carbon atom (usually terminal or sp) is... 3 One of the hydrogen atoms bonded to a 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. An arylalkyl group can contain 7 to 20 carbon atoms; for example, the alkyl portion may have 1 to 6 carbon atoms and the aryl portion may have 6 to 14 carbon atoms.

[0150] "Aryl alkenyl" refers to an acyclic alkenyl group in which the carbon atom (usually terminal or sp) is attached to the chain. 3 Carbon atom, but also sp 2 One of the hydrogen atoms bonded to a carbon atom is replaced by an aryl group. The aryl moiety of an aryl alkenyl group may include any aryl group disclosed herein, for example, and the alkenyl moiety of an aryl alkenyl group may include any alkenyl group disclosed herein, for example,. The aryl alkenyl group may contain 8 to 20 carbon atoms, the alkenyl moiety may contain 2 to 6 carbon atoms, and the aryl moiety may contain 6 to 14 carbon atoms.

[0151] "Arylynyl" refers to an acyclic ynyl group, in which the carbon atom (usually terminal or sp) is involved. 3 One of the hydrogen atoms bonded to a carbon atom (but also an sp carbon atom) is replaced by an aryl group. The aryl moiety of an arylynyl group may include any aryl group disclosed herein, for example, and the ynyl moiety of an arylynyl group may include any ynyl group disclosed herein, for example,. An arylynyl group may contain 8 to 20 carbon atoms, for example, the ynyl moiety may be 2 to 6 carbon atoms and the aryl moiety may be 6 to 14 carbon atoms.

[0152] The term "substituted" in relation to alkyl, alkylene, aryl, arylalkyl, alkoxy, heterocyclic, heteroaryl, and carbocyclic groups, such as "substituted alkyl," "substituted alkylene," "substituted aryl," "substituted arylalkyl," "substituted heterocyclic," and "substituted carbocyclic," respectively refers to alkyl, alkylene, aryl, arylalkyl, heterocyclic, and carbocyclic groups in which one or more hydrogen atoms are independently replaced by non-hydrogen substituents. Typical substituents include, but are not limited to, -X and -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)NR b 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 Independently, it can be H, alkyl, aryl, arylalkyl, heterocyclic, or a protecting group or prodrug moiety. Alkylene, alkenylene, and ynynylene can also be similarly substituted. Unless otherwise specified, when the term "substituted" is used with a group having two or more substituted moieties, such as arylalkyl, the substituent can be attached to the aryl moiety, the alkyl moiety, or both.

[0153] In the pharmaceutical field, "prodrugs" are defined as biologically inactive derivatives of drugs that are converted into biologically active parent drugs through certain chemical or enzymatic pathways after being administered to the human body.

[0154] Those skilled in the art will recognize that the substituents and other portions of compounds of formulas I-IV should be selected to provide sufficient stability to provide pharmaceutically useful compounds that can be formulated into acceptable and stable pharmaceutical compositions. Compounds of formulas I-IV possessing such stability are considered to fall within the scope of this invention.

[0155] "Heteroalkyl" refers to an alkyl group in which one or more carbon atoms are replaced by heteroatoms such as O, N, or S. For example, if the 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 group (e.g., -NHCH3, -N(CH3)2, etc.), or a thioalkyl group (e.g., -SCH3). If the 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). If the 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 alkyl mercapto group (e.g., -CH2CH2-SH). Heteroalkyl groups can have, for example, 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. C1-C6 heteroalkyl groups refer to heteroalkyl groups having 1 to 6 carbon atoms.

[0156] As used herein, “heterocyclic” or “heterocyclic group” includes, for example, but not limited to, Paquette, Leo A.; Principles of Modern Heterocyclic Chemistry In (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), particularly those heterocycles described in Volumes 13, 14, 16, 19 and 28; and J. Am. Chem. Soc. (1960) 82:5566. In one specific embodiment of the invention, "heterocycle" includes "carbon ring" 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 "heterocyclic group" includes saturated rings, partially unsaturated rings, and aromatic rings (i.e., heteroaromatic rings). Substituted heterocyclic groups include, for example, heterocycles substituted with any substituent disclosed herein (including carbonyl groups). Non-limiting examples of carbonyl-substituted heterocyclic groups are:

[0157]

[0158] Examples of heterocyclic compounds include, but are not limited to, pyridinyl, dihydropyridinyl, tetrahydropyridinyl (piperidinyl), thiazolyl, tetrahydrothiophene, sulfur-oxidized tetrahydrothiophene, pyrimidinyl, furanyl, thiophene, pyrroleyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthyl, indolyl, indololinyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidinoneyl, pyrrolidinyl, 2-pyrrolidoneyl, pyrrolinyl, tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,5,2-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl, thiophene, thiaanthryl, pyranyl, isobenzofuranyl, chromenyl, and tonyl. , phenoxazinyl, 2H-pyrroleyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indololinyl, isoyndolinyl, 3H-indolyl, 1H-indolyl, purineyl, 4H-quinazinyl, phthalazinyl, naphthidyl, quinoxolinyl, quinazolinyl, cinnaphthyl, pteridinyl, 4aH-carbazoyl, carbazoyl, β-carbazoyl, phenanthridineyl, acridineyl, pyrimidinyl, phenanthridineyl, phenazinyl, phenthiazinyl, furazinyl, phenoxazinyl, isochromyl, chromanyl, imidazoalkyl, imidazolinyl, pyrazolyl, piperazinyl, indololinyl, isodihydroindolyl, quininecycloyl, morpholinyl, oxazolyl, benzotriazolyl, benzoisooxazolyl, hydroxyindolyl, benzooxazolinyl, isatinoyl and bis-tetrahydrofuranyl:

[0159]

[0160] By way of example, but not limitation, the carbon-bonded heterocycle is bonded at the following positions: 2, 3, 4, 5, or 6 of pyridine, 3, 4, 5, or 6 of pyridazine, 2, 4, 5, or 6 of pyrimidine, 2, 3, 5, or 6 of pyrazine, 2, 3, 5, or 6 of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole, 2, 3, 4, or 5 of oxazole, imidazole, or thiazole, 3, 4, or 5 of isoxazole, pyrazole, or isothiazole, 2 or 3 of aziridine, 2, 3, or 4 of acridine, 2, 3, 4, 5, 6, 7, or 8 of quinoline, or 1, 3, 4, 5, 6, 7, or 8 of isoquinoline. More typically, carbon-bonded heterocycles include 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 5-pyridinyl, 6-pyridinyl, 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.

[0161] By way of example, but not limitation, nitrogen-bonded heterocycles are bonded at the following positions: aziridinium, azacyclic butane, pyrrole, pyrrolidine, 2-pyrrololine, 3-pyrroleoline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, dihydroindole, 1H-indazole at the 1-position, isoindole or isodihydroindole at the 2-position, morpholine at the 4-position, and carbazole or β-carbline at the 9-position. More typically, nitrogen-bonded heterocycles include 1-aziridinyl, 1-acetidinyl, 1-pyrroleyl, 1-imidazoyl, 1-pyrazolyl, and 1-piperidinyl.

[0162] "Heterocyclic alkyl" refers to a noncyclic alkyl group in which the carbon atom (usually terminal or sp) is involved. 3 One of the hydrogen atoms bonded to the carbon atom is replaced by a heterocyclic group (i.e., the heterocyclic-alkylene moiety). Typical heterocyclic alkyl groups include, but are not limited to, heterocyclic-CH2-, 2-(heterocyclic)ethane-1-yl, etc., wherein the "heterocyclic" moiety includes any of the above-mentioned heterocyclic groups, including... Principles of Modern Heterocyclic Chemistry Those described herein. Those skilled in the art will also understand that heterocyclic groups can be attached to the alkyl moiety of a heterocyclic alkyl group via carbon-carbon or carbon-heteroatom bonds, provided that the resulting group is chemically stable. Heterocyclic alkyl groups contain 3 to 20 carbon atoms; for example, the alkyl moiety of an aryl alkyl group has 1 to 6 carbon atoms and the heterocyclic moiety has 2 to 14 carbon atoms. Examples of heterocyclic alkyl groups include, but are not limited to, 5-membered sulfur-, oxygen-, and / or nitrogen-containing heterocycles such as thiazolylmethyl, 2-thiazolylethane-1-yl, imidazolylmethyl, oxadiazolylmethyl, thiadiazolylmethyl, etc., and 6-membered sulfur-, oxygen-, and / or nitrogen-containing heterocycles such as piperidinylmethyl, piperazinelmethyl, morpholinylmethyl, pyridizylmethyl, pyrimidinylmethyl, pyrazinelmethyl, etc.

[0163] "Heterocyclic alkenyl" refers to a noncyclic alkenyl group, in which the carbon atom (usually terminal or sp) is involved. 3 Carbon atom, but also sp 2 One of the hydrogen atoms bonded to the carbon atom is replaced by a heterocyclic group (i.e., the heterocyclic-alkenyl-part). The heterocyclic moiety of the heterocyclic chain alkenyl includes any heterocyclic group described herein, including... Principles of Modern Heterocyclic Chemistry The terms described herein refer to those alkenyl groups, and the alkenyl moiety of the heterocyclic alkenyl group includes any alkenyl group disclosed herein. Those skilled in the art will also understand that the heterocyclic group can be attached to the alkenyl moiety of the heterocyclic alkenyl group via carbon-carbon or carbon-heteroatom bonds, provided that the resulting group is chemically stable. The heterocyclic alkenyl group contains 4 to 20 carbon atoms; for example, the alkenyl moiety of the heterocyclic alkenyl group has 2 to 6 carbon atoms and the heterocyclic moiety has 2 to 14 carbon atoms.

[0164] "Heterocyclic ynyl group" refers to an acyclic ynyl group in which the atom is bonded to a carbon atom (usually terminal or sp). 3 A hydrogen atom of a carbon atom (but also an sp carbon atom) is surrounded by a heterocyclic group (i.e., a heterocyclic-ynylene-part). The heterocyclic moiety of a heterocyclic ynylene group includes any heterocyclic group described herein, including... Principles of Modern Heterocyclic Chemistry The terms described herein refer to those alkynyl groups, and the alkynyl moiety of a heterocyclic alkynyl group includes any alkynyl group disclosed herein. Those skilled in the art will also understand that a heterocyclic group can be attached to the alkynyl moiety of a heterocyclic alkynyl group via a carbon-carbon bond or a carbon-heteroatom bond, provided that the resulting group is chemically stable. A heterocyclic alkynyl group contains 4 to 20 carbon atoms; for example, the alkynyl moiety of a heterocyclic alkynyl group has 2 to 6 carbon atoms and the heterocyclic moiety has 2 to 14 carbon atoms.

[0165] "Heteroaryl" refers to an aromatic heterocyclic group having at least one heteroatom in its ring. Non-limiting examples of suitable heteroatoms that may be included in an aromatic ring include oxygen, sulfur, and nitrogen. Non-limiting examples of heteroaryl rings include all those aromatic rings listed in the definition of "heterocyclic group," including pyridinyl, pyrroloyl, oxazolyl, indolyl, isoyindolyl, purinyl, furanyl, thiophenyl, benzofuranyl, benzothiophenyl, carbazoyl, imidazoyl, thiazoyl, isoxazolyl, pyrazolyl, isothiazolyl, quinolinyl, isoquinolinyl, pyridazinyl, pyrimidinyl, pyrazinyl, etc.

[0166] "Carbocyclic" or "carbocyclic group" refers to a saturated (i.e., cycloalkyl), partially unsaturated (e.g., cycloalkenyl, cyclodienyl, etc.), or aromatic ring having 3 to 7 carbon atoms as a monocyclic ring, 7 to 12 carbon atoms as a bicyclic ring, or up to about 20 carbon atoms as a polycyclic ring. Monocyclic carbocyclic rings have 3 to 7 ring atoms, more commonly 5 or 6 ring atoms. Bicyclic carbocyclic rings have 7 to 12 ring atoms, for example, arranged in bicyclic [4,5], [5,5], [5,6], or [6,6] systems, or 9 or 10 ring atoms arranged in bicyclic [5,6] or [6,6] systems, or spirofused rings. Non-limiting examples of monocyclic carbocyclic rings 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 carbocyclic rings include naphthyl, tetrahydronaphthalene, and decahydronaphthalene.

[0167] "Carbocycloalkyl" refers to a non-cycloalkyl group in which one of the hydrogen atoms bonded to the carbon atom is replaced by a carbocyclo group as described herein. Typical, but not limiting, examples of carbocycloalkyl groups include cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclopentylmethyl, and cyclohexylmethyl.

[0168] "Arylhexaalkyl" refers to a heteroalkyl group as defined herein, wherein 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 arylhexaalkyl group provides a chemically stable moiety. For example, arylhexaalkyl groups may have the general formulas -alkylene-O-aryl, -alkylene-O-alkylene-aryl, -alkylene-NH-aryl, -alkylene-NH-alkylene-aryl, -alkylene-S-aryl, -alkylene-S-alkylene-aryl, etc. Furthermore, any alkylene moiety in the above general formulas may be further substituted with any substituents defined or exemplified herein.

[0169] "Heteroarylalkyl" refers to an alkyl group as defined herein, wherein the hydrogen atom has been replaced by a heteroaryl group as defined herein. Non-limiting examples of heteroarylalkyl groups include -CH2-pyridinyl, -CH2-pyrroloyl, -CH2-oxazolyl, -CH2-indolyl, -CH2-isoindolyl, -CH2-purinyl, -CH2-furanyl, -CH2-thienyl, -CH2-benzofuranyl, -CH2-benzothienyl, -CH2-carbazoyl, -CH2-imidazoyl, -CH2-thiazoyl, -CH2-isooxazolyl, -CH2-pyrazolyl, -CH2-isoquinolinyl, -CH2-isoquinolinyl, -CH2-pyridazinyl, -CH2-pyrazinyl, -CH(CH3)-pyridinyl, -CH(CH3)-pyrroloyl, -CH(CH3)- Oxazolyl, -CH(CH3)-indolyl, -CH(CH3)-isoindolyl, -CH(CH3)-purine, -CH(CH3)-furanyl, -CH(CH3)-thiophenyl, -CH(CH3)-benzofuranyl, -CH(CH3)-benzothiophenyl, -CH(CH3)-carbazoyl, -CH(CH3)-imidazoyl, -CH(CH3)-thiazoyl, -CH(CH3)-isooxazolyl, -CH(CH3)-pyrazolyl, -CH(CH3)-isothiazoyl, -CH(CH3)-quinolinyl, -CH(CH3)-isoquinolinyl, -CH(CH3)-pyridazinyl, -CH(CH3)-pyrimidinyl, -CH(CH3)-pyrazinyl, etc.

[0170] The term "optionally substituted" for a specific part (e.g., an optionally substituted aryl group) of a compound of formulas I-IV means that all substituents are hydrogen or that one or more hydrogens of that part may be replaced by substituents as defined below, such as "substituted".

[0171] Regarding specific portions of compounds of formulas I-IV (e.g., the carbon atoms of the (C1-C8) alkyl group may optionally be -O-, -S-, or -NR-), aThe term "optionally substituted" indicates 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.

[0172] The term "non-terminal carbon atom" in relation to alkyl, alkenyl, alkynyl, alkylene, alkenylene, or alkynyl moieties refers to a carbon atom located between the first and last carbon atoms of that moiety. Therefore, by example and not limitation, the C* atom in the alkyl moiety -CH2(C*)H2(C*)H2CH3 or the alkylene moiety -CH2(C*)H2(C*)H2CH2- would be considered a non-terminal carbon atom.

[0173] Some Q and Q1 substitutes are nitrogen oxides, for example + N(O)(R) or + N(O)(OR). These nitrogen oxides, as shown here, are bonded to carbon atoms and can also be represented by charge-dissociated groups, for example...

[0174]

[0175] And is intended to be equivalent to the above-described representation used to describe the purpose of the invention.

[0176] A "connector" or "linker" refers to a chemical part containing a covalent bond or a chain of atoms. Connectors include repeating alkoxy units (e.g., polyoxyethylene, PEG, polymethyleneoxy) and repeating alkylamino units (e.g., polyoxyethylene, Jeffamine). TM ); and esters and amides, including succinates, succinamides, diethylene glycol esters, malonates and hexamethylene amides.

[0177] Terms such as "oxygen-linked," "nitrogen-linked," "carbon-linked," "sulfur-linked," or "phosphorus-linked" refer to bonds formed between two parts that can be formed using more than one type of atom in the part, where the bond is formed by the specified atom. For example, a nitrogen-linked amino acid will be linked by the nitrogen atom of the amino acid, rather than by the oxygen or carbon atom.

[0178] In some embodiments of compounds of formulas I-IV, Z 1 or Z 2One or more nitrogen-linked groups in a naturally occurring α-amino acid ester are included. 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 may contain any of those described with respect to the substituent R, particularly those wherein R is an optionally substituted (C1-C8) alkyl group.

[0179] The terms "purine" or "pyrimidine" bases include, but are not limited to, adenine, N... 6 -alkylpurine, N 6 -Acylpurine (where the acyl group is C(O) (alkyl, aryl, alkylaryl or arylalkyl), N 6 -Benzylpurine, N 6 - Halopurines, N 6 -Vinylpurine, N 6 - Acetylpurine, 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-azapyrimidine, 2- and / or 4-mercaptopyrimidine, uracil, 5-halogenated uracil including 5-fluorouracil, C 5 -alkylpyrimidine, C 5 -Benzylpyrimidine, C 5 -Halogenated pyrimidines, C 5 -Vinylpyrimidine, C 5 -Ethynylpyrimidine, C 5 -Acylpyrimidine, C 5 -Hydroxyalkylpurine, C 5 - Acetaminopyrimidine, C 5 -Cyanopyrimidine, C 5 -5-Iodopyrimidine, C 6 -Iodopyrimidine, C 5 -Br-vinylpyrimidine, C 6 -Br-ethynylpyrimidine, C 5 -Nitropyrimidine, C 5 -aminopyrimidine, N 2 -alkylpurine, N 2-alkyl-6-thiopurine, 5-azacytidine, 5-azauracil, triazolopyridyl, imidazopyridyl, 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 formulas I-III are linked to ribose or their analogues via the nitrogen atom of the base. Functional oxygen and nitrogen groups on the bases may be protected if necessary or desired. Suitable protecting groups are well known to those skilled in the art and include trimethylsilyl, dimethylhexylsilyl, tert-butyldimethylsilyl and tert-butyldiphenylsilyl, triphenylmethyl, alkyl and acyl groups such as acetyl and propionyl, methanesulfonyl and p-toluenesulfonyl.

[0180] Unless otherwise stated, the carbon atoms in compounds of formulas I-IV are intended to have a tetravalent oxidation state. In some chemical structural representations where there are not enough carbon atoms connected in a sufficient number of variables to produce a tetravalent oxidation state, the remaining carbon substituents required to provide the tetravalent oxidation state should be considered as hydrogen. For example,

[0181]

[0182] They have the same meaning.

[0183] A “protecting group” is a part of a compound that masks or alters the properties of a functional group or the properties of the compound as a whole. The chemical substructures of protecting groups vary considerably. One function of protecting groups is as intermediates in the synthesis of parent drugs. 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 aid in the efficiency of desired chemical reactions, such as the orderly and planned creation and breaking of chemical bonds. In addition to the reactivity of the protected functional groups, the protection of a compound's functional groups alters other physical properties, such as polarity, lipophilicity (hydrophobicity), and other properties that can be measured using common analytical tools. Chemically protected intermediates may themselves be biologically active or inactive. A “hydroxyl protecting group” refers to those protecting groups used to protect the hydroxyl group (-OH).

[0184] Protected compounds may also exhibit altered, and in some cases optimized, in vitro and in vivo properties, such as resistance to cell membrane degradation or chelation. During this period, the protected compound with the intended therapeutic effect may be referred to as a prodrug. Another function of the protecting group is to convert the parent drug into a prodrug, thereby releasing the parent drug during the conversion of the prodrug in vivo. Because the active prodrug may be absorbed more efficiently than the parent drug, it may have greater potency in vivo. In vitro, in the case of chemical intermediates, or in vivo, in the case of prodrugs, the protecting group is removed. For chemical intermediates, it is not particularly important that the product obtained after deprotection, such as an alcohol, is physiologically acceptable, although it is generally preferable if the product is pharmacologically harmless.

[0185] The term "chirality" refers to molecules that have the property of non-overlapping mirror partners, while the term "achirality" refers to molecules that can overlap with their mirror partners.

[0186] The term "stereoisomer" refers to compounds that have the same chemical composition but differ in the spatial arrangement of their atoms or groups.

[0187] "Diabeta-isomers" are stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diabeta-isomers possess different physical properties, such as melting point, boiling point, spectral properties, reactivity, and biological properties. For example, when R... 7 yes And Z 1 and Z 2 At the same time, compounds of formulas I-IV can have chiral phosphorus atoms. When Z 1 or Z 2 When at least one of them also has a chiral center, such as Z 1 or Z 2 When the compound is a nitrogen-linked chiral, naturally occurring α-amino acid ester, then the compounds of formulas I-IV will exist as diastereomers because there are two chiral centers in the molecule. All such diastereomers and their uses described herein are included in this invention. Mixtures of diastereomers can be separated using high-resolution analytical procedures such as electrophoresis, crystallization, and / or chromatography. Diastereomers 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, enzyme stability, absorption, and metabolic stability.

[0188] "Enantiomers" refer to two stereoisomers of a compound that are mirror images of each other and cannot be superimposed.

[0189] The modifier “about” used with a quantity includes the value and has a meaning indicated by the context (e.g., the degree of error associated with the measurement of a particular quantity).

[0190] Unless otherwise stated, the term "treatment" as used herein means reversing, alleviating, suppressing the progression of the disorder or condition to which the term applies, or preventing the development of one or more symptoms of such disorder or condition. As used herein, the term "treatment" refers to the act of treatment, since "treatment" has just been defined above.

[0191] As used herein, the term "therapeutic effective amount" refers to the amount of a compound of formula I-IV present in the composition described herein that is intended to provide a desired level of the drug in the secretions and tissues of the airways and lungs, or, when such a composition is administered via a chosen route of administration, to produce a 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 those skilled in the art based on the information provided herein.

[0192] The term "physiological saline" refers to an aqueous solution containing 0.9% (w / v) NaCl.

[0193] The term "hypertonic saline" refers to an aqueous solution containing more than 0.9% (w / v) NaCl. For example, a 3% hypertonic saline solution would contain 3% (w / v) NaCl.

[0194] "Forming a reaction mixture" refers to the process of bringing at least two different substances into contact so that they mix together and can react. However, it should be understood that the resulting reaction products can be generated 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.

[0195] A "coupling agent" is a reagent capable of coupling two different compounds. Coupling agents can be catalytic or stoichiometric. For example, coupling agents can be lithium-based or magnesium-based, such as Grignard reagents. Exemplary coupling agents include, but are not limited to, n-BuLi, MgCl2, iPrMgCl, tBuMgCl, PhMgCl, or combinations thereof.

[0196] "Silane" refers to a silicon-containing group having the formula SiR4, where each R group can be alkyl, alkenyl, cycloalkyl, phenyl, or other silicon-containing groups. When a silane is attached to another compound, it is called a "silyl silane" and has the formula -SiR3.

[0197] "Halosilane" refers to a silane having at least one halogen group bonded to a silicon atom. Representative halosilanes have the formula halogen-SiR3, where each R group can be alkyl, alkenyl, cycloalkyl, phenyl, or other silicon-containing groups. Specific halosilanes include Cl-Si(CH3)3 and Cl-Si(CH3)2CH2CH2Si(CH3)2-Cl.

[0198] "Non-nucleophilic bases" refer to electron donor Lewis bases, such as nitrogen bases, including triethylamine, diisopropylethylamine, N,N-diethylaniline, pyridine, 2,6-dimethylpyridine, 2,4,6-trimethylpyridine, 4-dimethylaminopyridine, and quinine ring.

[0199] A "leaving group" is a group that retains the bonded electron pair during heterolytic bond cleavage. For example, leaving groups are readily replaced during nucleophilic substitution reactions. Suitable leaving groups include, but are not limited to, chloride ions, bromide ions, methanesulfonate ions, toluenesulfonate ions, trifluoromethanesulfonate ions, 4-nitrobenzenesulfonate ions, 4-chlorobenzenesulfonate ions, 4-nitrobenoxy ions, pentafluorophenoxy ions, etc. Other leaving groups that can be used in this invention will be recognized by those skilled in the art.

[0200] "Deprotecting agent" refers to any reagent capable of removing protecting groups. The type of 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 GMWuts and Theodora W. Greene, 4th edition, 2006.

[0201] II. Compounds of the present invention

[0202] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the appended description, structures, and formulas. While the 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. Rather, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the invention.

[0203] A method for treating arenaviridae infections in persons of need is provided, comprising administering a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt or ester thereof:

[0204]

[0205] in:

[0206] Each R 1 It is H or halogen;

[0207] Each R2 R 3 R 4 Or R 5 Independently for H, OR a 、N(R a 2, N3, CN, NO2, S(O) n R a Halogen, (C1-C8)alkyl, (C4-C8)carbocycloalkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)ynyl or (C2-C8)substituted ynyl;

[0208] Or any two R atoms on adjacent carbon atoms 2 R 3 R 4 Or R 5 Together they form -O(CO)O- or together with the ring carbon atoms they are attached to form a double bond;

[0209] R 6 Is it 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)carbocycloalkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)ynyl, (C2-C8)substituted ynyl or (C6-C 20 aryl (C1-C8) alkyl;

[0210] R 7 Choose from the following groups:

[0211] 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 ,

[0212] Each R 11 Or R 12 (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)ynyl or (C6-C 20 aryl (C1-C8) alkyl groups are independently optionally bonded by one or more halogens, hydroxyl groups, CN, N3, N(R) a )2 or OR a Substitution; and wherein one or more non-terminal carbon atoms of each of the (C1-C8) alkyl groups may optionally be replaced by -O-, -S- or -NR. a -replace,

[0213]

[0214] in:

[0215] R c Selected from phenyl, 1-naphthyl, 2-naphthyl,

[0216]

[0217] R d It is H or CH3;

[0218] R e1 and R e2 Each is independently H, (C1-C6)alkyl, or benzyl;

[0219] R f Selected from H, (C1-C8)alkyl, benzyl, (C3-C6)cycloalkyl, and -CH2-(C3-C6)cycloalkyl;

[0220] 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

[0221] n' is selected from 1, 2, 3, and 4; and

[0222] d) The following groups:

[0223]

[0224] in:

[0225] Q represents O, S, NR. + N(O)(R), N(OR), + N(O)(OR) or N-NR2;

[0226] Z 1 and Z 2 Together for -Q 1 (C(R y )2)3Q 1 -;

[0227] in

[0228] Each Q 1 Independently O, S, or NR; and

[0229] Each R y 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 Rs on the same carbon atom. y Together they form a carbon ring with 3 to 7 carbon atoms;

[0230] Each Q 2 Independently for O, S, NR, + N(O)(R), N(OR), + N(O)(OR) or N-NR2; or

[0231] Z 1 and Z 2 Each group is an independent group of formula Ia:

[0232]

[0233] in:

[0234] Each Q 3 Independently for bond, O, CR2, NR, + N(O)(R), N(OR), + N(O)(OR), N-NR2, S, SS, S(O) or S(O)2;

[0235] M2 is 0, 1, or 2;

[0236] Each R x Independently for R y Or the following formula:

[0237]

[0238] in:

[0239] Each M1a, Mlc, and M1d is independently 0 or 1;

[0240] M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0241] Z 3 It is Z 4 or Z 5 ;

[0242] Z 4 It is R, -C(Q) 2 )R y -C(Q) 2 )Z 5 -SO2R y or -SO2Z 5 ;and

[0243] Z 5 It is a carbon ring or a hetero ring, where Z 5 Independently controlled by 0 to 3 R y Group substitution;

[0244] R 8 It is 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)NR11 R 12 -C(=S)NR 11 R 12 -C(=O)OR 11 (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)ynyl, (C4-C8)carbocycloalkyl, (C6-C 20 Optionally substituted aryl groups, optionally substituted heteroaryl groups, -C(=O)(C1-C8)alkyl groups, -S(O) n (C1-C8)alkyl, (C6-C 20 aryl (C1-C8) alkyl, OR 11 or SR 11 ;

[0245] Each R 9 Or R 10 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 ;

[0246] Each R 11 Or R 12 Independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)ynyl, (C4-C8)carbocycloalkyl, (C6-C 20 Optionally substituted aryl groups, optionally substituted heteroaryl groups, -C(=O)(C1-C8)alkyl groups, -S(O) n (C1-C8)alkyl or (C6-C8)alkyl 20 ) aryl (C1-C8) alkyl; or R 11 and R 12 Together with nitrogen atoms that are all connected to them, they form 3-7 membered heterocycles, wherein any carbon atom of said heterocycle may optionally be -O-, -S-, or -NR. a-replace;

[0247] Each R a Independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)ynyl, (C6-C 20 )aryl (C1-C8)alkyl, (C4-C8)carbocycloalkyl, -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,

[0248] Each R is independently H, (C1-C8)alkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)ynyl, (C2-C8)substituted ynyl, (C6-C 20 )Aryl, (C6-C 20 ) substituted aryl, (C2-C 20 Heterocyclic groups, (C2-C) 20 ) substituted heterocyclic groups, (C6-C 20 aryl (C1-C8) alkyl or substituted (C6-C8) 20 aryl (C1-C8) alkyl;

[0249] Each n is independently 0, 1, or 2; and

[0250] Each R 2 R 3 R 5 R 6 R 11 Or R 12 Each (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) ynyl or (C6-C 20 aryl (C1-C8) alkyl groups are independently optionally bonded by one or more halogens, hydroxyl groups, CN, N3, N(R) a )2 or OR a Substitution; and wherein one or more non-terminal carbon atoms of each of the (C1-C8) alkyl groups may optionally be replaced by -O-, -S- or -NR. a -replace.

[0251] In another embodiment, a method for treating arenaviridae infections in persons in need is provided, comprising administering a therapeutically effective amount of a compound of formula II or a pharmaceutically acceptable salt or ester thereof:

[0252]

[0253] in,

[0254] R 1 R 3 R 5 R 7 R 8 and R 9 As defined above for equation I:

[0255] Each R 2 Is it OR a Or halogen; and

[0256] R 6 Is it OR a 、N(R a 2, N, 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)carbocycloalkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)ynyl or (C2-C8)substituted ynyl.

[0257] In one embodiment of a method for treating arenaviridae infections by administering a compound of formula II, R of formula II... 1 For H. In another aspect of this implementation, R of equation II 6 It can be N3, CN, halogen, (C1-C8)alkyl, (C1-C8)-substituted alkyl, (C2-C8)-alkenyl, (C2-C8)-substituted alkenyl, (C2-C8)-ynyl, or (C2-C8)-substituted ynyl. In another aspect of this embodiment, R of formula II... 6 It is CN, methyl, vinyl, or ethynyl. In another aspect of this embodiment, R of formula II... 6 It is CN. In another aspect of this implementation, R of formula II... 6 It is methyl. In another aspect of this embodiment, R of formula II... 5 For H. In another aspect of this implementation, R of equation II 2 OR a In another aspect of this implementation scheme, R of formula II2 It is OH. In another aspect of this embodiment, R of formula II... 2 It is F. In another aspect of this implementation scheme, R of formula II... 3 Is it OR a In another aspect of this implementation scheme, R of formula II 3 It is OH, -OC (=O)R 11 or -OC(=O)OR 11 In another aspect of this implementation scheme, R of formula II 3 It is OH. In another aspect of this embodiment, R of formula II... 8 It is NR 11 R 12 In another aspect of this implementation scheme, R of formula II 8 It is NH2. In another aspect of this embodiment, R of formula II... 8 Is it OR 11 In another aspect of this implementation scheme, R of formula II 8 It is OH. In another aspect of this embodiment, R of formula II... 9 For H. In another aspect of this implementation, R of equation II 9 For NR 11 R 12 In another aspect of this implementation scheme, R of formula II 9 It is NH2. In another aspect of this embodiment, R of formula II... 7 For H, -C(=O)R 11 -C(=O)OR 11 or

[0258]

[0259] In another aspect of this implementation scheme, R of Formula II 7 For H. In another aspect of this implementation, R of equation II 7 for

[0260]

[0261] In another embodiment of the method for treating arenaviridae infections, including the administration of 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 Lassa virus caused by strains selected from Josiah, NL, z148, Macenta, AV, and CSF.

[0262] In another aspect of this implementation plan, infection with the Arenaviridae family is caused by Allpahuayo virus (ALLV), Amapari virus (AMAV), Bear Canyon virus (BCNV), Katharina virus, Chapare virus, Cupiqui virus (CPXV), Dantonon virus, Flexal virus (FLEV), Citrulline virus (GTOV), Ippy virus (IPPYV), Junin virus (JUNV), Kodoko virus, Lassa virus (LASV), Latinovirus (LATV), Lymphocytic choroid plexus meningitis virus (LCMV), Lujo virus, Machupo virus (MACV), Mobala virus (MOBV), Morogoro virus, Mopeia virus (MOPV), Oliverio virus (OLVV), Parana virus (PARV), Picend virus (PICV), Pinhal virus, Pirital virus (PIRV), Sabia virus (SABV), Skinner box virus, Tacarib virus (TCRV), Tamiami virus (TAMV), or Whitewater Alloyo virus (WWAV).

[0263] In another embodiment, a method for treating arenaviridae infections in persons of need is provided, comprising administering a therapeutically effective amount of a compound of formula III represented by a compound of formula I or a pharmaceutically acceptable salt or ester thereof:

[0264]

[0265] in

[0266] R 6 R 7 R 8 and R 9 As defined above for Equation II;

[0267] Each R 2 Is it OR a Or F; and

[0268] Each R 3 Is it OR a .

[0269] In one embodiment of a method for treating arenaviridae infections including the administration of a compound of formula III, R of formula III... 6 It can be N3, CN, halogen, (C1-C8)alkyl, (C1-C8)-substituted alkyl, (C2-C8)-alkenyl, (C2-C8)-substituted alkenyl, (C2-C8)-ynyl, or (C2-C8)-substituted ynyl. In another aspect of this embodiment, R of formula III... 6It is CN, methyl, vinyl, or ethynyl. In another aspect of this embodiment, R of formula III... 6 It is CN. In another aspect of this implementation, R of formula III... 6 It is methyl. In another aspect of this embodiment, R of formula III... 2 Is it OR a In another aspect of this implementation scheme, R of Equation III 2 It is OH. In another aspect of this embodiment, R of formula III... 2 For F. In another aspect of this implementation, R of Equation III 3 For OH, -OC(=O)R 11 or -OC(=O)OR 11 In another aspect of this implementation scheme, R of Equation III 3 It is OH. In another aspect of this embodiment, R of formula III... 8 For NR 11 R 12 In another aspect of this implementation scheme, R of Equation III 8 It is NH2. In another aspect of this implementation, R of formula III... 8 Is it OR 11 In another aspect of this implementation scheme, R of Equation III 8 It is OH. In another aspect of this embodiment, R of formula III... 9 For H. In another aspect of this implementation, R of equation III 9 For NR 11 R 12 In another aspect of this implementation scheme, R of Equation III 9 It is NH2. In another aspect of this implementation, R of formula III... 7 For H, -C(=O)R 11 -C(=O)OR 11 or

[0270]

[0271] In another aspect of this implementation scheme, R of Formula III 7 For H. In another aspect of this implementation, R of equation III 7 for

[0272]

[0273] In another embodiment of a method for treating arenaviridae infections, including the administration of a compound of formula III, R of formula III... 6It 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 For NH2. In another aspect of this embodiment, R of formula III... 6 It is CN, methyl, vinyl, or ethynyl. In another aspect of this embodiment, R of formula III... 6 It is CN. In another aspect of this implementation, R of formula III... 6 It is methyl. In another aspect of this embodiment, R of formula III... 2 Is it OR a In another aspect of this implementation scheme, R of Equation III 2 For OH, -OC(=O)R 11 or -OC(=O)OR 11 In another aspect of this implementation scheme, R of Equation III 2 It is OH. In another aspect of this embodiment, R of formula III... 2 For F. In another aspect of this implementation, R of Equation III 3 For OH, -OC(=O)R 11 or -OC(=O)OR 11 In another aspect of this implementation scheme, R of Equation III 3 It is OH. In another aspect of this embodiment, R of formula III... 9 For H. In another aspect of this implementation, R of equation III 9 For NR 11 R 12 In another aspect of this implementation scheme, R of Equation III 9 It is NH2. In another aspect of this implementation, R of formula III... 7 For H, -C(=O)R 11 -C(=O)OR 11 or

[0274]

[0275] In another aspect of this implementation scheme, R of Formula III 7 For H. In another aspect of this implementation, R of equation III 7 for

[0276]

[0277] In another embodiment of a method for treating arenaviridae infections including the administration of a compound of formula III, R of formula III... 6For CN, methyl, vinyl or ethynyl, R 8 It is NH2, and R 9 It is H. In another aspect of this implementation, R of formula III... 6 It is CN. In another aspect of this implementation, R of formula III... 6 It is methyl. In another aspect of this embodiment, R of formula III... 2 Is it OR a In another aspect of this implementation scheme, R of Equation III 2 For OH, -OC(=O)R 11 or -OC(=O)OR 11 In another aspect of this implementation scheme, R of Equation III 2 It is OH. In another aspect of this embodiment, R of formula III... 2 For F. In another aspect of this implementation, R of Equation III 3 For OH, -OC(=O)R 11 or -OC(=O)OR 11 In another aspect of this implementation scheme, R of Equation III 3 It is OH. In another aspect of this embodiment, R of formula III... 7 For H, -C(=O)R 11 -C(=O)OR 11 or

[0278]

[0279] In another aspect of this implementation scheme, R of Formula III 7 For H. In another aspect of this implementation, R of equation III 7 for

[0280]

[0281] In another embodiment of the method for treating arenaviridae infections, including the administration of 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 Lassa virus induced by strains selected from Josiah, NL, z148, Macenta, AV, and CSF.

[0282] In another aspect of this implementation plan, arenavirus infections are caused by Allpahuayo virus (ALLV), Amapari virus (AMAV), Bear Canyon virus (BCNV), Katharina virus, Chapare virus, Cupiqui virus (CPXV), Dantonon virus, Flexal virus (FLEV), Citrulline virus (GTOV), Ippy virus (IPPYV), Junin virus (JUNV), Kodoko virus, Lassa virus (LASV), Latinovirus (LATV), Lymphocytic choroid plexus meningitis virus (LCMV), Lujo virus, Machupo virus (MACV), Mobala virus (MOBV), Morogoro virus, Mopeia virus (MOPV), Oliverio virus (OLVV), Parana virus (PARV), Picend virus (PICV), Pinhal virus, Pirital virus (PIRV), Sabia virus (SABV), Skinner box virus, Tacarib virus (TCRV), Tamiami virus (TAMV), or Whitewater Arroyo virus (WWAV).

[0283] In another embodiment, a method for treating arenaviridae infections in persons in need 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:

[0284]

[0285] Where R 7 As defined above for Equation I.

[0286] In another embodiment of the method for treating arenaviridae infections, a compound of formula IV, R, is administered. 7 It can be H. In another embodiment of a method for treating arenaviridae infections, it includes administering a compound of formula IV, R. 7 Selected from the group as defined in equation I, such as a), b), or c).

[0287] In another embodiment of the method for treating arenaviridae infections, a compound of formula IV, R, is administered. 7 for

[0288]

[0289] Z 1 and Z 2 Each of these groups independently has the following structure:

[0290]

[0291] And Z3 It is Z 5 .

[0292] In another embodiment of the method for treating arenaviridae infections, a compound of formula IV, R, is administered. 7 for

[0293]

[0294]

[0295] Z 1 and Z 2 Each of these groups independently has the following structure:

[0296]

[0297] And Z 3 It is Z 5 .

[0298] In another embodiment of the method for treating arenaviridae infections, a compound of formula IV, R, is administered. 7 for

[0299]

[0300] Each Q 3b Independently O or N(R). In another implementation, each Q 3b It is O and each R x Independently:

[0301]

[0302] Where M12c is 1, 2, or 3 and each Q 3 It can be a bond, O, CR2, or S independently.

[0303] In some implementation schemes, R e1 and R e2 Each can be independently H, C1-C6 alkyl, or benzyl. In some embodiments, R e1 It can be H, C1-C6 alkyl or benzyl, and R e2 It can be H or C1-C6 alkyl. In some embodiments, R e1 and R e2 Each can be independently H or C1-C6 alkyl. In some embodiments, R e1 and R e2 Each can be H or benzyl independently. In some embodiments, R e1 It can be H, methyl, or benzyl, and R e2It can be H or methyl. In some embodiments, R e1 It can be H or methyl, and R e2 It can be H or methyl. In some embodiments, R e1 It can be methyl, and R e2 It can be H or methyl. In some embodiments, R e1 It can be H or benzyl, and R e2 It can be H or methyl.

[0304] In another embodiment of the method for treating arenaviridae infections, a compound of formula IV, R, is administered. 7 yes

[0305]

[0306] In another embodiment of the method for treating arenaviridae infections, a compound of formula IV, R, is administered. 7 for

[0307]

[0308] In another embodiment of the method for treating arenaviridae infections, a compound of formula IV, R, is administered. 7 for

[0309]

[0310] Where R f Selected from H, C1-C8 alkyl, benzyl, C3-C6 cycloalkyl, and -CH2-C3-C6 cycloalkyl. In another embodiment of the compound of formula IV, R... f It is a C1-C8 alkyl group. In another embodiment of the compound of formula IV, R f It is 2-ethylbutyl.

[0311] In another embodiment of the method for treating arenaviridae infections, a compound of formula IV, R, is administered. 7 yes

[0312]

[0313] in,

[0314] R f Selected from H, C1-C8 alkyl, benzyl, C3-C6 cycloalkyl and -CH2-C3-C6 cycloalkyl; and

[0315] R gIt is selected from C1-C8 alkyl, -O-C1-C8 alkyl, benzyl, -O-benzyl, -CH2-C3-C6 cycloalkyl, -O-CH2-C3-C6 cycloalkyl and CF3.

[0316] In another embodiment of the method for treating arenaviridae infections, an administration of an IV compound, R, is included. 7 for

[0317]

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

[0319] In another embodiment of the method for treating arenaviridae infections, an administration of an IV compound, R, is included. 7 for:

[0320]

[0321] Where R g The compound is selected from 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, Rf is a C1-C8 alkyl. In another embodiment of the compound of formula IV, R... f It is a C1-C6 alkyl group.

[0322] In another embodiment of the method for treating arenaviridae infections, a compound of formula IV, R, is administered. 7 Selected from the following groups:

[0323]

[0324] In another embodiment of the method for treating arenaviridae infections, an administration of an IV compound, R, is included. 7 for

[0325]

[0326] In another embodiment of the method for treating arenaviridae infections, a compound of formula IV, Z, is administered. 1 and Z 2 Each could be:

[0327]

[0328] In another embodiment, a method for treating arenaviridae infections in persons in need is provided, comprising administering a therapeutically effective amount of a compound of formulas I-IV, wherein R 11 Or R 12 Independently, it can be H, (C1-C8)alkyl, (C1-C8)alkenyl, (C2-C8)ynyl, (C4-C8)carbocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -C(=O)(C1-C8)alkyl, or -S(O). n (C1-C8)alkyl or aryl(C1-C8)alkyl. In another embodiment, R 11 and R 12 Together with nitrogen atoms that are all connected to them, they form 3-7 membered heterocycles, wherein any carbon atom of the heterocycle may optionally be -O-, -S-, or -NR. a - Instead. Therefore, -NR is used as an example rather than a limitation. 11 R 12 The group can be represented by the following heterocycles:

[0329] wait.

[0330] In another embodiment, a method for treating arenaviridae infections in persons in need is provided, comprising administering a therapeutically effective amount of a compound of formulas I-IV, wherein each R 3 R 4 R 5 R 6 R 11 Or R 12 Independently (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)ynyl or aryl(C1-C8)alkyl, wherein the (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)ynyl or aryl(C1-C8)alkyl is independently optionally influenced by one or more halogens, hydroxyl groups, CN, N3, N(R) a )2 or OR a Replacement. Therefore, R is used as an example rather than a limitation. 3 R 4 R 5 R 6 R 11 Or R 12 It can represent groups such as -CH(NH2)CH3, -CH(OH)CH2CH3, -CH(NH2)CH(CH3)2, -CH2CF3, -(CH2)2CH(N3)CH3, -(CH2)6NH2, etc.

[0331] In another embodiment, a method for treating arenaviridae infections in persons in need is provided, 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 It is a (C1-C8) alkyl group, wherein one or more non-terminal carbon atoms of each (C1-C8) alkyl group may optionally be -O-, -S- or -NR-. a - Instead. Therefore, R is used as an example rather than a limitation. 3 R 4 R 5 R 6 R 11 Or R 12 It can represent groups such as -CH2OCH3, -CH2OCH2CH3, -CH2OCH(CH3)2, -CH2SCH3, -(CH2)6OCH3, -(CH2)6N(CH3)2, etc.

[0332] In another embodiment of the method for treating arenaviridae infections, the method includes administering a compound of formula I, said compound being

[0333]

[0334]

[0335] Or its pharmaceutically acceptable salts or esters.

[0336] In another embodiment of the method for treating arenaviridae infections, the method includes administering a compound of formula I, said compound being

[0337]

[0338]

[0339]

[0340] Or its pharmaceutically acceptable salts or esters.

[0341] In another embodiment of the method for treating arenaviridae infections, an administration of a compound of formula IV is included, said compound being:

[0342]

[0343] Or its pharmaceutically acceptable salts or esters.

[0344] In another embodiment of the method for treating arenaviridae infections, an administration of a compound of formula IV is included, said compound being:

[0345]

[0346] Or its pharmaceutically acceptable salts or esters.

[0347] In another embodiment of a method for treating arenaviridae infections, the method comprises administering a compound of formula I-IV, said compound being

[0348]

[0349]

[0350] Or its pharmaceutically acceptable salts or esters.

[0351] In another embodiment of a method for treating arenaviridae infections, the method includes administering a compound of formula I-IV, said compound being

[0352]

[0353] Or its pharmaceutically acceptable salts or esters.

[0354] A method for treating coronavirus infections in individuals requiring this treatment is provided, comprising administering a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt or ester thereof:

[0355]

[0356] in:

[0357] Each R 1 It is H or halogen;

[0358] Each R 2 R 3 R 4 Or R 5 Independently for H, OR a 、N(R a 2, N3, CN, NO2, S(O) n R a Halogen, (C1-C8)alkyl, (C4-C8)carbocycloalkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)ynyl, (C2-C8)substituted ynyl;

[0359] Or any two R atoms on adjacent carbon atoms 2 R 3 R 4 Or R5 Together they form -O(CO)O- or together with the ring carbon atoms they are attached to form a double bond;

[0360] R 6 Is it 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)carbocycloalkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)ynyl, (C2-C8)substituted ynyl or (C6-C 20 aryl (C1-C8) alkyl;

[0361] R 7 Choose from the following groups:

[0362] 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 ,

[0363] Each R 11 Or R 12 (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)ynyl or (C6-C 20 aryl (C1-C8) alkyl groups are independently optionally bonded by one or more halogens, hydroxyl groups, CN, N3, N(R) a )2 or OR aSubstitution; and wherein one or more non-terminal carbon atoms of each of the (C1-C8) alkyl groups may optionally be replaced by -O-, -S- or -NR. a -replace,

[0364] b)

[0365]

[0366] in:

[0367] R c Selected from phenyl, 1-naphthyl, 2-naphthyl,

[0368]

[0369] R d It is H or CH3;

[0370] R e1 and R e2 Each is independently H, (C1-C6)alkyl, or benzyl;

[0371] R f Selected from H, (C1-C8)alkyl, benzyl, (C3-C6)cycloalkyl, and -CH2-(C3-C6)cycloalkyl;

[0372] 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

[0373] n' is selected from 1, 2, 3, and 4; and

[0374] d) The following groups:

[0375]

[0376] in:

[0377] Q represents O, S, NR. + N(O)(R), N(OR), + N(O)(OR) or N-NR2;

[0378] Z 1 and Z 2 Together for -Q 1 (C(R y )2)3Q 1 -;

[0379] in

[0380] Each Q 1 Independently O, S, or NR; and

[0381] Each R y 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 Rs on the same carbon atom. y Together they form a carbon ring with 3 to 7 carbon atoms;

[0382] Each Q 2 Independently for O, S, NR, + N(O)(R), N(OR), + N(O)(OR) or N-NR2; or

[0383] Z 1 and Z 2 Each group is an independent group of formula Ia:

[0384]

[0385] Formula Ia

[0386] in:

[0387] Each Q 3 Independently for bond, O, CR2, NR, + N(O)(R), N(OR), + N(O)(OR), N-NR2, S, SS, S(O) or S(O)2;

[0388] M2 is 0, 1, or 2;

[0389] Each R x Independently for R y Or the following formula:

[0390]

[0391] in:

[0392] Each M1a, Mlc, and M1d is independently 0 or 1;

[0393] M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0394] Z 3 It is Z 4 or Z 5 ;

[0395] Z 4 It is R, -C(Q) 2 )R y -C(Q) 2 )Z 5 -SO2R y or -SO2Z 5 ;and

[0396] Z 5 It is a carbon ring or a hetero ring, where Z 5 Independently controlled by 0 to 3 R y Group substitution;

[0397] R 8 It is 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)ynyl, (C4-C8)carbocycloalkyl, (C6-C 20 Optionally substituted aryl groups, optionally substituted heteroaryl groups, -C(=O)(C1-C8)alkyl groups, -S(O) n (C1-C8)alkyl, (C6-C 20 aryl (C1-C8) alkyl, OR 11 or SR11 ;

[0398] Each R 9 Or R 10 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 ;

[0399] Each R 11 Or R 12 Independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)ynyl, (C4-C8)carbocycloalkyl, (C6-C 20 Optionally substituted aryl groups, optionally substituted heteroaryl groups, -C(=O)(C1-C8)alkyl groups, -S(O) n (C1-C8)alkyl or (C6-C8)alkyl 20 ) aryl (C1-C8) alkyl; or R 11 and R 12 Together with nitrogen atoms that are all connected to them, they form 3-7 membered heterocycles, wherein any carbon atom of said heterocycle may optionally be -O-, -S-, or -NR. a -replace;

[0400] Each R a Independently H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)ynyl, (C6-C 20 )aryl (C1-C8)alkyl, (C4-C8)carbocycloalkyl, -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,

[0401] Each R is independently H, (C1-C8)alkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)ynyl, (C2-C8)substituted ynyl, (C6-C 20 )Aryl, (C6-C 20 ) substituted aryl, (C2-C 20 Heterocyclic groups, (C2-C) 20 ) substituted heterocyclic groups, (C6-C 20 aryl (C1-C8) alkyl or substituted (C6-C8) 20 aryl (C1-C8) alkyl;

[0402] Each n is independently 0, 1, or 2; and

[0403] Each R 2 R 3 R 5 R 6 R 11 Or R 12 Each (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) ynyl or (C6-C 20 aryl (C1-C8) alkyl groups are independently optionally bonded by one or more halogens, hydroxyl groups, CN, N3, N(R) a )2 or OR a ; and wherein one or more non-terminal carbon atoms of each of the (C1-C8) alkyl groups may optionally be -O-, -S- or -NR. a -replace.

[0404] In another embodiment, a method for treating a person in need of a coronavirus infection is provided, comprising administering a therapeutically effective amount of a compound of formula II represented by a compound of formula I or a pharmaceutically acceptable salt or ester thereof:

[0405]

[0406] in,

[0407] R 1 R 3 R 5 R 7 R 8 and R 9 As defined above for equation I:

[0408] Each R 2 Is it OR a Or halogen; and

[0409] R 6 Is it OR a 、N(Ra 2, N, 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)carbocycloalkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)ynyl or (C2-C8)substituted ynyl.

[0410] In one embodiment of a method for treating coronavirus infections by administering a compound of formula II, R of formula II... 1 For H. In another aspect of this implementation, R of equation II 6 It can be N3, CN, halogen, (C1-C8)alkyl, (C1-C8)-substituted alkyl, (C2-C8)-alkenyl, (C2-C8)-substituted alkenyl, (C2-C8)-ynyl, or (C2-C8)-substituted ynyl. In another aspect of this embodiment, R of formula II... 6 It is CN, methyl, vinyl, or ethynyl. In another aspect of this embodiment, R of formula II... 6 It is CN. In another aspect of this implementation, R of formula II... 6 It is methyl. In another aspect of this embodiment, R of formula II... 5 For H. In another aspect of this implementation, R of equation II 2 OR a In another aspect of this implementation scheme, R of formula II 2 It is OH. In another aspect of this embodiment, R of formula II... 2 It is F. In another aspect of this implementation scheme, R of formula II... 3 Is it OR a In another aspect of this implementation scheme, R of formula II 3 It is OH, -OC (=O)R 11 or -OC(=O)OR 11 In another aspect of this implementation scheme, R of formula II 3 It is OH. In another aspect of this embodiment, R of formula II... 8 It is NR11 R 12 In another aspect of this implementation scheme, R of formula II 8 It is NH2. In another aspect of this embodiment, R of formula II... 8 Is it OR 11 In another aspect of this implementation scheme, R of formula II 8 It is OH. In another aspect of this embodiment, R of formula II... 9 For H. In another aspect of this implementation, R of equation II 9 For NR 11 R 12 In another aspect of this implementation scheme, R of formula II 9 It is NH2. In another aspect of this embodiment, R of formula II... 7 For H, -C(=O)R 11 -C(=O)OR 11 or

[0411]

[0412] In another aspect of this implementation scheme, R of Formula II 7 For H. In another aspect of this implementation, R of equation II 7 for

[0413]

[0414] In another embodiment of the method for treating coronavirus infections including the administration of a compound of formula II, the coronavirus infection is caused by a coronavirus. In another aspect of this embodiment, the coronavirus is a MERS virus or a SARS virus. In another aspect of this embodiment, the coronavirus is a MERS virus. In another aspect of this embodiment, the coronavirus is a SARS virus. In another aspect of this embodiment, the coronavirus is caused by a MERS virus caused by a strain selected from known strains.

[0415] In another embodiment, a method for treating a person in need of a coronavirus infection 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:

[0416]

[0417] in,

[0418] R 6 R 7 R 8 and R 9 As defined above for Equation II;

[0419] Each R 2 Is it OR a Or F; and

[0420] Each R 3 Is it OR a .

[0421] In one embodiment of a method for treating coronavirus infections including the administration of a compound of formula III, R of formula III... 6 It can be N3, CN, halogen, (C1-C8)alkyl, (C1-C8)-substituted alkyl, (C2-C8)-alkenyl, (C2-C8)-substituted alkenyl, (C2-C8)-ynyl, or (C2-C8)-substituted ynyl. In another aspect of this embodiment, R of formula III... 6 It is CN, methyl, vinyl, or ethynyl. In another aspect of this embodiment, R of formula III... 6 It is CN. In another aspect of this implementation, R of formula III... 6 It is methyl. In another aspect of this embodiment, R of formula III... 2 Is it OR a In another aspect of this implementation scheme, R of Equation III 2 It is OH. In another aspect of this embodiment, R of formula III... 2 For F. In another aspect of this implementation, R of Equation III 3 For OH, -OC(=O)R 11 or -OC(=O)OR 11 In another aspect of this implementation scheme, R of Equation III 3 It is OH. In another aspect of this embodiment, R of formula III... 8 For NR 11 R 12 In another aspect of this implementation scheme, R of Equation III 8 It is NH2. In another aspect of this implementation, R of formula III... 8 Is it OR 11 In another aspect of this implementation scheme, R of Equation III 8 It is OH. In another aspect of this embodiment, R of formula III... 9 For H. In another aspect of this implementation, R of equation III 9 For NR 11 R 12 In another aspect of this implementation scheme, R of Equation III 9 It is NH2. In another aspect of this implementation, R of formula III... 7 For H, -C(=O)R 11 -C(=O)OR 11or

[0422]

[0423] In another aspect of this implementation scheme, R of Formula III 7 For H. In another aspect of this implementation, R of equation III 7 for

[0424]

[0425] In another embodiment of a method for treating coronavirus infections, including the administration of a compound of formula III, R of formula III... 6 It 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 For NH2. In another aspect of this embodiment, R of formula III... 6 It is CN, methyl, vinyl, or ethynyl. In another aspect of this embodiment, R of formula III... 6 It is CN. In another aspect of this implementation, R of formula III... 6 It is methyl. In another aspect of this embodiment, R of formula III... 2 Is it OR a In another aspect of this implementation scheme, R of Equation III 2 For OH, -OC(=O)R 11 or -OC(=O)OR 11 In another aspect of this implementation scheme, R of Equation III 2 It is OH. In another aspect of this embodiment, R of formula III... 2 For F. In another aspect of this implementation, R of Equation III 3 For OH, -OC(=O)R 11 or -OC(=O)OR 11 In another aspect of this implementation scheme, R of Equation III 3 It is OH. In another aspect of this embodiment, R of formula III... 9 For H. In another aspect of this implementation, R of equation III 9 For NR 11 R 12 In another aspect of this implementation scheme, R of Equation III 9 It is NH2. In another aspect of this implementation, R of formula III... 7 For H, -C(=O)R 11 -C(=O)OR 11 or

[0426]

[0427] In another aspect of this implementation scheme, R of Formula III 7 For H. In another aspect of this implementation, R of equation III 7 for

[0428]

[0429] In another embodiment of a method for treating coronavirus infections that includes administering a compound of formula III, R of formula III... 6 For CN, methyl, vinyl or ethynyl, R 8 It is NH2, and R 9 It is H. In another aspect of this implementation, R of formula III... 6 It is CN. In another aspect of this implementation, R of formula III... 6 It is methyl. In another aspect of this embodiment, R of formula III... 2 Is it OR a In another aspect of this implementation scheme, R of Equation III 2 For OH, -OC(=O)R 11 or -OC(=O)OR 11 In another aspect of this implementation scheme, R of Equation III 2 It is OH. In another aspect of this embodiment, R of formula III... 2 For F. In another aspect of this implementation, R of Equation III 3 For OH, -OC(=O)R 11 or -OC(=O)OR 11 In another aspect of this implementation scheme, R of Equation III 3 It is OH. In another aspect of this embodiment, R of formula III... 7 For H, -C(=O)R 11 -C(=O)OR 11 or

[0430]

[0431] In another aspect of this implementation scheme, R of Formula III 7 For H. In another aspect of this implementation, R of equation III 7 for

[0432]

[0433] In another embodiment of the method for treating coronavirus infections, including the administration of a compound of formula III, the coronavirus infection is caused by a coronavirus. In another aspect of this embodiment, the coronavirus is a MERS virus or a SARS virus. In another aspect of this embodiment, the coronavirus is a MERS virus. In another aspect of this embodiment, the coronavirus is a SARS virus. In another aspect of this embodiment, the coronavirus is caused by a MERS virus caused by a strain selected from known strains.

[0434] In another embodiment, a method for treating a person with a coronavirus infection in need is provided, comprising administering a therapeutically effective amount of a compound of formula IV or a pharmaceutically acceptable salt or ester thereof:

[0435]

[0436] Formula IV

[0437] Where R 7 As defined above for Equation I.

[0438] In another embodiment of a method for treating coronavirus infections that includes an administration of a compound of formula IV, R 7 It could be H. In another embodiment of a method for treating coronavirus infections, it includes administering a compound of formula IV, R. 7 Selected from the group as defined in equation I, such as a), b), or c).

[0439] In another implementation of a method for treating coronavirus infections, a compound of formula IV, R, is included. 7 for

[0440]

[0441] Z 1 and Z 2 Each of these groups independently has the following structure:

[0442]

[0443] And Z 3 It is Z 5 .

[0444] In another embodiment of a method for treating coronavirus infections that includes an administration of a compound of formula IV, R 7 for

[0445]

[0446] Z 1 and Z2 Each of these groups independently has the following structure:

[0447]

[0448] And Z 3 It is Z 5 .

[0449] In another embodiment of a method for treating coronavirus infections that includes an administration of a compound of formula IV, R 7 for

[0450]

[0451] Each Q 3b Independently O or N(R). In another implementation, each Q 3b It is O and each R x Independently:

[0452]

[0453] Where M12c is 1, 2, or 3 and each Q 3 It can be a bond, O, CR2, or S independently.

[0454] In some implementation schemes, R e1 and R e2 Each can be independently H, C1-C6 alkyl, or benzyl. In some embodiments, R e1 It can be H, C1-C6 alkyl or benzyl, and R e2 It can be H or C1-C6 alkyl. In some embodiments, R e1 and R e2 Each can be independently H or C1-C6 alkyl. In some embodiments, R e1 and R e2 Each can be H or benzyl independently. In some embodiments, R e1 It can be H, methyl, or benzyl, and R e2 It can be H or methyl. In some embodiments, R e1 It can be H or methyl, and R e2 It can be H or methyl. In some embodiments, R e1 It can be methyl, and R e2 It can be H or methyl. In some embodiments, R e1 It can be H or benzyl, and R e2 It can be H or methyl.

[0455] In another embodiment of a method for treating coronavirus infections that includes an administration of a compound of formula IV, R7 yes

[0456]

[0457] In another embodiment of a method for treating coronavirus infections that includes an administration of a compound of formula IV, R 7 for

[0458]

[0459] In another embodiment of a method for treating coronavirus infections that includes an administration of a compound of formula IV, R 7 for

[0460]

[0461] Where R f Selected from H, C1-C8 alkyl, benzyl, C3-C6 cycloalkyl, and -CH2-C3-C6 cycloalkyl. In another embodiment of the compound of formula IV, R... f It is a C1-C8 alkyl group. In another embodiment of the compound of formula IV, R f It is 2-ethylbutyl.

[0462] In another embodiment of a method for treating coronavirus infections that includes an administration of a compound of formula IV, R 7 yes

[0463]

[0464] in,

[0465] R f Selected from H, C1-C8 alkyl, benzyl, C3-C6 cycloalkyl and -CH2-C3-C6 cycloalkyl; and

[0466] R g It is selected from C1-C8 alkyl, -O-C1-C8 alkyl, benzyl, -O-benzyl, -CH2-C3-C6 cycloalkyl, -O-CH2-C3-C6 cycloalkyl and CF3.

[0467] In another embodiment of a method for treating coronavirus infections that includes the administration of an IV compound, R 7 for

[0468]

[0469] Where R f Selected from H, C1-C8 alkyl, benzyl, C3-C6 cycloalkyl, and -CH2-C3-C6 cycloalkyl. In another embodiment of the compound of formula IV, R... fIt is a C1-C8 alkyl group. In another embodiment of the compound of formula IV, R f It is a C1-C6 alkyl group. In another embodiment of the compound of formula IV, R f It is 2-ethylbutyl.

[0470] In another embodiment of a method for treating coronavirus infections including the administration of an IV compound, R 7 for:

[0471]

[0472] Where 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. In another embodiment of the compound of formula IV, R f It is a C1-C8 alkyl group. In another embodiment of the compound of formula IV, R f It is a C1-C6 alkyl group.

[0473] In another embodiment of a method for treating coronavirus infections that includes an administration of a compound of formula IV, R 7 Selected from:

[0474]

[0475] In another embodiment of a method for treating coronavirus infections that includes the administration of an IV compound, R 7 for

[0476]

[0477] In another embodiment of a method for treating coronavirus infections including the administration of a compound of formula IV, Z 1 and Z 2 Each could be:

[0478]

[0479] In another embodiment, a method for treating coronavirus infections in people in need is provided, comprising administering a therapeutically effective amount of a compound of formulas I-IV, wherein R 11 Or R 12 Independently, it can be H, (C1-C8)alkyl, (C1-C8)alkenyl, (C2-C8)ynyl, (C4-C8)carbocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -C(=O)(C1-C8)alkyl, or -S(O). n (C1-C8)alkyl or aryl(C1-C8)alkyl. In another embodiment, R11 and R 12 Together with nitrogen atoms that are all connected to them, they form 3-7 membered heterocycles, wherein any carbon atom of the heterocycle may optionally be -O-, -S-, or -NR. a - Instead. Therefore, as an example and not a limitation, the group -NR 11 R 12 It can be represented by the following heterocycles:

[0480] wait.

[0481] In another embodiment, a method for treating coronavirus infections in people in need is provided, comprising administering a therapeutically effective amount of a compound of formulas I-IV, wherein each R 3 R 4 R 5 R 6 R 11 Or R 12 Independently (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)ynyl or aryl(C1-C8)alkyl, wherein the (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)ynyl or aryl(C1-C8)alkyl is independently optionally influenced by one or more halogens, hydroxyl groups, CN, N3, N(R) a )2 or OR a Replacement. Therefore, R is used as an example rather than a limitation. 3 R 4 R 5 R 6 R 11 Or R 12 It can represent groups such as -CH(NH2)CH3, -CH(OH)CH2CH3, -CH(NH2)CH(CH3)2, -CH2CF3, -(CH2)2CH(N3)CH3, -(CH2)6NH2, etc.

[0482] In another embodiment, a method of treating coronavirus infections in people in need is provided, 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 It is a (C1-C8) alkyl group, wherein one or more non-terminal carbon atoms of each (C1-C8) alkyl group may optionally be -O-, -S- or -NR-. a - Instead. Therefore, R is used as an example rather than a limitation. 3 R 4 R 5 R6 R 11 Or R 12 It can represent groups such as -CH2OCH3, -CH2OCH2CH3, -CH2OCH(CH3)2, -CH2SCH3, -(CH2)6OCH3, -(CH2)6N(CH3)2, etc.

[0483] In another embodiment of the method for treating coronavirus infections, the method includes administering a compound of formula I, said compound being

[0484]

[0485] Or its pharmaceutically acceptable salts or esters.

[0486] In another embodiment of the method for treating coronavirus infections, the method includes administering a compound of formula I, said compound being

[0487]

[0488]

[0489]

[0490] Or its pharmaceutically acceptable salts or esters.

[0491] In another embodiment of the method for treating coronavirus infections, an administration of a compound of formula IV is included, said compound being:

[0492]

[0493]

[0494] Or its pharmaceutically acceptable salts or esters.

[0495] In another embodiment of the method for treating coronavirus infections, an administration of a compound of formula IV is included, said compound being:

[0496]

[0497] Or its pharmaceutically acceptable salts or esters.

[0498] In another embodiment of a method for treating coronavirus infections, the method includes administering a compound of formula I-IV, said compound being

[0499]

[0500]

[0501] Or its pharmaceutically acceptable salts or esters.

[0502] In another embodiment of a method for treating coronavirus infections, the method includes administering a compound of formula I-IV, said compound being

[0503]

[0504] Or its pharmaceutically acceptable salts or esters.

[0505] The treatments described in this article include methods for treating human coronavirus infections, including those caused by alpha coronavirus 229E (HCoV-229E) and NL63 (HCoV-NL63, New Haven coronavirus), beta coronavirus OC43 (HCoV-OC43), HKU1, SARS-CoV (the coronavirus that causes severe acute respiratory syndrome (SARS)) and MERS-CoV (the coronavirus that causes MERS), formerly known as novel coronavirus 2012 and HCoV-EMC.

[0506] The names of the compounds disclosed herein were provided using ACD / nomenclature software (Advanced Chemistry Development, Inc., Toronto, Canada). Other compounds or groups may be named using common names, systematic names, or non-systematic names. The naming and numbering of the compounds of this invention are illustrated using representative compounds of Formula I:

[0507]

[0508] It is named (2S)--2-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propionic acid-2-ethylbutyl ester. Other compounds of the present invention include:

[0509]

[0510] It was named (S)-2-(((S)-(((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-2-ethylbutyl ester, and

[0511]

[0512] It is named (S)-2-((R)-(((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-2-ethylbutyl ester.

[0513] Any reference to the compounds of the invention described herein also includes reference to their physiologically acceptable salts. Examples of physiologically acceptable salts of the compounds of the invention include those derived from suitable bases (e.g., alkali metals or alkaline earth metals, e.g., Na). + Li + K + Ca +2 and Mg +2 ), ammonium and NR4 + (where R is as defined in this paper) salt. 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, aminosulfonic acid, phosphoric acid, nitric acid, etc.; (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, ethanesulfonic 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-naphthylcarboxylate, dihydroxynaphthylcarboxylate, salicylic acid, stearic acid, phthalic acid, mandelic acid, lactic acid, ethanesulfonic acid, lysine, arginine, glutamic acid, glycine, serine, threonine, alanine, isoleucine, leucine, etc.; and (c) salts formed by elemental anions, such as chlorine, bromine, and iodine. Physiologically acceptable salts of hydroxyl compounds include the anion of the compound with a suitable cation such as Na+. + and NR4 + The combination of .

[0514] Compounds of formulas I-IV and their pharmaceutically acceptable salts may exist in different polymorphs or pseudopolymorphs. As used herein, crystalline polymorphism refers to the ability of a crystalline compound to exist in different crystal structures. Crystalline polymorphism may be caused by differences in crystal packing (packing polymorphism) or differences in packing between different conformational isomers of the same molecule (conformational polymorphism). As used herein, crystalline pseudopolymorphism refers to the ability of a compound's hydrates or solvates 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 conformational isomers of the same molecule (conformational pseudopolymorphism). The present invention comprises all polymorphs and pseudopolymorphs of compounds of formulas I-III and their pharmaceutically acceptable salts.

[0515] Compounds of formulas 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 are no positions of long-range ordered atoms. This definition also applies when the crystal size is 2 nanometers or smaller. Additives, including solvents, can be used to produce the amorphous forms of the present invention. The present invention includes all amorphous forms of compounds of formulas I-IV and their pharmaceutically acceptable salts.

[0516] For therapeutic purposes, the salts of the active ingredients of the compounds of this 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 this invention.

[0517] Finally, it should be understood that the compositions herein comprise the unionized and zwitterionic forms of the compounds of the present invention, and a combination with a stoichiometric amount of water in the hydrate.

[0518] It should be noted that all enantiomers, diastereomers, racemic mixtures, tautomers, polymorphs, pseudopolymorphs, and pharmaceutically acceptable salts of compounds within the range of formulas I-IV are included in this invention. All mixtures of these enantiomers and diastereomers are within the scope of this invention.

[0519] The compounds of the present invention, illustrated by examples of Formulas I-IV, may have a chiral center, such as a chiral carbon or phosphorus atom. Therefore, the compounds of the present invention comprise racemic mixtures of all stereoisomers, including enantiomers, diastereomers, and transisomers. Additionally, the compounds of the present invention contain enriched or resolved 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 diastereomer mixtures, as well as isolated or synthesized individual optical isomers (substantially free of their enantiomeric or diastereomeric partners), are all within the scope of the present invention. Racemic mixtures are isolated into their respective substantially optically pure isomers using well-known techniques, such as isolating diastereomers formed with optically active auxiliaries (e.g., acids or bases) and then converting them back to the optically active substance. In most cases, the desired optical isomers are synthesized via stereooriented reactions, starting from suitable stereoisomers of the desired starting material.

[0520] The stereochemical definitions and conventions used in this article generally follow 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 an optically active form, meaning they possess the ability to rotate the plane of polarized light. In describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule with respect to its chiral center. The prefixes d and 1, D and L or (+) and (-) are used to indicate the rotation sign of the compound with respect to plane polarized light; S, (-), or 1 indicates that the compound is levorotatory, while 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 can also be called enantiomers, and mixtures of these isomers are generally called enantiomeric mixtures. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur in chemical reactions or processes where there is no stereoselectivity or stereodirection. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomers that is not optically active.

[0521] The compounds of this invention may also exist in tautomer forms under certain circumstances. Although only one delocalized resonance structure can be described, all such forms are within the scope of this invention. For example, purine, pyrimidine, imidazole, guanidine, amidine, and tetrazolium systems can exist as en-amine tautomers, and all their possible tautomer forms are within the scope of this invention.

[0522] Any formula (including compounds of formula I) or structure given herein is intended to represent both unlabeled forms and isotopically labeled compounds. Isotopically labeled compounds have the structures described by the formulas given herein, except that one or more atoms are replaced by atoms having selected atomic weights or mass numbers. Examples of isotopes that can be incorporated into the compounds of this disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as, but not limited to, 2H (deuterium, D) and 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 disclosed herein, such as those incorporating radioactive isotopes like... 3 H, 13 C and 14Compounds of C. These isotope-labeled compounds can be used in metabolic studies, reaction kinetic studies, detection or imaging techniques such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT), including the detection of drug or substrate tissue distribution or radiotherapy of patients.

[0523] This disclosure also includes compounds of Formula I, wherein one to n hydrogen atoms bonded to carbon atoms are replaced by deuterium, where n is the number of hydrogen atoms in the molecule. Such compounds exhibit enhanced metabolic resistance and can therefore be used to increase the half-life of any Formula I compound administered to mammals, particularly humans. See, for example, Foster, “Deuteriu MIsotope Effects in Studies of Drug Metabolism”, Trends Pharmacol. Sci. 5(12):524-527 (1984). Such compounds are synthesized by methods known in the art, for example by using starting materials in which one or more hydrogen atoms are replaced by deuterium.

[0524] The deuterium-labeled or substituted therapeutic compounds disclosed herein may have improved DMPK (drug metabolism and pharmacokinetics) properties involving distribution, metabolism, and excretion (ADME). Substitution with a heavier isotope such as deuterium can provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life, reduced dose requirement, and / or improved therapeutic index. 18 F-labeled compounds can be used in PET or SPECT studies. The isotopically labeled compounds and their prodrugs disclosed herein can generally be prepared by replacing non-isotopically labeled reagents with readily available isotopically labeled reagents using the methods disclosed in the embodiments and preparations described below. It should be understood that deuterium is considered a substituent in the compounds of formula I in this context.

[0525] The concentration of such heavier isotopes, particularly deuterium, can be defined by the isotope enrichment factor. In the compounds disclosed herein, any atom not specifically designated as a particular isotope is intended to represent any stable isotope of that atom. Unless otherwise stated, when a position is explicitly designated as “H” or “hydrogen,” that position is understood to be hydrogen with its naturally occurring isotopic composition. Therefore, in the compounds disclosed herein, any atom specifically designated as deuterium (D) means to represent deuterium.

[0526] Whenever the compounds described herein are referred to by more than one of the same designated groups, such as "R" or "R", 1 "If it is replaced, then it should be understood that these groups can be the same or different, that is, each group is chosen independently. (Wave line)" It indicates the covalent bond connection point with an adjacent substructure, group, part, or atom.

[0527] The selected substituents in compounds I-IV are present in a recursive manner. In this context, a "recursive substituent" refers to another instance of a substituent that can refer to itself. Due to this recursive nature of the substituents, theoretically, a large number of compounds can exist in any given embodiment. For example, R x Includes R y Substituent. R y It can be R. R can be Z. 3 Z 3 It could be Z 4 And Z 4 It can be R or contain R y Substituents. Or, Z 3 It could be Z 5 It can contain R y Substituents. Those skilled in the art of medicinal chemistry will understand that the total number of such substituents is reasonably limited by the desired properties of the compound. By way of example, and not limitation, these properties include physical properties such as molecular weight, solubility, or log P; applied properties such as activity against the intended target; and practical properties such as ease of synthesis.

[0528] As an example rather than a limitation, in some implementations, Z 3 and R y These are recursive substituents. 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 fewer times. Even more typically, in a given embodiment, each recursive substituent may independently occur 3 times or fewer times. For example, in a given embodiment, Z... 3 It will appear 0 to 8 times, R y It will occur 0 to 6 times. Even more typically, in a given implementation, Z... 3 It will appear 0 to 6 times, and R y It will appear 0 to 4 times.

[0529] Recursive substituents are a contemplated aspect of this invention. Those skilled in the art of medicinal chemistry will understand the versatility of such substituents. In one embodiment of this invention, where recursive substituents are present, the total number will be determined as described above.

[0530] 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.

[0531] A. Substitution forms of compounds

[0532] Compounds of formulas I-IV may contain a phosphate ester 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 Independently, it can be H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)ynyl, (C4-C8)carbocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, -C(=O)(C1-C8)alkyl, or -S(O). n (C1-C8)alkyl or aryl(C1-C8)alkyl; or R 11 and R 12 Together with nitrogen atoms that are all connected to them, they form 3-7 membered heterocycles, wherein any carbon atom of the heterocycle may optionally be -O-, -S-, or -NR. a -replace;

[0533] Each R a Independently, it is H, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)ynyl, aryl(C1-C8)alkyl, (C4-C8)carbocycloalkyl, -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;

[0534] Each R is independently H, (C1-C8)alkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)ynyl, (C2-C8)substituted ynyl, C6-C 20 Aryl, C6-C 20 Substituted aryl, C2-C 20 Heterocyclic group, C2-C 20 Substituted heterocyclic groups, arylalkyl groups, or substituted arylalkyl groups; and

[0535] Each R 11Or R 12 Each (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, or aryl (C1-C8)alkyl group is independently and optionally resorbed by one or more halogens, hydroxyl groups, CN, N3, N(R) a )2 or OR a Substitution; and wherein one or more non-terminal carbon atoms of each of the (C1-C8) alkyl groups may optionally be replaced by -O-, -S- or -NR. a -replace,

[0536] b)

[0537]

[0538] in:

[0539] R c Selected from phenyl, 1-naphthyl, 2-naphthyl,

[0540]

[0541] R d It is H or CH3;

[0542] R e1 and R e2 Each is independently H, C1-C6 alkyl, or benzyl;

[0543] R f Selected from H, C1-C8 alkyl, benzyl, C3-C6 cycloalkyl and -CH2-C3-C6 cycloalkyl;

[0544] 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

[0545] n' is selected from 1, 2, 3, and 4; and

[0546] d) The following groups:

[0547]

[0548] in,

[0549] Each Q 1 Independently O, S, or NR; and

[0550] Each R y 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 atoms on the same carbon atom y Together they form a carbon ring of 3-7 carbon atoms;

[0551] Each Q 2 Independently for O, S, NR, + N(O)(R), N(OR), + N(O)(OR) or N-NR2; or

[0552] Z 1 and Z 2 Each group is an independent group of formula Ia:

[0553]

[0554] in:

[0555] Each Q 3 Independently for bond, O, CR2, NR, + N(O)(R), N(OR), + N(O)(OR), N-NR2, S, SS, S(O) or S(O)2;

[0556] M2 is 0, 1, or 2;

[0557] Each R x Independently for R y Or the following formula:

[0558]

[0559] in:

[0560] Each M1a, Mlc, and M1d is independently 0 or 1;

[0561] M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0562] Z 3 It is Z 4 or Z 5 ;

[0563] Z 4 It is R, -C(Q) 2 )R y -C(Q) 2 )Z 5 -SO2R y or -SO2Z 5 ;and

[0564] Z 5 It is a carbon ring or a hetero ring, where Z 5 Independently controlled by 0 to 3 R y Group substitution.

[0565] Z 5 Carbon rings and Z 5 Heterocyclic rings can be independently divided by 0 to 3 R groups. y Group substitution. Z 5 It can be a saturated, unsaturated, or aromatic ring containing monocyclic or bicyclic carbon rings or heterocyclic rings. 5 It can have 3 to 10 ring atoms, for example, 3 to 7 ring atoms. When it contains 3 ring atoms, Z 5 The ring is saturated; Z is present when there are 4 ring atoms. 5 The ring can be saturated or monounsaturated; Z is defined as having 5 ring atoms. 5 The ring can be saturated, monounsaturated, or doubly unsaturated; when it contains 6 ring atoms, Z... 5 The rings are saturated, monounsaturated, or diunsaturated, or aromatic.

[0566] Z 5 Heterocycles can be monocyclic with 3-7 ring members (2-6 carbon atoms and 1-3 heteroatoms selected from N, O, P, and S) or bicyclic with 7-10 ring members (4-9 carbon atoms and 1-3 heteroatoms selected from N, O, P, and S). 5 Heterocyclic monocyclic rings 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). 5Heterocyclic bicyclic structures 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 Heterocycles can be linked to Q via stable covalent bonds from carbon, nitrogen, sulfur, or other atoms. 2 Bonding.

[0567] Z 5 Heterocyclic compounds include, for example, pyridinyl, dihydropyridinyl isomers, piperidine, pyridazinyl, pyrimidinyl, pyrazinyl, mesazinyl, oxazolyl, imidazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, furanyl, thiofuranyl, thiophenyl, and pyrroleyl. 5 This also includes, but is not limited to, the following examples:

[0568]

[0569] As defined above, Z 5 Carbocyclic and heterocyclic rings can be independently substituted by 0 to 3 R groups. For example, substituted Z... 5 Carbon rings include:

[0570]

[0571] Examples of substituted phenyl carbocyclic rings include:

[0572]

[0573] In another embodiment, the Z of compounds of formulas I-IV 5 It is a carbon ring or a hetero ring, where Z 5 Independently controlled by 0 to 3 R z Group substitution, wherein each R z 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.

[0574] Compounds of formulas I-IV The implementation scheme includes substructures, such as:

[0575]

[0576] Each Q 3b Independently, it is either O or N(R). In another aspect of this implementation, each Q... 3b It is O and each R x Independently:

[0577]

[0578] Where M12c is 1, 2, or 3 and each Q 3 Independently, it can be a bond, O, CR2, or S. In another aspect of this embodiment, a Q... 3b -R x It is NH(R), and the other Q 3b -R x Is it OR x , where R x yes:

[0579]

[0580] Where M12c is 2. In another aspect of this implementation, each Q... 3b It is O and each R x Independently:

[0581]

[0582] Where M12c is 2. In another aspect of this implementation, each Q 3b It is O and each R x Independently:

[0583]

[0584] Where M12c is 1 and Q 3 For the bond, O or CR2.

[0585]

[0586] Each Q 3Independently, it is either O or N(R). In another aspect of this implementation, each Q... 3 It is O. In another aspect of this implementation, the substructure is:

[0587]

[0588] Where R y As defined in this article, R 5 .

[0589] Formulas I-IV Another implementation scheme includes a substructure:

[0590]

[0591] Each Q 2c Independently for O, N(R) y ) or S.

[0592] Compounds of formulas I-IV Another implementation scheme includes Z 1 or Z 2 One of them with 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 compounds selected from the following formula Ib:

[0593]

[0594] Formula 1b

[0595] In another aspect of the implementation of formula Ib, each Q and Q 3 It is O. In another aspect of the implementation of formula Ib, Z 1 or Z 2 It is Q 3b -R x ; Each Q, Q 3 and Q 3b It is O and R x yes:

[0596]

[0597] Where M12c is 1, 2, or 3 and each Q 3 Independently, it can be a bond, O, CR2, or S. In another aspect of the implementation of formula Ib, Z 1 or Z 2 It is Q 3b -R x Each Q, Q3 and Q 3b It is O and R x yes:

[0598]

[0599] Where M12c is 2. In another aspect of the implementation of equation Ib, Z 1 or Z 2 It is Q 3b -R x ; Each Q, Q 3 and Q 3b It is O and R x yes:

[0600]

[0601] Where M12c is 1 and Q 3 For the bond, O or CR2.

[0602] Compounds of formulas I-IV Another implementation scheme includes a substructure:

[0603]

[0604] Z 5 It is a carbocyclic ring, such as a phenyl or a substituted phenyl group. In another aspect of this embodiment, the substructure is:

[0605]

[0606] Q 3b It is O or N(R) and the phenyl carbide ring is substituted with 0 to 3 R groups. In another aspect of this embodiment of the substructure, R x yes:

[0607]

[0608] Where M12c is 1, 2, or 3 and each Q 3 It can be a bond, O, CR2, or S independently.

[0609] Formulas I-IV Another implementation scheme includes a substructure:

[0610]

[0611] The chiral carbons of amino acids and lactate ester moieties can be R or S configurations or racemic mixtures.

[0612] I-IV Another implementation scheme is a substructure

[0613]

[0614] Each Q 3 Independently -O- or -NH-. In another aspect of this embodiment, R y It 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... y R is (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 It is (C1-C8)alkyl, (C1-C8)substituted alkyl, (C2-C8)alkenyl, (C2-C8)substituted alkenyl, (C2-C8)ynyl, or (C2-C8)substituted ynyl; R is CH3; and each Q 3 It is -NH-. In another aspect of this implementation, Z 1 and Z 2 A naturally occurring amino acid or a naturally occurring amino acid ester that is independently linked to nitrogen. In another aspect of this embodiment, Z 1 and Z 2 It 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 a 2-hydroxyl group.

[0615] Formulas I-IV Another implementation scheme is a substructure:

[0616]

[0617] In one aspect of this implementation scheme, each R x Independently, each R is a (C1-C8) alkyl group. In another aspect of this embodiment, each R... x Independently for C6-C 20 Aryl or C6-C 20 Substituted aryl groups.

[0618] In a preferred embodiment, Selected from

[0619]

[0620]

[0621] R x The implementation scheme includes esters, carbamates, carbonates, thioesters, amides, thioamides, and urea groups:

[0622]

[0623] B. Metabolites of the compounds of this invention

[0624] Also falling within the scope of this invention are the in vivo metabolites of the compounds described herein, provided that these products are novel and not obvious compared to the prior art. Such products can result from, for example, oxidation, reduction, hydrolysis, amidation, esterification, etc., of the applied compound, primarily due to enzymatic processes. Therefore, this invention includes novel and non-obvious compounds produced by a method comprising exposing the compounds of this invention to mammals for a time sufficient to produce their metabolites. Typically, this is achieved by preparing radiolabeled (e.g.,) compounds of this invention. 14 C or 3 The compound (H) is 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), allowing sufficient time for metabolism (typically about 30 seconds to 30 hours), and its metabolites are isolated from urine, blood, or other biological samples for identification. These products are easily separated because they are labeled (others are separated by using antibodies capable of binding to epitopes present in the metabolites). The metabolite structures are determined in a conventional manner, such as by MS or NMR analysis. Typically, the 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 metabolites are not present in the body, they are useful in the diagnostic determination of therapeutic doses of the compounds of the present invention, even if they themselves do not possess anti-arenaviridae activity.

[0625] Formulations and methods for determining the stability of compounds in alternative gastrointestinal secretions are known. Compounds are defined herein as stable in the gastrointestinal tract, wherein less than about 50 molar percentage of the protected group is deprotected in alternative intestinal or gastric juice after incubation at 37°C for 1 hour. The fact that compounds are stable in the gastrointestinal tract does not mean that they cannot be hydrolyzed in vivo. The prodrugs of this 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 cells in general.

[0626] III. Pharmaceutical Preparations

[0627] The compounds of the present invention are formulated using conventional carriers and excipients, which will be selected according to conventional practice. Tablets will contain excipients, flow aids, fillers, binders, etc. Aqueous formulations are prepared aseptically and are intended for delivery, typically isotonic, other than orally. 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 typically from about 7 to 10. In some embodiments, the pH range of the formulation is from about 2 to about 5, but typically from about 3 to 4.

[0628] Although the active ingredients can be administered alone, they may be preferably presented as pharmaceutical formulations. The formulations of the present invention for veterinary and human use comprise at least one active ingredient as defined above, as well as one or more acceptable carriers and optional other therapeutic ingredients, particularly those discussed herein. The carrier must be “acceptable” from the perspective of compatibility with other components of the formulation and physiological harmlessness to the recipient.

[0629] This formulation includes those suitable for the routes of administration described above. The formulation can be conveniently presented in unit dose form and can be prepared by any method well known in the pharmaceutical field. Techniques and formulations are generally available at Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). These methods involve the step of binding the active ingredient to a carrier constituting one or more excipients. Generally, formulations are prepared by uniformly and tightly binding the active ingredient to a liquid carrier or a finely fragmented solid carrier, or both, and then shaping the product if desired.

[0630] The formulations of the present invention suitable for oral administration can be presented in discrete unit form, such as capsules, pouches, or tablets, each containing a predetermined amount of the active ingredient; as powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water emulsion or a water-in-oil emulsion. The active ingredient can also be administered as a bolus, saccharin, or paste.

[0631] Tablets are prepared by compression or molding, optionally containing one or more excipients. Compressed tablets can be prepared by compressing a free-flowing form of active ingredient, such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant, or dispersant, in a suitable machine. Molded tablets can be prepared by molding a mixture of powdered active ingredients moistened with an inert liquid diluent in a suitable machine. Tablets may optionally be coated or scored and optionally formulated to provide a slow or controlled release of the active ingredient therefrom.

[0632] For infections of the eyes or other external tissues such as the mouth and skin, the formulation is preferably a topical ointment or cream containing an active ingredient in an amount, 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 can be used with a paraffin or water-miscible ointment base. Alternatively, the active ingredient can be formulated as a cream using an oil-in-water emulsion base.

[0633] If desired, the aqueous phase of the cream base may include, for example, at least 30% w / w of polyols, i.e., alcohols having two or more hydroxyl groups, such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerin, and polyethylene glycol (including PEG 400) and mixtures thereof. Topical formulations may ideally include compounds that enhance the absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogues.

[0634] The oil phase of the emulsion of the present invention can be composed of known ingredients in a known manner. Although this phase may contain only emulsifiers (also referred to as emulsifiers), it ideally contains at least one emulsifier with fats or oils, or with a mixture of both fats and oils. Preferably, hydrophilic emulsifiers are included together with lipophilic emulsifiers that act as stabilizers. Oils and fats are also preferred. In summary, the emulsifiers, with or without stabilizers, constitute so-called emulsified waxes, and these waxes, together with the oils, constitute a so-called emulsified ointment matrix, which forms the oily dispersed phase of the ointment formulation.

[0635] The emulsifiers and emulsion stabilizers suitable for the formulations of this invention include Cetearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearate, and sodium lauryl sulfate. Other emulsifiers and emulsion stabilizers suitable for use in the formulations of this invention include...

[0636] The selection of suitable oils or fats for formulations is based on achieving the desired cosmetic properties. Creams should preferably be non-greasy, non-staining, and washable products with a suitable consistency to prevent leakage from tubes or other containers. Straight-chain or branched mono- or dialkyl esters can be used, such as diisohexyl adipate, isohexadecanyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate, or mixtures of branched esters known as Crodamol CAP, the latter three being preferred esters. These can be used alone or in combination depending on the desired properties. Alternatively, high-melting-point fats, such as white soft paraffin and / or liquid paraffin or other mineral oils, can be used.

[0637] Pharmaceutical formulations according to the invention comprise the combination according to the invention, one or more pharmaceutically acceptable carriers or excipients, and optionally other therapeutic agents. Pharmaceutical formulations containing the active ingredient can be in any form suitable for the intended method of administration. For example, when intended for oral use, they can be prepared as tablets, lozenges, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups, or elixirs. Compositions intended for oral use can be prepared according to any method known in the art for manufacturing pharmaceutical compositions, such compositions may contain one or more agents, including sweeteners, flavoring agents, coloring agents, and preservatives, to provide a palatable formulation. Tablets containing the active ingredient and non-toxic, pharmaceutically acceptable excipients suitable for tablet manufacturing are acceptable. These excipients can be, for example, inert diluents such as calcium carbonate or sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrants such as corn starch or alginate; binders such as starch, gelatin, or gum arabic; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets may be uncoated or coated using known techniques, including microencapsulation, to delay disintegration and absorption in the gastrointestinal tract, thereby providing sustained action over a longer period. For example, delay-release materials, such as glyceryl monostearate or glyceryl distearate, may be used alone or in combination with waxes.

[0638] Oral formulations may also be provided in hard gelatin capsules, wherein the active ingredient is mixed with an inert solid diluent such as calcium phosphate or kaolin, or in soft gelatin capsules, wherein the active ingredient is mixed with an aqueous or oily medium such as peanut oil, liquid paraffin or olive oil.

[0639] The aqueous suspension of the present invention contains an active substance mixed with excipients suitable for manufacturing aqueous suspensions. Such excipients include suspending agents such as sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and gum arabic, and dispersants or wetting agents such as naturally occurring phospholipids (e.g., lecithin), condensation products of ethylene oxide and fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide and long-chain fatty alcohols (e.g., heptadecanethoxycetyl alcohol), and condensation products of ethylene oxide and esters derived from fatty acids and hexyl anhydrides (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propylparaben, 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).

[0640] Oil suspensions can be formulated by suspending the active ingredient in vegetable oils such as peanut oil, olive oil, sesame oil, or coconut oil, or in mineral oils such as liquid paraffin. Oral suspensions may contain thickeners such as beeswax, hard paraffin, or cetyl alcohol. Sweeteners (such as those mentioned above) and flavoring agents can be added to provide a palatable oral formulation. These compositions can be preserved by adding antioxidants such as ascorbic acid.

[0641] The dispersible powders and granules of the present invention, suitable for preparing aqueous suspensions by adding water, provide an active ingredient that can be mixed 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, flavoring agents, and coloring agents may also be present.

[0642] The pharmaceutical compositions of the present invention may also be in the form of an oil-in-water emulsion. The oil phase may 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 tragacanth, naturally occurring phospholipids such as soybean lecithin, esters or metaesters derived from fatty acids and hexitan anhydrides such as sorbitan monooleate, and condensation products of these metaesters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweeteners and flavoring agents. Syrups and elixirs may be formulated with sweeteners such as glycerin, sorbitol, or sucrose. Such formulations may also contain modifiers, preservatives, flavoring agents, or coloring agents.

[0643] The pharmaceutical compositions of the present invention can be in the form of sterile injectable formulations, such as sterile aqueous or oily suspensions for injection. These suspensions can be formulated using suitable dispersants or wetting agents and suspending agents mentioned above, according to known techniques. Sterile injectable formulations can also be sterile injectable solutions or suspensions in non-toxic, parenteral diluents or solvents, such as solutions in 1,3-butanediol or prepared as lyophilized powders. Water, Ringer's solution, and isotonic sodium chloride solution can be used in acceptable carriers and solvents. Additionally, sterile non-volatile oils can generally be used as solvents or suspension media. For this purpose, any mild non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Furthermore, fatty acids such as oleic acid can also be used in the preparation of injectable formulations. Water, Ringer's solution, isotonic sodium chloride solution, and hypertonic sodium chloride solution can be used in acceptable carriers and solvents.

[0644] 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 administration method. For example, a time-release formulation intended for oral administration in humans may contain about 1-1000 mg of the active substance mixed with a suitable and convenient amount of a carrier material, which may comprise about 5-95% (by weight) of the total composition. Pharmaceutical compositions can be prepared to provide easily measurable dosages. For example, an aqueous solution for intravenous infusion may contain about 3-500 μg of the active ingredient per milliliter of solution so that a suitable volume can be infused at a rate of about 30 mL / hour.

[0645] Formulations suitable for topical ocular administration also include eye drops, wherein 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 formulations at a concentration of 0.5-20 w / w, advantageously 0.5-10 w / w, and particularly about 1.5 w / w.

[0646] Formulations suitable for topical oral administration include tablets containing the active ingredient in a flavoring matrix, typically sucrose and gum arabic or tragacanth; tablets containing the active ingredient in an inert matrix such as gelatin and glycerin or sucrose and gum arabic; and mouthwashes containing the active ingredient in a suitable liquid carrier.

[0647] Formulations for rectal administration can be presented as suppositories having a suitable matrix containing, for example, cocoa butter or salicylates.

[0648] Formulations suitable for intrapulmonary or intranasal administration have particle sizes ranging from, for example, 0.1 to 500 micrometers, such as 0.5, 1, 30, 35 micrometers, and are administered via rapid inhalation through the nasal passage or 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 together with other therapeutic agents, such as compounds previously used to treat or prevent infections of the following Arenaviridae family.

[0649] Preparations suitable for vaginal administration may be in the form of vaginal suppositories, tampons, creams, gels, pastes, foams or sprays, and contain, in addition to the active ingredient, a suitable carrier known in the art.

[0650] Preparations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions, which may contain antioxidants, buffers, antibacterial agents, and solutes that make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners.

[0651] The formulations are presented in single-dose or multi-dose containers (e.g., sealed ampoules and vials) and can be stored under freeze-drying (lyophilization) conditions, requiring only the addition of a sterile liquid carrier (e.g., water for injection) immediately before use. Immediate-use solutions and suspensions are prepared from the aforementioned types of sterile powders, granules, and tablets. Preferred single-dose formulations are those containing the active ingredient at the daily dose or a unit daily sub-dose or a suitable portion thereof as described above.

[0652] It should be understood that, in addition to the ingredients specifically mentioned above, the formulations of the present invention may include other agents conventional in the art for the type of formulation discussed, such as flavoring agents for formulations suitable for oral administration.

[0653] The present invention further provides a veterinary composition comprising at least one active ingredient as defined above and a veterinary carrier.

[0654] A veterinary carrier is a material used for the purpose of administering the composition and can be a solid, liquid, or gaseous material that is inert or acceptable in the veterinary field and compatible with the active ingredient. These veterinary compositions can be administered orally, parenterally, or via any other desired route.

[0655] The compounds of the present invention are used to provide controlled-release pharmaceutical formulations (“controlled-release formulations”) containing one or more of the compounds of the present invention as active ingredients, wherein the release of the active ingredient is controlled and modulated to allow for less frequent administration or to improve the pharmacokinetic or toxicological characteristics of a given active ingredient.

[0656] IV. Route of Administration

[0657] One or more of the compounds of the present invention (referred to herein as the active ingredients) may be administered via any route suitable for the condition to be treated. Suitable routes include oral, rectal, nasal, pulmonary, local (including oral and sublingual), vaginal, and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural). It is understood that preferred routes may vary depending on, for example, the recipient's condition. An advantage of the compounds of the present invention is that they have oral bioavailability and can be administered orally.

[0658] In the method of the present invention for treating arenaviroid infections, the compounds of the present invention can be administered at any time to persons who may have come into contact with or who already have arenaviroid infections. In some embodiments, the compounds of the present invention can be administered prophylactically to persons who have come into contact with persons who have arenaviroid infections. In some embodiments, the compounds of the present invention can be administered to persons who test positive for arenaviroid infection but have not yet shown symptoms of arenaviroid infection. In some embodiments, the compounds of the present invention can be administered to humans at the onset of arenaviroid infection symptoms.

[0659] The effective dose of the active ingredient depends at least on the nature of the condition being treated, its toxicity, whether the compound is being used prophylactically (at a lower dose) or against an active viral infection, the method of delivery, and the pharmaceutical formulation, and will be determined by clinicians using routine dose-escalation studies. A dose of approximately 0.0001 to approximately 100 mg per kilogram of body weight per day is expected; typically, it is approximately 0.01 to approximately 10 mg per kilogram of body weight per day; more typically, it is approximately 0.01 to approximately 5 mg per kilogram of body weight per day; most typically, it is approximately 0.05 to approximately 0.5 mg per kilogram of body weight per day. For example, a candidate daily dose for an adult weighing approximately 70 kg would be in the range of 1 mg to 1000 mg, preferably in the range of 5 mg to 500 mg, and could be administered in single or multiple doses.

[0660] The effective dose of the compound of the present invention for treating arenaviroid infections can depend on whether the dose is for prophylactic use or for treating a person already infected with arenaviroid infection. Furthermore, the dose can depend on whether the person with arenaviroid infection is asymptomatic or has already shown symptoms of arenaviroid infection. Higher doses are necessary for treating individuals who test positive for arenaviroid infection and those showing symptoms of arenaviroid infection compared to those receiving prophylactic treatment.

[0661] Any suitable time period is contemplated for administering the compounds of the present invention. For example, administration may be from 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 may also be from 1 week to 15 weeks, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 weeks. Longer durations of administration are also considered. The timing of administration may depend on whether the compound is administered prophylactically or to treat a person with an arenaviroid infection. For example, prophylactic administration may be within a suitable timeframe after a person has been in frequent contact with another person with an arenaviroid infection and after the last contact with a person with an arenaviroid infection. For a person already infected with an arenaviroid infection, the timing of administration may be any length of time required to treat the patient, and within a suitable timeframe after a negative test for arenaviroid infection to ensure that the arenaviroid infection does not recur.

[0662] V. Combination Therapy

[0663] The compositions of the present invention are also used in combination with other active ingredients. For the treatment of arenaviridae virus infections, preferably, other active therapeutic agents are active against arenaviridae virus 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 to patients suffering from infections of the Arenaviridae family, including: parenteral fluids (including glucose 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, and medications for other common illnesses in the affected population, such as antimalarial drugs (including artemether and artesunate-benfluridine 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 Shigella infection.

[0664] Any compound of the present invention can also be combined with one or more other active therapeutic agents in unit dosage forms and administered to a patient simultaneously or sequentially. Combination therapy can be administered as a simultaneous or sequential regimen. When administered sequentially, the combination can be given in two or more doses.

[0665] Co-administration of the compound of the present invention with one or more other active therapeutic agents generally refers to the simultaneous or sequential administration of the compound of the present invention and one or more other active therapeutic agents, such that therapeutically effective amounts of the compound of the present invention and one or more other active therapeutic agents are present in the patient's body.

[0666] Co-administration includes administering a unit dose of the compound of the present invention before or after administering a unit dose of one or more other active therapeutic agents, for example, administering the compound of the present invention within seconds, minutes, or hours after administering one or more other active therapeutic agents. For example, a unit dose of the compound of the present invention may be administered first, followed by a unit dose of one or more other active therapeutic agents within seconds or minutes. Alternatively, a unit dose of one or more other therapeutic agents may be administered first, followed by a unit dose of the compound of the present invention within seconds or minutes. In some cases, it may be necessary to administer a unit dose of the compound of the present invention first, followed by a unit dose of one or more other active therapeutic agents several hours (e.g., 1-12 hours). In other cases, it may be necessary to administer a unit dose of one or more other active therapeutic agents first, followed by a unit dose of the compound of the present invention several hours (e.g., 1-12 hours).

[0667] Combination therapy can provide “synergistic effects” and “synergistic enhancement effects,” meaning that the effect obtained when active ingredients are used together is greater than the sum of the effects of using the compounds alone. Synergistic effects are obtained when the active ingredients: (1) are co-formulated and administered or delivered simultaneously in a combination formulation; (2) are delivered alternately or in parallel as separate formulations; or (3) are administered via other regimens. When delivered as an alternating therapy, a synergistic effect is obtained when the compounds are administered or delivered sequentially, for example, in separate tablets, pills, or capsules, or by different injections in separate syringes. Typically, during alternating therapy, the effective dose of each active ingredient is administered sequentially, i.e., continuously, while in combination therapy, the effective doses of two or more active ingredients are administered together. Synergistic antiviral effects refer to antiviral effects that are greater than the predicted pure additive effect of the individual compounds in the combination.

[0668] In yet another embodiment, this application provides a method for inhibiting isopiridae polymerase in cells, comprising: contacting isopiridae-infected cells with an effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, solvate and / or ester thereof, thereby inhibiting the isopiridae polymerase.

[0669] In yet another embodiment, this application provides a method for inhibiting isopiridae polymerase in cells, comprising: contacting isopiridae-infected cells 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 the isopiridae polymerase.

[0670] In yet another embodiment, this application provides a method for inhibiting isopiraviridae polymerase in cells, comprising: contacting cells infected with isopiraviridae viruses 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 alternative active therapeutic agent.

[0671] In yet another embodiment, this application provides a method for treating human infection with a human arenaviridae virus, comprising: administering to a patient a therapeutically effective amount of a compound of formula I-IV or a pharmaceutically acceptable salt, solvate, and / or ester thereof.

[0672] In yet another embodiment, this application provides a method for treating human infection with a isonaviridae virus, comprising: administering to a 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 isonaviridae polymerase is inhibited.

[0673] In yet another embodiment, this application provides a method for treating human infection with a human arenaviridae virus, comprising: administering to a 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.

[0674] Kits are also provided that comprise a compound of Formula I or a pharmaceutically acceptable salt, pharmaceutically acceptable ester, stereoisomer, mixture of stereoisomers, or tautomer thereof. In individual embodiments, individual kits are provided that comprise compounds selected from each group of formulas herein, as well as each subgroup and embodiment thereof, including Formulas II, II, 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, pharmaceutically acceptable esters, stereoisomers, mixtures of stereoisomers, or tautomers thereof. In one aspect, the kit comprises a compound of Formula I or a pharmaceutically acceptable salt thereof. Each individual kit described herein may include a label and / or instructions for use to treat a disease or condition in an individual (e.g., a human) in need of the compound. In some embodiments, the disease or condition is a human isonaviridae virus infection, including lassa virus infection or juning virus infection. In other embodiments, each individual kit may also include instructions for use of an additional drug in combination with a compound of Formula I for the treatment of a disease or condition in an individual (e.g., a human) in need. In some of these embodiments, the disease or condition is a human isonaviridae virus infection, including lassa virus infection or juning virus infection. In each kit described herein, there is another embodiment in which the kit contains a single dose of the compound described herein or a pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, tautomer, polymorph, pseudopolymorph, amorphous form, hydrate, or solvate thereof. Examples of individual dose units may include pills, tablets, capsules, pre-filled syringes or syringe cartridges, intravenous infusion bags, etc., each containing a therapeutically effective amount of the compound or a pharmaceutically acceptable salt, racemic, enantiomer, diastereomer, tautomer, polymorph, pseudopolymorph, amorphous form, hydrate, or solvate thereof. In some embodiments, the kit may contain a single dose unit, and in other embodiments, multiple dose units may be present, such as the number of dose units required for a particular regimen or period.

[0675] Articles comprising compounds of formula I or thereof, pharmaceutically acceptable salts, pharmaceutically acceptable esters, stereoisomers, mixtures of stereoisomers or tautomers thereof; and containers. In one aspect, the article comprises compounds of formulas I, II, III, 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 containers. In individual embodiments, the container for the article may be a vial, can, ampoule, pre-loaded syringe, blister pack, tin, can, bottle, box, or intravenous bag.

[0676] Also provided as separate embodiments is the use of compounds selected from the various formulas 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 embodiments herein, including compounds 1-32 or their pharmaceutically acceptable salts, solvates and / or esters.

[0677] VI. Methods for inhibiting arenaviridae polymerase

[0678] Another aspect of the invention relates to a method for inhibiting the activity of Arenaviridae polymerases, comprising the step of treating a sample suspected of containing Arenaviridae with a compound or composition of the invention.

[0679] The arenaviridae family, which can be treated using the method of this invention, are single-stranded negative-sense RNA viruses that typically infect primates. In fact, arenaviruses can replicate in all cell types.

[0680] Based on studies of non-human primates infected with Lassa virus, the first cells infected appear to be dendritic cells in lymphoid tissue. The infection progresses to infect Kupffer cells in the liver, parenchymal cells in the liver and adrenal glands, endothelial cells in various tissues including nerve tissue, and finally the epithelium. Evidence has been documented in humans of liver infection leading to hepatitis (Hensley, L., 2011, Virology Journal; Yun, NE, 2012 Viruses).

[0681] Currently, 30 species of arenaviruses have been identified: Allpahuayo virus (ALLV), Amapari virus (AMAV), Bear Canyon virus (BCNV), Catalina virus, Chapare virus, Cupiqui virus (CPXV), Dantonon virus, Flexal virus (FLEV), Guanarido virus (GTOV), Ippy virus (IPPYV), Junin virus (JUNV), Kodoko virus, and Lassa virus (LASV; six strains - Josiah, NL, z148, Macenta, ... The virus includes various viral types, such as AV and CSF, Latino virus (LATV), lymphocytic choriomeningitis virus (LCMV), Lujo virus, Machupo virus (MACV), Mobala virus (MOBV), Morogoro virus, Mopeia virus (MOPV), Oliverio virus (OLVV), Parana virus (PARV), Pichind virus (PICV), Pinhal virus, Pirital virus (PIRV), Sabia virus (SABV), Skinner Tank virus, Tacarib virus (TCRV), Tamiami virus (TAMV), and Whitewater Arroyo virus (WWAV). Sand virions are heterogeneous, ranging in diameter from 40 nm to over 200 nm, and consist of a nucleocapsid surrounded by a lipid envelope. Due to the incorporation of host cell ribosomes into the viral particle during assembly, electron micrographs of the virion's interior show a characteristic granular appearance. The genome of arenavirus consists of two single-stranded RNA segments, small (S) and large (L). Both segments are ambiguously organized and encode two genes in opposite directions. The L RNA (~7kb) encodes the viral RNA-dependent RNA polymerase (L) and the small ring finger zinc-binding protein (Z). The S RNA (-3.4kb) encodes the glycoprotein precursor protein (GPC) and the nucleoprotein (NP). The GPC is cleaved post-translationally to produce two envelope glycoproteins, GP1 and GP2, as well as a stable signal peptide (SSP) (Yun, NE, 2012 Viruses).

[0682] The compositions of the present invention can be used as inhibitors of arenavir polymerase, as intermediates for such inhibitors, or for other uses as described below. The inhibitor binds to sites on the surface or within cavities of the arenaviridae polymerase, which has a unique geometry. Compositions binding arenaviridae polymerase can bind reversibly to varying degrees. Those compounds that bind substantially irreversibly are ideal candidates for use in this method of the present invention. Once labeled, substantially irreversibly bound compositions can be used as probes to detect arenaviridae polymerase. Therefore, the present invention relates to a method for detecting arenaviridae polymerase in samples suspected of containing arenaviridae polymerase, comprising the steps of: treating a sample suspected of containing arenaviridae polymerase with a composition containing a labeling compound of the present invention; and observing the effect of the sample on the activity of the label. Suitable labels are well known in the diagnostic field and include stable free radicals, fluorophores, radioisotopes, enzymes, chemiluminescent groups, and chromophores. The compounds described herein are labeled in a conventional manner using functional groups such as hydroxyl, carboxyl, thiol, or amino groups.

[0683] Within the scope of this 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 synthesizing the desired glycoprotein; and so on. Typically, the sample will be from an organism suspected of producing arenaviridae polymerases, usually a pathogenic organism such as arenaviridae viruses. 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.

[0684] The processing steps of this invention include adding the composition of this invention to a sample, or include adding a precursor of the composition to a sample. The addition step includes any administration method as described above.

[0685] If desired, the activity of arenaviridae polymerases 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 can all be considered. One of the screening methods described above is typically used; however, any other method, such as observing the physiological characteristics of living organisms, is also applicable.

[0686] Organisms containing arenaviridae polymerases include arenaviridae viruses. The compounds of this invention can be used to treat or prevent arenaviridae infections in animals or humans.

[0687] However, when screening for compounds that can inhibit human arenaviridae viruses, it should be remembered that enzyme assay results may not be correlated with cell culture assays. Therefore, cell-based assays should be the primary screening tool.

[0688] In another embodiment, this application provides a method for treating human isopiroid virus infection, comprising: administering to a 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 isopiroid infection is caused by an isopiroid virus. In some embodiments, the isopiroid infection is caused by a Junin virus. In some embodiments, the isopiroid infection is caused by Lassa virus strains Josiah, NL, z148, Macenta, AV, or CSF. In some embodiments, an isopiroid polymerase is inhibited.

[0689] The compounds of this invention can be used to treat individuals already infected with arenaviridae, or can be administered prophylactically to reduce or prevent the occurrence of arenaviridae infection. Physical examination of patients infected with arenaviridae after fever often reveals purulent pharyngitis, bilateral conjunctival hemorrhage, facial edema, and general abdominal tenderness. Macroscopic pathological changes may include pleural effusion, pulmonary edema, ascites, and hemorrhagic manifestations in the gastrointestinal mucosa. The mortality rate for hospitalized patients varies between 5 and 10%.

[0690] VII. Screening for Arenaviridae polymerase inhibitors

[0691] The compositions of the present invention are screened for inhibitory activity against arenaviridae polymerases using any conventional techniques for assessing enzyme activity. Within the scope of the present invention, compositions that inhibit arenaviridae polymerases in vitro are typically screened first, followed by screening for in vivo activity of compositions exhibiting inhibitory activity. The inhibitory activity is less than about 5 × 10⁻⁶. -6 M is preferably less than about 1×10 -7 Compositions of M with in vitro Ki (inhibition constant) are preferably used in vivo.

[0692] Useful in vitro screening methods have already been described in detail and will not be repeated here. However, these examples illustrate suitable in vitro assays.

[0693] VIII. Preparation of Compounds

[0694] 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 partially protected nucleoside can then be reacted with a suitable prodrug moiety to modify the nucleoside, and then the protecting group can be removed to obtain the compounds of the present invention.

[0695] A. Preparation of nucleosides via iodine-base method

[0696] In some embodiments, the present invention provides a method for preparing compound V:

[0697]

[0698] The method for preparing compound V includes forming a compound of formula VI under conditions suitable for preparing compound V: (The method involves a coupling agent, a halosilane, and the compound itself.)

[0699]

[0700] The reaction mixture of compound VII:

[0701]

[0702] Each PG is independently a hydroxyl protecting group, or two PG groups on adjacent carbons can combine to form -C(R) 19 ) 2- Group, R 10 It is H or silyl, and R 19 It is H, C1-C8 alkyl, phenyl, or substituted phenyl.

[0703] Any suitable coupling agent can be used in the method for preparing compound 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 magnesium-based coupling agent, such as, but not limited to, MgCl2, iPrMgCl, tBuMgCl, PhMgCl, or combinations 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 combinations thereof. In some embodiments, the coupling agent can be n-BuLi. In some embodiments, the coupling agent can be both PhMgCl and iPrMgCl.

[0704] 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 the compound of formula V, such as 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 the compound of formula V, such as 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 the 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 the compound of formula V.

[0705] Any suitable halosilane can be used in the method for preparing compound 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 alkyl, alkenyl, alkynyl, cycloalkyl, or phenyl. 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.

[0706] R 10 The silyl group can be any suitable group, but it can depend on the choice of the halosilane. For example, when the halosilane is TMS-C1, the silyl group can be trimethylsilyl.

[0707] The silane haloalkane can be present in any suitable amount. For example, the silane haloalkane can be present in an amount of at least 1.0 equivalents (mol / mol) of the compound of formula V, such as about 1.0, 2, 3, 4, 5, 6, 7, 8, 9, or about 10.0 equivalents (mol / mol). The silane haloalkane can also be present in an amount of about 1.0 to about 10.0 equivalents (mol / mol) of the compound of formula V, such as 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 silane haloalkane can be present in an amount of about 1.0 to about 5.0 equivalents (mol / mol) of the compound of formula V. In some embodiments, the silane haloalkane can be present in an amount of about 1.0 to about 2.0 equivalents (mol / mol) of the compound of formula V.

[0708] The hydroxyl protecting group can be any protecting group suitable for a hydroxyl functional group. Representative hydroxyl 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, neopentanoyl, or benzoyl. In some embodiments, the hydroxyl protecting group can be trimethylsilane (TMS), tert-butyldimethylsilane (TBDMS), tert-butyldiphenylsilane (TBDPS), methyl-methoxy (MOM), tetrahydropyran (THP), tert-butyl, allyl, benzyl, acetyl, neopentanoyl, or benzoyl. In some embodiments, the hydroxyl protecting group can be benzyl.

[0709] A hydroxyl group on an adjacent carbon, referred to as 1,2-hydroxyl, can react with a diether ketone to form a cyclic protecting group called an acetone compound. Exemplary acetone compounds include, but are not limited to, acetone compounds and benzyl acetal. In some embodiments, the hydroxyl protecting groups on adjacent carbons can combine to form an acetone compound.

[0710] When R 19 When the group is a C1-C8 alkyl group, 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 can be methyl.

[0711] Any suitable solvent may be used in the method of the present invention. Representative solvents include, but are not limited to, pentane, pentane derivatives, hexane, hexane derivatives, heptane, heptane, petroleum ether, cyclopentane, cyclohexane, benzene, toluene, xylene, trifluoromethylbenzene, halogenated benzenes such as chlorobenzene, fluorobenzene, dichlorobenzene and difluorobenzene, dichloromethane, chloroform, acetone, ethyl acetate, diethyl ether, tetrahydrofuran, or combinations thereof. In some embodiments, the solvent may be tetrahydrofuran. Other representative solvents include, but are not limited to, 2-methyltetrahydrofuran, dibutyl ether, methyl tert-butyl ether, dimethoxyethane, dioxane (1,4-dioxane), N-methylpyrrolidone (NMP), or combinations thereof.

[0712] The reaction mixture of this 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.

[0713] The reaction mixture of this method can be subjected to 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 an atmospheric atmosphere or an inert gas such as nitrogen or argon.

[0714] The method of the present invention can provide compound V in any suitable yield. For example, compound V can be prepared in yields of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least about 95%.

[0715] The method of the present invention can provide compounds of formula V with any suitable purity. For example, compounds of formula V can be prepared with 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 with a purity of at least 95%. In some embodiments, compounds of formula V can be prepared with a purity of at least 98%. In some embodiments, compounds of formula V can be prepared with a purity of at least 99%.

[0716] In some embodiments, the method includes preparing compound V:

[0717]

[0718] This method includes forming compounds of formula VI under conditions suitable for preparing compounds of formula V: TMS-Cl, PhMgCl, iPrMgCl

[0719]

[0720] The reaction mixture of compound VII:

[0721]

[0722] In some embodiments, the present invention provides compounds

[0723]

[0724] B. Addition of the prodrug portion

[0725] The present invention also provides a method for coupling a prodrug moiety to a nucleoside to provide the compounds of the present invention. In some embodiments, the present invention provides a method for preparing compounds of formula VIII:

[0726]

[0727] The method described herein includes forming a compound of formula IX, comprising a coupling agent, a non-nucleophilic base, under conditions suitable for forming a compound of formula VIII:

[0728]

[0729] The reaction mixture of compound X:

[0730]

[0731] Each R a It can be 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 is independently H, C1-C6 alkyl or benzyl, R f H, C1-C8 alkyl, benzyl, C3-C6 cycloalkyl, or -CH2-C3-C6 cycloalkyl, R 19 It is H, C1-C8 alkyl, phenyl or substituted phenyl, and LG is a leaving group.

[0732] As described above regarding the method for preparing compound V, any suitable coupling agent can be used in the method for preparing compound VIII. In some embodiments, the coupling agent may be a magnesium coupling agent. In some embodiments, the coupling agent may be MgCl2, iPrMgCl, tBuMgCl, PhMgCl, or a combination thereof. In some embodiments, the coupling agent may be MgCl2.

[0733] Any suitable nonnucleophilic base can be used in the method for preparing compound VIII. Representative nonnucleophilic bases include, but are not limited to, triethylamine, diisopropylethylamine, N,N-diethylaniline, pyridine, 2,6-dimethylpyridine, 2,4,6-trimethylpyridine, 4-dimethylaminopyridine, and quinine ring. In some embodiments, the nonnucleophilic base may be diisopropylethylamine (DIPEA).

[0734] As described above regarding the method for preparing compound V, the protecting group PG can be any suitable hydroxyl protecting group. An exemplary protecting group PG can be benzyl, or the PG group can combine to form an acetone compound. Exemplary acetone compounds include, but are not limited to, acetone compounds and benzyl acetal. In some embodiments, hydroxyl protecting groups on adjacent carbons can combine to form an acetone compound. In some embodiments, the PG group combines to form -C(R 19 ) 2- In some implementations, each R a It is the PG group that combines to form the protecting group PG of -C(Me)2.

[0735] When R e When the group is C1-C8 alkyl, each R eIt can be methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, neohexyl, heptyl, or octyl. In some embodiments, each R e The group can be methyl.

[0736] When R f When the group is a C1-C8 alkyl group, R f 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 f The group can be methyl, ethyl, isopropyl, tert-butyl, or isohexyl. When R... f When the group is a C3-C6 cycloalkyl group, R f It can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In some embodiments, R f It can be cyclobutyl, cyclopentyl, or cyclohexyl.

[0737] When R 19 When the group is a C1-C8 alkyl group, R 19 It 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 can be methyl.

[0738] The leaving group can be any suitable leaving group. Suitable leaving groups LG include, but are not limited to, chlorides, bromides, methanesulfonates, toluenesulfonates, trifluoromethanesulfonates, 4-nitrobenzenesulfonates, 4-chlorobenzenesulfonates, 4-nitrobenzenoxy, pentafluorophenoxy, etc. In some embodiments, the leaving group LG can be 4-nitrobenzenoxy or pentafluorophenoxy. In some embodiments, the leaving group LG can be 4-nitrobenzenoxy.

[0739] In some implementations, each R a It is the combination of PG groups that forms C(R) 19 )2-of-PG, R f It is a C1-C8 alkyl group, R 19 It is a C1-C8 alkyl group, and the leaving group LG is 4-nitrophenoxy or pentafluorophenoxy.

[0740] In some implementations, the coupling agent is MgCl2, and the non-nucleophilic base is diisopropylethylamine.

[0741] In some implementations, the compound of formula VIII can be

[0742]

[0743] In some implementations, the compound of formula VIII can be

[0744]

[0745] In some implementations, the compound of formula VIII can be

[0746]

[0747] In some embodiments, the method for preparing compound VIII includes forming a reaction mixture comprising MgCl2, DIPEA, compound IX, and compound X under conditions suitable for forming compound VIII:

[0748]

[0749] When the R of compound VIII a When the group is a hydroxyl protecting group PG, the method may include removing the protecting group to form each R a This is an additional step in preparing compounds of formula VIII. In some embodiments, the method for preparing compounds of formula VIII includes a process suitable for forming each R... a The formation of a compound of formula VIII containing a deprotecting agent and a compound of formula VIII (where each R) is a compound of formula VIII under the condition of H. a The second reaction mixture is a protecting group (PG). The deprotecting agent can be any suitable reagent that removes the protecting group PG, such as hydrogen and a hydrogenation catalyst or acid. For example, if the protecting group PG is benzyl, the deprotecting agent can be hydrogen and platinum on carbon. Alternatively, when the protecting group PG is an acetone compound, 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, etc. In some embodiments, the method for preparing compound VIII includes a reaction mixture suitable for forming each R... a The formation of a compound of formula VIII containing an acid and a compound of formula VIII (where R is H) is achieved under the conditions of H. a Groups combine to form C(R) 19 ) 2- The second reaction mixture. In some embodiments, the acid may be hydrochloric acid.

[0750] Any suitable solvent can be used in the method of this invention. Representative solvents include, but are not limited to, pentane, pentane derivatives, hexane, hexane derivatives, heptane, heptane derivatives, petroleum ether, cyclopentane, cyclohexane, benzene, toluene, xylene, trifluorotoluene, halogenated benzenes such as chlorobenzene, fluorobenzene, dichlorobenzene and difluorobenzene, dichloromethane, chloroform, acetone, ethyl acetate, diethyl ether, tetrahydrofuran, acetonitrile, or combinations thereof. In some embodiments, the solvent may be acetonitrile.

[0751] The reaction mixture of this 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.

[0752] The reaction mixture of this method can be subjected to 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 an atmospheric atmosphere or an inert gas such as nitrogen or argon.

[0753] The method of the present invention can provide compounds of formula VIII in any suitable yield. For example, compounds of formula VIII can be prepared in yields of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least about 95%.

[0754] The method of the present invention can provide compounds of formula VIII of any suitable purity. For example, compounds of formula VIII can be prepared with 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 with a purity of at least 95%. In some embodiments, compounds of formula VIII can be prepared with a purity of at least 98%. In some embodiments, compounds of formula VIII can be prepared with a purity of at least 99%.

[0755] In some embodiments, the present invention provides compounds

[0756]

[0757] IX. Implementation Examples

[0758] Certain abbreviations and acronyms are used to describe experimental details. While most of these will be understood by those skilled in the art, Table 1 contains a list of many of these abbreviations and acronyms.

[0759] Table 1. List of Abbreviations and Acronyms

[0760] Abbreviations and meanings

[0761] Ac2O Acetic anhydride

[0762] AIBN 2,2'-azobis(2-methylpropionitrile)

[0763] Bn benzyl

[0764] BnBr benzyl bromide

[0765] BSA bis(trimethylsilyl)acetamide

[0766] BzCl benzoyl chloride

[0767] CDI carbonyl diimidazole

[0768] DABCO 1,4-diazabicyclo[2.2.2]octane

[0769] DBN 1,5-diazabicyclo[4.3.0]non-5-ene

[0770] DDQ 2,3-Dichloro-5,6-dicyano-1,4-p-benzoquinone

[0771] DBU 1,5-diazabicyclo[5.4.0]undec-5-ene

[0772] DCA dichloroacetamide

[0773] DCC dicyclohexylcarbodiimide

[0774] DCM dichloromethane

[0775] DMAP 4-Dimethylaminopyridine

[0776] DME 1,2-dimethoxyethane

[0777] DMTC1 dimethoxytriphenylmethyl chloride

[0778] DMSO dimethyl sulfoxide

[0779] DMTR4,4'-Dimethoxytriphenylmethyl

[0780] DMF dimethylformamide

[0781] Ethyl acetate (EtOAc)

[0782] ESI Electrospray Ionization

[0783] HMDS (Hexamethyldisilazane)

[0784] HPLC (High-Performance Liquid Chromatography)

[0785] LDA diisopropylaminolithium

[0786] LRMS low-resolution mass spectrometry

[0787] MCPBA (m-chloroperbenzoic acid)

[0788] MeCN acetonitrile

[0789] MeOH methanol

[0790] MMTC monomethoxytriphenylmethyl chloride

[0791] m / z or m / e mass-to-charge ratio

[0792] MH + Quality +1

[0793] MH - Quality minus 1

[0794] MsOH methanesulfonic acid

[0795] MS or ms mass spectrometry

[0796] NBS N-bromosuccinimide

[0797] Phphenyl

[0798] rt or rt room temperature

[0799] TBAF tetrabutylammonium fluoride

[0800] TMSCl Trimethylchlorosilane

[0801] TMSBr trimethylbromosilane

[0802] TMSI Trimethyliodosilane

[0803] TMSOTf (trimethylsilyl) trifluoromethanesulfonate

[0804] TEA Triethylamine

[0805] TBA Tributylamine

[0806] TBAP Tributylammonium Pyrophosphate

[0807] TBSCl tert-butyldimethylsilyl chloride

[0808] TEAB triethylammonium bicarbonate

[0809] TFA (trifluoroacetic acid)

[0810] TLC or TLC thin-layer chromatography

[0811] Tr triphenylmethyl

[0812] Tol 4-methylbenzoyl

[0813] Turbo Grignard 1:1 mixture of isopropyl magnesium chloride and lithium chloride

[0814] δ from tetramethylsilane to parts per million of the lower field

[0815] A. Preparation of compounds

[0816] Example 1. Ethyl (2S)-2-(chloro(phenoxy)phosphorylamino)propionate (chloride A)

[0817]

[0818] 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) with stirring. Phenyl dichlorophosphate (1.49 mL, 10 mmol) was added, followed by dropwise addition of Et₃N over 10 min. The reaction mixture was then slowly warmed to room temperature and stirred for 12 h. Anhydrous Et₂O (50 mL) was added, and the mixture was stirred for 30 min. The solids formed were removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was subjected to silica gel chromatography, eluting with a 0–50% EtOAc solution in hexane to give 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.

[0819] Example 2. (2S)-2-(chloro(phenoxy)phosphorylamino)propionic acid-2-ethylbutyl ester (chloride B)

[0820]

[0821] Alanine chlorophosphoamino-2-ethylbutyl ester B was prepared using the same procedure as chloride A, except that ethyl alanine was substituted with 2-ethylbutyl alanine. This crude product was used for the next reaction. Treatment with methanol or ethanol formed the substitution product with the desired LCMS signal.

[0822] Example 3. Isopropyl (2S)-2-(chloro(phenoxy)phosphorylamino)propionate (chloride C)

[0823]

[0824] Alanine chlorophosphoaminoisopropyl ester C was prepared using the same procedure as chloride A, except that isopropyl alanine was used instead of ethyl alanine. This substance was used as a crude product for the next reaction. Treatment with methanol or ethanol formed the substitution product with the desired LCMS signal.

[0825] Example 4. (2R,3R,4S,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-3,4-dihydroxy-5-(hydroxymethyl))tetrahydrofuran-2-carboxynitrile (Compound 1)

[0826]

[0827] The preparation of (2R,3R,4S,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carboxynitrile is described below.

[0828]

[0829] Commercially available lactose (10 g, 23.8 mmol) was dissolved in anhydrous DMSO (30 mL) under N2(g). 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 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, eluting with a 25% hexane solution of EtOAc to give a lactone (9.55 g, 96%). 1 ¹H NMR (400MHz, 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] - Tr = 2.82 min. HPLC Tr = 4.59 [2-98% ACN in H₂] within 5 min at a flow rate of 2 ml / min.

[0830]

[0831] Bromopyrazole (prepared according to WO2009 / 132135) (0.5 g, 2.4 mmol) was suspended in anhydrous THF (10 mL) under N2 (g). The suspension was stirred and TMSCl (0.67 mL, 5.28 mmol) was added. The mixture was stirred at room temperature for 20 min, then cooled to -78 °C, and 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 min, and then the lactone (1 g, 2.4 mmol) was added via 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 with anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel chromatography and eluted with a hexane solution of 0-50% EtOAc to give the product as a 1:1 mixture of end-group isomers (345 mg, 26% yield). LCMS m / z 553 [M+H].

[0832]

[0833] Hydroxynucleotide (1.1 g, 2.0 mmol) was dissolved in anhydrous CH2Cl2 (40 mL) and the solution was cooled to 0 °C with stirring under N2 (g). TMSCN (0.931 mL, 7 mmol) was added and the mixture was stirred for another 10 min. TMSOTf (1.63 mL, 9.0 mmol) was slowly added to the reaction and the mixture was stirred for 1 h. The reaction mixture was then diluted with CH2Cl2 (120 mL) and quenched with an aqueous solution of NaHCO3 (120 mL). The reaction mixture was stirred for another 10 min, and the organic layer was separated. The aqueous layer was extracted with dichloromethane (150 mL), and the combined organic extracts were dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was dissolved in a minimal amount of CH2Cl2 and subjected to silica gel chromatography with a gradient elution of 0–75% EtOAc and hexane to give tribenzylcyanonucleotide, a mixture of terminal isomers (0.9 g, 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].

[0834]

[0835] Tribenzylcyanonucleotide (70 mg, 0.124 mmol) was dissolved in anhydrous CH₂Cl₂ (2 mL) and cooled to -78 °C under N₂ (g). A solution of BCl₃ (1 N 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 complete by LC / MS, MeOH was added to quench the reaction. The reaction mixture was warmed to room temperature, and the solvent was removed under reduced pressure. The residue was subjected to C18 reversed-phase HPLC, eluting with H₂O (0.1% TFA) for 5 min, followed by gradient elution with a 0-70% MeCN H₂O solution (0.1% TFA) over 35 min for the α-terminal isomer (20 mg, 37%) and β-terminal isomer 1 (20 mg, 37%). (α-terminal isomer) 1 ¹H NMR (300MHz, 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-terminal isomer) 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.

[0836] Example 5. (2R,3R,4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carboxynitrile (Compound 2)

[0837]

[0838] The preparation of (2R,3R,4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carboxynitrile is described below.

[0839]

[0840] 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), eluting with a 0-100% EtOAc solution 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). 1HNMR (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].

[0841]

[0842] (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 used for treatment. The resulting mixture was stirred for 24 hours and then filtered through a diatomaceous earth pad. The eluent was concentrated under reduced pressure, and the residue was subjected to silica gel chromatography (120 g SiO2 HP Gold Combiflash Column) and 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 H NMR(300MHz, CDCl3) δ7.37(m,10H),5.45(dd,J=49,5.7,Hz,1H),4.85(d,J=11 .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) R = 5.29 min. Phenomenex Synergi 4M Hydro-RP 80A, 50 × 4.60 mm, 4 microns; flow rate 2 mL / min.

[0843]

[0844] (3R,4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-ol. A solution of 7-bromopyrrolo[1,2-f][1,2,4]-triazine-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 hexane, 1.09 mL, 1.09 mmol). The solution was stirred for 30 minutes, then treated dropwise with a solution of (3R,4R,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorodihydrofuran-2(3H)-one (106 mg, 0.319 mmol) in 1.4 mL of THF. The resulting mixture was stirred for 30 minutes, then AcOH (83 μL, 1.44 mmol) in 1.0 mL of THF was added to quench the reaction. The mixture was warmed to room temperature and then concentrated under reduced pressure. The residue was diluted with 100 mL of EtOAc and washed with 50 mL of saturated NaCl solution. The organic layer was dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel chromatography (40 g SiO2 HP Gold Combiflash Column), eluted with a 0-100% hexane solution of EtOAc, followed by elution with a 0-100% gradient of EtOAc solutions (20% MeOH in EtOAc solution), to give (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 (282.2MHz, CDCl3) δ-211 (m). LCMS m / z 465 [M+H]. HPLC (6-98% MeCN-H2O gradient, 0.05% TFA modifier) R = 4.37 minutes (α-isomer), 4.54 minutes (β-isomer).

[0845]

[0846] (3R,4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-carboxynitrile: 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 20 drops of saturated NaHCO3 solution, 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), eluted with 0-100% EtOAc in hexane, to give (3R,4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-carboxynitrile, as a white solid (110 mg, 55%, 60 / 40 α / β isomers). Data for the two isomers: Rf = 0.53 (EtOAc). f =0.53(EtOAc). 1 HNMR (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 (282.2MHz, CDCl3) δ -196(m), -203(m). LCMS m / z 474 [M+H]. HPLC (6-98% MeCN-H2O gradient, 0.05% TFA modifier) R =4.98min.

[0847]

[0848] (2R,3R,4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carboxylonitrile (2) Dissolve (3R,4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-(benzyloxy)-5-(benzyloxymethyl)-3-fluorotetrahydrofuran-2-carboxylonitrile (110 mg, 0.23 mmol) in CH2Cl2 (1.5 mL) and cool to 0 °C. Treat the reaction mixture with BCl3 (1.0 M CH2Cl2 solution, 766 μL, 0.77 mmol) and stir for 2 hours. The mixture was then cooled to -78°C and treated with Et3N (340 μL, 2.44 mmol), followed by treatment with MeOH (2 mL), and then heated to RT. The reaction was concentrated under reduced pressure and then co-evaporated with MeOH (3 × 5 mL). The residue was then suspended in H2O (5 mL) and treated with NaHCO3 (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) through a porous glass funnel (coarse), and the eluent was concentrated under reduced pressure. The residue was subjected to reversed-phase HPLC (a gradient of 6-98% MeCN in H2O solution containing 0.05% TFA modifier) ​​to give (2R,3R,4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carboxynitrile 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 solution). 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 (282.2MHz, CDCl3) δ-197 (m). LCMS m / z 294 [M+H]. HPLC (2–98% MeCN–H2O gradient, 0.05% TFA modifier) R = 1.49 min.

[0849] Example 6. (2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-4-fluoro-2- (Hydroxymethyl)-5-methyltetrahydrofuran-3-ol (Compound 3)

[0850]

[0851] The preparation of (2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-4-fluoro-2-(hydroxymethyl)-5-methyltetrahydrofuran-3-ol is described below.

[0852]

[0853] 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) (3 N THF solution) was added, and the resulting mixture was stirred overnight. Acetic acid (7 mmol) was added to quench the reaction, and the solvent was removed by rotary evacuation under reduced pressure. The residue was redissolved in CH2Cl2, and the solution was subjected to a silica gel stopper to separate the product (0.355 g) as the crude mixture. LC / MS (m / z: 480, M +1 The crude substance 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 adding Et3N (3.5 mmol). The mixture was concentrated under reduced pressure and the residue was subjected to silica gel chromatography to give a methyl-substituted nucleoside (0.174 g, 0.377 mmol, 44% yield) as a 4:1 mixture of β- and α-terminal isomers. 1 ¹H NMR (300MHz, CD3CN) major end-group isomers: 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 end-group isomer δ-207 (m, 1F). LCMS m / z 463 [M+H].

[0854]

[0855] The benzylated nucleoside (0.134 g, 0.290 mmol), Degussa catalyst (0.268 g), and AcOH (30 mL) were mixed together. H2 (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. The residue was dissolved in a minimal amount of H2O and subjected to reversed-phase HPLC (C18 hydrORP column) to separate the β-terminal isomer 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).

[0856] The characteristics of a small number of α-terminal isomers are 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).

[0857] Example 7. (2R)-2-((((2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7- Isopropyl 4-fluoro-3-hydroxy-5-methyltetrahydrofuran-2-yl)methoxy)-(phenoxy)phosphorylamino)propionate (chemical) Compound 4)

[0858]

[0859] Nucleoside 3 (0.011 g, 0.04 mmol) was dissolved in trimethyl phosphate (2 mL) and cooled to 0 °C. The mixture was stirred under a nitrogen atmosphere (g) and 1-methylimidazole (0.320 mL, 5 mmol) was added, followed by alanine monoisopropyl phenol phosphoryl chloride C (0.240 mL, 4.4 mmol). The reaction mixture was stirred at 0 °C for 2 hours and then slowly warmed to room temperature. Monitoring was performed by LC / MS. When LC / MS was complete, the reaction mixture was treated with H₂O (5 mL) and then concentrated under reduced pressure. The residue was dissolved in CH₂Cl₂ and subjected to silica gel chromatography, eluting with 0-100% EtOAc in hexane. The product fraction was collected and concentrated. The residue was subjected to preparative HPLC to give alanine isopropyl monoamide prodrug 4, a mixture of isomers (4.7 mg, 0.003 mmol, 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].

[0860] Example 8. (2R)-2-((((2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7- ethyl propionate (compound)-4-fluoro-3-hydroxy-5-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propionate 5)

[0861]

[0862] 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% hexane solution of EtOAc. The product fraction was collected and concentrated. The crude product was eluted with a 0-100% hexane solution of EtOAc. The crude product was collected and concentrated under reduced pressure. The residue was subjected to preparative HPLC to give 5 (2.0 mg, 4% yield). LCMS m / z 538 [M+H].

[0863] Example 9. ((2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-4-fluoro- 3-Hydroxy-5-methyltetrahydrofuran-2-yl)methyltetrahydrotriphosphate (Compound 6)

[0864]

[0865] 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 to >80% monophosphate formation was determined by monitoring with 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 an aqueous solution of 1N 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 title product 6 (1.7 mg, 6% yield). LCMS m / z 521 [MH]. Tr = 0.41. HPLC ion exchange TR = 9.40 min.

[0866] Example 10. (2R,3R,5S)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-3-hydroxy-5- (hydroxymethyl)-tetrahydrofuran-2-carboxynitrile (compound 7)

[0867]

[0868] The preparation of (2R,3R,5S)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-3-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-carboxynitrile is described below.

[0869]

[0870] ((3αR,5S,6αR)-2,2-dimethyl-tetrahydrofurano[2,3-d][1,3]dioxolane-5-yl)methanol. Acetate feedstock (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). 1 N NaOH (aqueous solution) (10 mL) was added until pH 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 a 0-70% hexane solution of EtOAc 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).

[0871]

[0872] (3αR,5S,6αR)-5-(benzyloxymethyl)-2,2-dimethyl-tetrahydrofurano[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 in portions over 5 min to the sodium hydride mixture. The resulting mixture was stirred for 20 min. Then benzyl bromide (892 μL, 7.46 mmol) was added. The reaction mixture was stirred for 2 h 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 then re-extracted with EtOAc (30 mL). The combined organic extracts were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel chromatography and eluted with a hexane solution of 0-40% EtOAc 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).

[0873]

[0874] (3R,5S)-5-(benzyloxymethyl)-tetrahydrofuran-2,3-diol. 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 subjected to silica gel chromatography, eluting with a 0-70% EtOAc solution in hexane 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).

[0875]

[0876] (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 diatomaceous earth (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 subjected to silica gel chromatography, eluting with a 0–70% EtOAc solution 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).

[0877]

[0878] (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 treatment with 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. Additional benzyl bromide (50 μL) and silver oxide (150 mg) were 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, eluting with a 0–20% hexane solution of EtOAc 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).

[0879]

[0880] (3R,5S)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-3-(benzyloxy)-5-(benzyloxymethyl)-tetrahydrofuran-2-ol. 7-Bromopyrrolo[1,2-f][1,2,4]triazine-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 mixture 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). A 2.5 M n-butyllithium solution 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. Then, it was quenched with AcOH until the pH reached 5-6. The mixture was heated to RT 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 subjected to silica gel chromatography, 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].

[0881]

[0882] (3R,5S)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-3-(benzyloxy)-5-(benzyloxymethyl)-tetrahydrofuran-2-carboxynitrile. An alcohol (250 mg, 0.56 mmol) was dissolved in anhydrous CH₂Cl₂ (10 mL) and stirred at -15 °C with Ar(g). TMSCN (448 μL, 3.36 mmol) was added dropwise, and the mixture was stirred for 10 min. TMSOTf (466 μL, 2.58 mmol) was added dropwise over 10 min, and the resulting mixture was stirred at -15 °C for 90 min. Additional TMSCN (224 μL, 3 equivalents) and TMSOTf (202 μL, 2 equivalents) were added, and stirring continued for 5 h. A saturated aqueous solution of NaHCO₃ was added to quench the reaction, and the mixture was stirred for 10 min. The organic layer was separated and washed with saturated NaHCO3 aqueous solution and saturated NaCl solution, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel chromatography, eluted with 0-70% EtOAc in hexane solution, to give the title product (150 mg, 59%). LCMS m / z 456.3 [M+H], 454.1 [M–H].

[0883]

[0884] (2R,3R,5S)2-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-3-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-carboxynitrile (7). Benzyl ether (150 mg, 0.329 mmol) was dissolved in anhydrous CH2Cl2 (2 mL), and the mixture was stirred at -20 °C under Ar (g). A 1 M BCl3 solution (724 μL, 0.724 mmol) in CH2Cl2 was added dropwise, and the resulting mixture was stirred for 2 h. An additional 1 M BCl3 solution in CH2Cl2 (724 μL, 0.724 mmol) was added, and stirring continued for 2 h. The mixture was then cooled to -78 °C and slowly treated with a 2:1 mixture of Et3N and MeOH (3 mL). The mixture was stirred for 10 min, then treated with MeOH (10 mL). The reaction was warmed to room temperature and then concentrated under reduced pressure. The residue was dissolved in MeOH and concentrated under reduced pressure. The residue was then dissolved again in MeOH and treated with solid NaHCO3. 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].

[0885] Example 11. (2S)-2-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7- Isopropyl propionate (Cyclo-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)-phosphorylamino)propionate (Chemical) Object 8)

[0886]

[0887] Nucleoside 1 (45 mg, 0.15 mmol) was dissolved in anhydrous trimethyl phosphate (0.5 mL), and the solution was stirred at 0 °C under N2 (g). Methylimidazole (36 μL, 0.45 mmol) was added to the solution. Chlorophosphatidylcholine C (69 mg, 0.225 mmol) was dissolved in anhydrous THF (0.25 mL) and added dropwise to the nucleoside mixture. When the reaction was complete by LCMS, the reaction mixture was diluted with EtOAc and washed with saturated NaHCO3 aqueous solution and saturated NaCl, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel chromatography, eluted with 0–5% MeOH in CH2Cl2 solution, and then subjected to preparative HPLC to give the product (20.9 mg, 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 / z 561.0[M+H],559.0[MH].

[0888] 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)propionic acid Ester (Compound 9)

[0889] Compound 9 can be prepared by the following methods.

[0890] Program 1

[0891]

[0892] Compound 1 and chloride B were prepared using the same preparation method used for compound 8.

[0893] 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].

[0894] Program 2

[0895]

[0896] (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)amino)propionate-2-ethylbutyl ester. 2-Ethylbutyl (2S)-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propionate (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]triazine-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carboxynitrile (350 mg, 1.2 mmol) was added to the reaction mixture in one step. Then, a THF solution of tert-butylmagnesium chloride (1 M, 1.8 mL, 1.8 mmol) was added dropwise to the reaction mixture over 10 minutes. The reaction mixture was stirred for 2 hours, during which time the reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated sodium bicarbonate aqueous solution (3 × 15 mL), followed by washing with saturated sodium chloride aqueous solution (15 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The oily substance was purified by silica gel column chromatography (0-10% MeOH in DCM solution) to give (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)propionate-2-ethylbutyl ester (311 mg, 43%, a 1:0.4 diastereomer mixture 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 (162MHz, CD3OD) δ 3.71, 3.65. LCMS m / z 603.1 [M+H], 600.9 [MH]. HPLC (2–98% MeCN–H2O gradient over 8.5 min, containing 0.1% TFA modifier, 1.5 mL / min, column: Phenomenex Kinetex C18, 2.6 μm). 4.6 x 100 mm)t R = 5.544 min, 5.601 min.

[0897] Separation of (S) and (R) diastereomers

[0898] (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)propionic acid-2-ethylbutyl ester 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 for the first eluted diastereomer was 17.4 min, and the retention time for the second eluted diastereomer was 25.0 min.

[0899] The first eluted diastereomer is (S)-2-(((R)-(((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)propionate-2-ethylbutyl ester:

[0900]

[0901] 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 PNMR (162MHz, CD3OD) δ 3.71 (s). HPLC (2–98% MeCN–H2O gradient over 8.5 min, containing 0.1% TFA modifier, 1.5 mL / min, column: Phenomenex Kinetex C18, 2.6 μm). 4.6x100mm)t R =5.585min.

[0902] The second eluted diastereomer is (S)-2-(((S)-(((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)propionate-2-ethylbutyl ester:

[0903]

[0904] 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). 31PNMR (162MHz, CD3OD) δ 3.73 (s). HPLC (2–98% MeCN–H2O gradient over 8.5 min, containing 0.1% TFA modifier, 1.5 mL / min, column: Phenomenex Kinetex C18, 2.6 μm). 4.6 x 100 mm)t R =5.629min.

[0905] Example 13. (2S)-2-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7- ethyl propionate (compound)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propionate 10)

[0906]

[0907] The preparation of ethyl (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)propionate is described below.

[0908] Procedure 1. Preparation via chloride A

[0909]

[0910] Compound 1 and chloride A were prepared using the same preparation method used for compound 8.

[0911] 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 PNMR(121.4MHz,CD3OD)δ3.7.LCMS m / z 547.0[M+H],545.0[MH].

[0912] Procedure 2. Preparation of nitrobenzene compound L

[0913]

[0914] Compound 1 (50 mg, 0.17 mmol) was dissolved in NMP-THF (1:1 mL) and cooled in an ice bath. Then, tBuMgCl (0.257 mL, 0.257 mmol) was added over 5 minutes. The resulting mixture was heated to room temperature and stirred for 30 minutes. Then, a solution of compound L (prepared according to US20120009147, 74.6 mg, 0.189 mmol) in THF (2 mL) was added. After 30 minutes, the reaction mixture was purified by HPLC (10–80% acetonitrile aqueous solution) 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%, 2.5:1 diastereomer mixture). 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 PNMR(162MHz,CD3OD)δ3.73,3.68.MS m / z=547(M+1)+.

[0915] Example 14. (2S)-2-((((2R,3R,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7- ethyl propionate (compound)-5-cyano-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propionate 11)

[0916]

[0917] 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 / z 549.0[M+H],547.1[MH].

[0918] Example 15. (2S,2'S)-2,2'-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]) Triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)dipropionic acid Ethyl ester (compound 12)

[0919]

[0920] 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 min. 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 min. Then, Et3N (70 μL, 0.5 mmol) was added to obtain a solution with pH 9–10. The mixture was stirred for 2 h. It was then diluted with EtOAc, washed with saturated NaHCO3 aqueous solution, and then washed with 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 give 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].

[0921] Example 16. (2S,3R,4S,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-2-acetylene 5-(hydroxymethyl)tetrahydrofuran-3,4-diol (compound 13)

[0922]

[0923] The preparation of (2S,3R,4S,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-2-ethynyl-5-(hydroxymethyl)tetrahydrofuran-3,4-diol is described below.

[0924]

[0925] Nucleotidol (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, then treated with a 0.5 N solution of ethynyl magnesium bromide in THF (17.2 mL, 17.2 mmol). The reaction mixture was stirred overnight at room temperature. AcOH (1.5 mL) was added to quench the reaction. The mixture was concentrated under reduced pressure and the residue was redissolved in CH2Cl2. The solution was passed through a silica gel stopper and eluted with a 0–80% EtOAc solution in hexane to give the title product, a crude mixture. LCMS m / z 579 [M+H].

[0926]

[0927] Crude ethynyl 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 the reaction was 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 a 0-75% EtOAc solution in hexane to give ethynyl nucleosides as a mixture of terminal isomers (0.200 g, 33%, 2 steps). LCMS m / z 561 [M+H].

[0928]

[0929] Tribenzyl nucleotide (0.650 g, 1.16 mmol) was dissolved in anhydrous CH₂Cl₂ (30 mL) and cooled to -78 °C under N₂ (g). A solution of boron tribromide (1 N in CH₂Cl₂, 5.5 mL) 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 rise to room temperature. The mixture was concentrated under reduced pressure and subjected to preparative HPLC to provide the α-terminal isomer (20 mg) and the β-terminal isomer 13 (110 mg). (β-terminal isomer) 1¹H NMR (300MHz, 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-terminal isomer) 1 HNMR (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].

[0930] Example 17. (2R,3R,4R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-1,3,4-triazine (Benzyloxy)hexane-2,5-diol (Compound 14)

[0931]

[0932] The preparation of (2R,3R,4R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-1,3,4-tris(benzyloxy)hexane-2,5-diol is described below.

[0933]

[0934] Tribenzyl alcohol (0.250 g, 0.453 mmol) synthesized from 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 THF solution of 3.0 N methyl magnesium chloride (1.2 mL, 3.62 mmol) was added. The reaction mixture was stirred overnight at room temperature. The reaction was quenched with acetic acid (1.5 mL), and the mixture was concentrated under reduced pressure. The residue was redissolved in CH2Cl2 and passed through a silica gel stopper, eluted with a 0–80% EtOAc solution in hexane. The crude product (0.452 g) was then used for the next step without further purification. LCMS m / z 569 [M+H].

[0935]

[0936] Crude methyl nucleotide (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 subjected to silica gel chromatography, eluting with a 0–75% hexane solution of EtOAc to give the product as a mixture of terminal isomers (0.20 g, 46% in 2 steps). LCMS m / z 551 [M+H].

[0937]

[0938] Tribenzyl nucleotide (0.20 g, 0.364 mmol) was dissolved in AcOH (30 mL). Pd / C (Degussa) (400 mg) was added. The stirred mixture was washed three times with N2 (g), 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 the α-terminal isomer and the β-terminal isomer 14. (α-terminal isomer) 1 ¹H NMR (300 MHz, D₂O) δ 7.81 (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-terminal isomer) 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].

[0939] Example 18. S,S'-2,2'-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]tri (azine-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)

[0940]

[0941] 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 phosphine (Nucleoside, Nucleotides, Nucleic acids; 14; 3-5; 1995; 763-766. Lefebvre, Isabelle; Pompon, Alain; Perigaud, Christian; Girardet, Jean-Luc; Gosselin, Gilles; et al.) (87 mg, 0.192 mmol) was added. The reaction was stirred for 2 hours. Then it was quenched with 30% hydrogen peroxide (0.120 mL). The mixture was stirred at room temperature for 30 minutes and then treated with a saturated aqueous solution of sodium thiosulfate (1 mL). The mixture was stirred for 10 minutes. Then it was concentrated under reduced pressure. The residue was subjected to preparative HPLC to separate 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].

[0942] Example 19. S,S'-2,2'-((((2R,3S,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]tri (-7-yl)-5-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)phosphoryl)bis(oxy)bis(ethane-2,1-di) (2,2-Dimethylpropionic acid thioester) (Compound 16)

[0943]

[0944] 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 / z658.9[M+].Tr=2.31

[0945] Example 20. ((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-5-cyano 3,4-Dihydroxytetrahydrofuran-2-yl)methyltetrahydrotriphosphate (compound 17)

[0946]

[0947] 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 (121.4MHz, D2O) δ -5.4 (d, 1P), -10.8 (d, 1P), -21.1 (t, 1P). LCMS m / z 530 [MH], 531.9 [M+H] Tr=0.22min. HPLC ion exchange Tr=9.95min.

[0948] Example 21. ((2R,3S,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-5-ethyl alkynyl-3,4-dihydroxytetrahydrofuran-2-yl)methyltetrahydrotriphosphate (compound 18)

[0949]

[0950] Compound 18 was prepared from compound 13 using a similar procedure as 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.

[0951] Example 22. ((2R,3S,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-3,4- Dihydroxy-5-methyltetrahydrofuran-2-yl)methyltetrahydrotriphosphate (compound 19)

[0952]

[0953] Compound 19 was prepared from compound 14 using a procedure similar to that used to prepare compound 6. 1HNMR(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].

[0954] Example 23. ((2R,3R,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7-yl)-5-cyano 4-Fluoro-3-hydroxytetrahydrofuran-2-yl)methyltetrahydrotriphosphate (Compound 20)

[0955]

[0956] Compound 20 was prepared from compound 2 using a procedure similar to that used to prepare compound 6. 1 HNMR(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 (161MHz, 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.

[0957] 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 Ester (21)

[0958]

[0959] The preparation of ethyl (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-phenylpropionate is described below.

[0960] Preparation of (S)-2-amino-3-phenylpropionic acid ethyl ester hydrochloride.

[0961]

[0962] L-phenylalanine (5 g, 30 mmol) was dissolved in EtOH (30 mL). TMSCl (6.915 mL, 54 mmol) was added to the reactants at room temperature. The reaction vessel was equipped with a reflux condenser, and the reactants were placed in a bath at 80 °C. The reaction was stirred overnight. The next day, the reactants were cooled to room temperature, concentrated under reduced pressure, and the resulting residue was dissolved in Et2O. The resulting slurry was filtered, and the separated solid was further washed with Et2O. The washed solid was placed under high vacuum to give example (S)-2-amino-3-phenylpropionate ethyl hydrochloride (6.86 g, 99%). 1 HNMR(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).

[0963] (2S)-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)-3-phenylpropionate ethyl ester (compound D) preparation

[0964]

[0965] Ethyl (S)-2-amino-3-phenylpropionate hydrochloride (1.01 g, 4.41 mmol) was dissolved in DCM (50 mL). The solution was cooled to 0 °C, and PhOP(O)Cl2 (0.656 mL, 4.41 mmol) was added, followed by the slow addition of Et3N (1.62 mL, 11.5 mmol) over 5 minutes. The cooling bath was removed, and the reaction was allowed to warm to room temperature and stirred for 80 minutes. p-NO2PhOH (0.583 g, 4.19 mmol) was added, followed by Et3N (0.3 mL, 2.1 mmol). The reaction progress was monitored by LC / MS. After the reaction was complete, the solution was diluted with Et2O, and the resulting solid was removed by filtration. The concentrated filtrate was separated by silica gel column chromatography (25 g dry support column, 120 g column; eluent: 100% hexane to 55% EtOAc in hexane solution) to separate compound D (1.25 g, 60%, as a mixture of diastereomers). 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].

[0966] (2S)-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano Ethyl 3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropionate (compound) 21) Preparation

[0967]

[0968] Compound 1 (0.030 g, 0.103 mmol) was dissolved in DMF (1 mL), and then THF (0.5 mL) was added. t-BuMgCl (1 M / THF, 154.5 μL, 0.154 μmol) was added dropwise to the reaction mixture under vigorous stirring. The resulting white slurry was stirred at room temperature for 30 minutes. A solution of compound D (0.058 g, 0.124 mmol) in THF (1 mL) was added dropwise to the reactants at room temperature. The reaction progress was monitored by LC / MS. When the reaction reached 50% conversion, the reactants were cooled in an ice bath and quenched with glacial acetic acid (70 μL). The reaction was concentrated, and compound 21 (22 mg, 34%, a 2.6:1 mixture of diastereomers) was separated from the residue by reversed-phase HPLC. 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].

[0969] 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-methylbutyric acid ethyl Ester (22)

[0970]

[0971] The preparation of ethyl (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-methylbutyrate is described below.

[0972] (2S)-3-methyl-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)ethyl butyrate (compound E) preparation

[0973]

[0974] Ethyl (S)-2-amino-3-methylbutyrate (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 ice bath was removed, and the reaction was heated 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 the reaction was complete, the mixture was diluted with Et2O, and the resulting solid was removed by filtration. The filtrate was concentrated and compound E (0.642 g, 79%, a mixture of diastereomers) was separated by silica gel column chromatography (12 g dry support 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].

[0975] (2S)-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano ethyl 3-methylbutyrate (compound)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-methylbutyrate 22) Preparation

[0976]

[0977] Compound 1 (0.040 g, 0.137 mmol) was dissolved in NMP (1.5 mL), and then THF (0.25 mL) was added. The solution was cooled in an ice bath and t-BuMgCl (1 M / THF, 425.7 μL, 0.426 μmol) was added dropwise with vigorous stirring. The ice bath was removed, and the resulting white slurry was stirred at room temperature for 15 min. A THF (0.5 mL) solution of compound E (0.081 g, 0.192 mmol) was added dropwise to the reaction mixture at room temperature. The reaction progress was monitored by LC / MS. When the reaction reached 50% conversion, the reactants were cooled in an ice bath and quenched with glacial acetic acid (70 μL). The reaction mixture was concentrated, and compound 22 (22 mg, 34%) was partially purified from the residue by reversed-phase HPLC. The semi-pure substance was further purified by silica gel column chromatography (12 g dry support column, 40 g column; eluent: 100% EtOAc to 10% MeOH in EtOAc) to give compound 22 (0.034 g, 43%, a mixture of diastereomers in a 1.8:1 ratio). 1 HNMR(400MHz,DMSO-d6)δ7.91(d,J=1.6Hz,1H),7.88(brs,2H),7.32(m,2H),7. 15(m,3H),6.90(t,J=4.2Hz,1H),6.84(d,J=4.8Hz,1H),6.26(dd,J=13.4,6.2H z,1H),5.87(quart.J=11.2Hz,1H),5.35(m,1H),4.64(m,1H),4.25(m,2H),3.9 3-4.15(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].

[0978] Example 26. (S)-2-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7- Isobutyl propionate (23)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propionate

[0979]

[0980] The preparation of isobutyl (S)-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)propionate isobutyl ester is as follows.

[0981]

[0982] Compound 1 (60.0 mg, 206 μmol) was dissolved in NMP (0.28 mL). THF (0.2 mL) was added, followed by the addition of tert-butylmagnesium chloride (1.0 M tetrahydrofuran solution, 0.309 mL) at room temperature and under an argon atmosphere. After 20 minutes, a THF (0.2 mL) solution of compound F (prepared according to Cho, A. et al J. Med. Chem. 2014, 57, 1812-1825., 81 mg, 206 μmol) was added, and the resulting mixture was heated 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%, 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 1250U HPLC; 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 5μC18110A, 50 × 4.6 mm; Solvent: ACN containing 0.1% TFA, water containing 0.1% TFA; Gradient: 0 min - 5.0 min 2-98% ACN, 5.0 min - 6.0 min 98% ACN, 2 mL / min.

[0983] 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)cyclobutyl propionate (24)

[0984]

[0985] The preparation of (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)cyclobutyl propionate is described below.

[0986] (2S)-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)cyclobutyl propionate (compound G)

[0987]

[0988] Phenyl dichlorophosphate (1.49 mL, 10 mmol) was dissolved in 10 mL of anhydrous DCM and stirred in an ice bath under nitrogen atmosphere. L-alanine isobutyl hydrochloride (0.9 g, 5 mmol) was added in one go. Then triethylamine (765 μL, 5.5 mmol) was added dropwise. The reaction was stirred for 1 hour. More triethylamine (765 μL, 5.5 mmol) was added dropwise and the reaction was stirred for 45 minutes. p-Nitrophenol (1.25 g, 9 mmol) was added in one go and the reaction was stirred for 30 minutes. Triethylamine (765 μL, 5.5 mmol) was added and the reaction mixture was stirred for 2 hours. Then additional p-nitrophenol (1.25 g, 9 mmol) and triethylamine (765 μL, 5.5 mmol) were added, and the reaction was stirred for another 2 hours. The reaction mixture was concentrated under reduced pressure. The resulting crude product was diluted with EtOAc and washed twice with 5% citric acid aqueous solution, followed by washing 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 chromatography (0-20-50% EtOAc in hexane solution) to give compound G (1.48 g, 70% yield, 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].

[0989] (2S)-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano Cyclobutyl propionate (compound 24) (-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)cyclobutyl propionate

[0990]

[0991] 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 in a single batch. 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 h. The reaction mixture was cooled to room temperature, diluted with EtOAc, and washed with 5% citric acid aqueous solution, followed by washing 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 chromatography (0-2-5% MeOH in DCM solution) to give compound 24 (42 mg, 37% yield, a mixture of diastereomers). 1 ¹H NMR (400MHz, 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].

[0992] Example 28. (2S)-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7- (2-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropionic acid isopropyl) Ester (25)

[0993]

[0994] The preparation of isopropyl (2S)-(((((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-phenylpropionate isopropyl ester is described below.

[0995] (2S)-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoic acid isopropyl Ester (compound H) Preparation

[0996]

[0997] Phenyl dichlorophosphate (718 μL, 4.8 mmol) was dissolved in 10 mL of anhydrous DCM and stirred in an ice bath under nitrogen atmosphere. L-phenylalanine isopropyl hydrochloride (1 g, 4.1 mmol) was added in one go. Another 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 736 μL, 5.3 mmol of triethylamine 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 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.

[0998] The reaction mixture was then concentrated under reduced pressure, diluted with EtOAc, and washed twice with 5% citric acid aqueous solution, followed by washing with saturated sodium chloride aqueous solution. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (0-15% EtOAc in hexane solution) to give compound H (1.57 g, yield 68%, 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].

[0999] (2S)-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano Isopropyl 3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropionate (compound) Preparation of substance 25)

[1000]

[1001] Compound 1 (58 mg, 0.2 mmol) and compound H (116 mg, 0.24 mmol) were mixed and 2 mL of anhydrous DMF was added. The reaction mixture was stirred at room temperature under a nitrogen atmosphere. A 1 M tBuMgCl THF solution (300 μL, 0.3 mmol) was added dropwise over 3 minutes, and the reaction mixture was stirred for 16 hours. The reaction mixture was 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 was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (0–5% MeOH in DCM solution) to give compound 25 (40 mg, 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].

[1002] Example 29. (S)-2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]tri Methyl methoxy(phenoxy)phosphoryl(amino)propionate (7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy(phenoxy)phosphoryl(amino)propionate (26)

[1003]

[1004] The preparation of methyl propionate (S)-2-(((S)-(((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)propionate is described below.

[1005]

[1006] Compound 1 (100 mg, 0.34 mmol) was dissolved in THF (2 mL) and cooled in an ice-water bath. Then, 1 Mt-BuMgCl (0.52 mL, 0.77 mmol) was slowly added dropwise. The resulting mixture was stirred at room temperature for 30 minutes. Then, a solution of compound I (prepared according to WO2012142085, 29 mg, 0.52 mmol) in THF (2 mL) was added over 5 minutes, and the resulting mixture was stirred at room temperature for 24 hours. The reaction mixture was then diluted with EtOAc, cooled in an ice-water bath, washed with an aqueous solution of NaHCO3 (2 mL), washed with brine, dried over sodium sulfate, and concentrated under vacuum. The mixture was purified by silica gel column chromatography (MeOH 0-20% DCM solution) and preparative HPLC (acetonitrile 10-80% aqueous solution) to give compound 26 (12 mg, 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) + .

[1007] Example 30. (S)-2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]] tri Neopentyl propionate (-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propionate (27)

[1008]

[1009] The preparation of neopentyl propionate (S)-2-(((S)-(((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)propionate is described below.

[1010]

[1011] Compound 1 (100 mg, 0.34 mmol) was dissolved in THF (2 mL) and cooled in an ice-water bath. Then, 1 M t-BuMgCl (0.52 mL, 0.77 mmol) was slowly added dropwise. The resulting mixture was stirred at room temperature for 30 minutes. Then, compound J (prepared according to WO2012075140, 248 mg, 0.52 mmol) was added over 5 minutes, and the mixture was stirred at room temperature for 24 hours. The mixture was diluted with EtOAc, cooled in an ice-water bath, treated with an aqueous solution of NaHCO3 (2 mL), washed with brine, dried over sodium sulfate, and concentrated under vacuum. The mixture was purified by silica gel column chromatography (MeOH 0-20% DCM solution) and preparative HPLC (acetonitrile 10-80% aqueous solution) to give compound 27 (12 mg, 10%, a single diastereomer). 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) + .

[1012] Example 31. (2S)-2-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7- Cyclopentyl propionate (28)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)cyclopentyl propionate

[1013]

[1014] The preparation of (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)cyclopentyl propionate is described below.

[1015]

[1016] Compound 1 (100 mg, 0.34 mmol) was dissolved in THF (2 mL) and cooled in an ice-water bath. Then, 1 M t-BuMgCl (0.52 mL, 0.77 mmol) was slowly added dropwise. The resulting mixture was stirred at room temperature for 30 minutes. Then, a solution of compound K (prepared according to WO2012075140, 247 mg, 0.52 mmol) in THF (2 mL) was added over 5 minutes, and the resulting mixture was stirred at room temperature for 24 hours. It was diluted with EtOAc, cooled in an ice-water bath, treated with an aqueous solution of NaHCO3 (2 mL), washed with brine, dried over sodium sulfate, and concentrated under vacuum. The mixture was purified by silica gel column chromatography (MeOH 0-20% DCM solution) and preparative HPLC (acetonitrile 10-80% aqueous solution) to give Example 28 (47 mg, 23%, a 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) + .

[1017] 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)cyclohexyl propionate (29)

[1018]

[1019] The preparation of (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)cyclohexyl propionate is described below.

[1020]

[1021] Diisopropylethylamine (0.075 mL, 0.43 mmol) was added dropwise over 5 minutes at 0 °C 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). The resulting mixture was stirred at 50 °C for 1 h. The reaction mixture was then cooled in an ice-water bath, treated with 1 M citric acid (0.5 mL), and purified directly by preparative HPLC (ACN 0-70% aqueous solution) to give compound 29 (20 mg, 19%, 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)+.

[1022] Example 33.2-(((((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)-2-methylpropionate ethyl ester (30)

[1023]

[1024] The preparation of ethyl 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)-2-methylpropionate is described below.

[1025] Preparation of ethyl 2-((tert-Butoxycarbonyl)amino)-2-methylpropionate

[1026]

[1027] Triphenylphosphine (6.18 g, 25.00 mmol) was dissolved in THF (30 mL). Next, DIAD (4.92 mL, 25.00 mmol) was added and the mixture was stirred at room temperature for 10 minutes. 2-((tert-butoxycarbonyl)amino)-2-methylpropionic acid (5.08 g, 25.00 mmol) was dissolved in THF (20 mL) and added to the reaction mixture, followed by ethanol (2.19 mL, 37.49 mmol). The reaction was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the crude product was dissolved in 1:1 Et₂O:hexane (120 mL). The solid triphenylphosphine was filtered off, and the solvent was removed under reduced pressure. The crude product was dissolved in a minimal amount of CH₂Cl₂ and purified by silica gel chromatography (0-50% EtOAc / Hex) to give ethyl 2-((tert-butoxycarbonyl)amino)-2-methylpropionic acid (2.71 g, 47%). 1 ¹H NMR (400MHz, chloroform-d) δ 4.18 (q, J = 7.1Hz, 2H), 1.49 (s, 6H), 1.43 (s, 9H), 1.27 (t, J = 7.1Hz, 3H).

[1028] Preparation of ethyl 2-amino-2-methylpropionate hydrochloride

[1029]

[1030] Ethyl 2-((tert-butoxycarbonyl)amino)-2-methylpropionate (2.71 g, 11.72 mmol) was dissolved in CH₂Cl₂ (25 mL), and a dioxane solution of 4N HCl (25 mmol) was slowly added while stirring at room temperature. After 1 hour, the reaction was confirmed to be complete by TLC. The solvent was removed under reduced pressure, and the crude product was co-evaporated twice with Et₂O, then placed under high vacuum to give ethyl 2-amino-2-methylpropionate 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).

[1031] Preparation of ethyl 2-methyl-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propionate (compound N)

[1032]

[1033] Phenyl dichlorophosphate (0.97 mL, 6.50 mmol) and ethyl 2-amino-2-methylpropionate 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 cold bath was removed and the reaction mixture was stirred at room temperature. After 2 hours, the mixture was subjected to...31 P NMR confirmed the completion of amino acid addition. p-Nitrophenol (0.860 g, 6.17 mmol) was added, followed by TEA (0.87 g, 7.69 mmol). The reaction was stirred at room temperature. After 2 hours, the completion of the reaction was confirmed by LC-MS. The reaction mixture 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 1250U HPLC; 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.

[1034] 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)-2-methylpropionate ethyl ester (compound 30) preparation

[1035]

[1036] 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 min, 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 mixture was placed in a preheated oil bath at 50 °C. 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 reversed-phase HPLC without modifiers to give compound 30 (32 mg, 25%, 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 1250U HPLC; 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.

[1037] Example 34.2-((((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)-2-methylpropionic acid isopropyl ester (31)

[1038]

[1039] The preparation of isopropyl 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)-2-methylpropionate isopropyl ester is described below.

[1040] Preparation of isopropyl 2-((tert-butoxycarbonyl)amino)-2-methylpropionate

[1041]

[1042] Triphenylphosphine (6.17 g, 25.00 mmol) was dissolved in THF (30 mL). Next, DIAD (4.92 mL, 25.00 mmol) was added and the mixture was stirred at room temperature for 10 minutes. 2-((tert-butoxycarbonyl)amino)-2-methylpropionic acid (5.07 g, 25.00 mmol) was dissolved in THF (20 mL) and added to the reaction mixture, followed by isopropanol (1.91 mL, 25.00 mmol). The reaction was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the crude product was dissolved in 1:1 Et₂O:hexane (120 mL). The solid triphenylphosphine was filtered off, and the solvent was removed under reduced pressure. The crude product was dissolved in a minimal amount of CH₂Cl₂ and purified by silica gel chromatography (0–50% EtOAc / Hex) to provide isopropyl 2-((tert-butoxycarbonyl)amino)-2-methylpropionic acid (4.09 g, 67%). 1 ¹H NMR (400MHz, chloroform-d) δ 5.03 (p, J = 6.2Hz, 1H), 1.48 (s, 6H), 1.40 (d, J = 6.2Hz, 9H), 1.24 (d, J = 6.3Hz, 6H).

[1043] Preparation of isopropyl 2-amino-2-methylpropionate hydrochloride

[1044]

[1045] Isopropyl 2-((tert-butoxycarbonyl)amino)-2-methylpropionate (4.09 g, 16.67 mmol) was dissolved in CH₂Cl₂ (50 mL), and a solution of 4N HCl in dioxane (50 mmol) was slowly added while stirring at room temperature. The reaction was confirmed to be complete by TLC after 1 hour. The solvent was removed under reduced pressure, and the crude product was co-evaporated twice with Et₂O, then placed under high vacuum to give isopropyl 2-amino-2-methylpropionate 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).

[1046] Preparation of 2-methyl-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propionate isopropyl ester (compound O) Preparation

[1047]

[1048] Phenyl dichlorophosphate (0.83 mL, 5.58 mmol) and isopropyl 2-amino-2-methylpropionic acid 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 cold bath was removed and the reaction mixture was stirred at room temperature. After 2 hours, the reaction mixture was... 31 P NMR confirmed the completion of amino acid addition. p-Nitrophenol (0.74 g, 5.30 mmol) was added, followed by TEA (0.81 g, 5.84 mmol). The reaction was stirred at room temperature. After 2 hours, the completion of the reaction was confirmed by LC-MS. The reaction mixture 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 1250U HPLC; 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.

[1049] 2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3, Isopropyl 4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-2-methylpropionate (compound 31) Preparation

[1050]

[1051] 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 min, 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 mixture was placed in a preheated oil bath at 50 °C. After 2 hours, the reaction mixture was cooled to room temperature and quenched with acetic acid and methanol. The crude product was concentrated and purified by reversed-phase HPLC without modifiers to give 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 1250U HPLC; MS system: Thermo LCQ Fleet; 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.

[1052] Example 35. (S)-2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]tri Azine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propionic acid-2-ethyl Butyl ester (32)

[1053]

[1054] The preparation of (S)-2-2-((S)-(((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-ethyl butyl ester is described below.

[1055] (3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)dihydrofuran-2(3H-) Preparation of ketones.

[1056]

[1057] (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), an aqueous solution of K₂HPO₄ (2.5 M, 14.3 mL), and TEMPO (56 mg). The mixture was cooled to approximately 1 °C. An aqueous bleach solution (7.9 wt%) was slowly added in portions until the starter 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 MgSO₄ and concentrated under reduced pressure to give a solid product.

[1058] Preparation of (4-amino-7-iodopyrrolo[2,1-f][1,2,4]triazine)

[1059]

[1060] N-iodosuccinimide (17.01 g; 75.6 mmol) was added fractionally 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) while maintaining the contents at about 0 °C. After the reaction was complete (about 3 hours at about 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 about 20–30 °C. The resulting slurry was stirred at about 22 °C for 1.5 hours and then filtered. The solid was washed with water (50 mL) and dried under vacuum at about 50 °C to give solid 4-amino-7-iodopyrrolo[2,1-f][1,2,4]triazine. 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). 13C NMR (101MHz, DMSO-d6) δ155.7,149.1,118.8,118.1,104.4,71.9.MS m / z=260.97[M+H].

[1061] (3R,4R,5R)-2-(4-aminopyrrole) was prepared from (4-amino-7-iodopyrrolo[2,1-f][1,2,4]triazine. [2,1-f][1,2,4]triazine-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol

[1062]

[1063] Iodine base 2 (81 g) and THF (1.6 LV) were added to a reactor under a nitrogen atmosphere. The resulting solution was cooled to approximately 5 °C, and TMSCl (68 g) was added. PhMgCl (345 mL, 1.8 M THF solution) was then slowly added while maintaining the internal temperature at approximately ≤5 °C. The reaction mixture was stirred at approximately 0 °C for 30 minutes, and then cooled to approximately -15 °C. iPrMgCl-LiCl (311 mL, 1.1 M THF solution) was then slowly added while maintaining the internal temperature below approximately -12 °C. After stirring at approximately -15 °C for approximately 10 minutes, the reaction mixture was cooled to approximately -20 °C, and a THF (400 mL) solution of lactone 1 (130 g) was added. The reaction mixture was then stirred at approximately -20 °C for approximately 1 hour and quenched with AcOH (57 mL). The reaction mixture was heated to approximately 0 °C and adjusted to pH 7–8 with an aqueous solution of NaHCO3 (5 wt%, 1300 mL). The reaction mixture was then diluted with EtOAc (1300 mL) to separate the organic and aqueous layers. The organic layer was washed with 1N HCl (1300 mL), NaHCO3 aqueous solution (5 wt%, 1300 mL), and brine (1300 mL), then dried over anhydrous Na2SO4 and concentrated to dryness. The product was purified by silica gel column chromatography using a gradient consisting of a mixture of MeOH and EtOAc to give the product.

[1064] ((2S)-2-2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propionic acid-ethyl butyl ester)(Sp and Rp of Preparation of the mixture:

[1065]

[1066] L-alanine 2-ethylbutyl hydrochloride (5.0 g, 23.84 mmol) was combined with dichloromethane (40 mL), cooled to approximately -78 °C, and phenyl dichlorophosphate (3.65 mL, 23.84 mmol) was added. Triethylamine (6.6 mL, 47.68 mmol) was added over approximately 60 minutes at approximately -78 °C, and the resulting mixture was stirred at ambient temperature for 3 hours. The reaction mixture was cooled to approximately 0 °C, and pentafluorophenol (4.4 g, 23.84 mmol) was added. Triethylamine (3.3 mL, 23.84 mmol) was added over approximately 60 minutes. The mixture was stirred at ambient temperature for approximately 3 hours and concentrated under reduced pressure. The residue was dissolved in EtOAc, washed several times with aqueous sodium carbonate solution, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of EtOAc and hexane (0–30%). The fraction containing the product was concentrated under reduced pressure to obtain (2S)-2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propionic acid-2-ethylbutyl ester in solid form. 1 ¹H NMR (400MHz, 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 (162MHz, chloroform-d) δ -1.52. 19 FNMR(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].

[1067] Preparation of the title compound (a mixture of Sp and Rp):

[1068]

[1069] At ambient temperature, nucleoside (29 mg, 0.1 mmol), phosphonamide (60 mg, 0.12 mmol), and N,N-dimethylformamide (2 mL) were combined. Tert-butylmagnesium chloride (1 M THF solution, 0.15 mL) was slowly added. After approximately 1 hour, the reaction mixture was diluted with ethyl acetate and washed with aqueous citric acid solution (5 wt%), saturated NaHCO3 solution, and saturated salt solution. The organic phase was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of methanol and CH2Cl2 (0–5%). The fraction containing the product was concentrated under reduced pressure to provide the product.

[1070] (3aR,4R,6R,6aR)-4-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-6-(hydroxymethyl)-2, 2-Dimethyltetrahydrofurano[3,4-d][1,3]dioxacyclopentane-4-carboxynitrile:

[1071]

[1072] Sulfuric acid (18M, 1.44 mL) was added to a mixture of (2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carboxynitrile (5.8 g, 0.02 mol), 2,2-dimethoxypropane (11.59 mL, 0.09 mol), and acetone (145 mL) at ambient temperature. The mixture was heated 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 × 50 mL). The combined organic phases were dried with sodium sulfate and concentrated under reduced pressure to give crude (2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carboxynitrile. 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].

[1073] (2S)-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano Preparation of 2-ethylbutyl 3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propionate Note:

[1074]

[1075] Acetonitrile (100 mL) was combined with (2S)-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propionate-2-ethylbutyl ester (9.6 g, 21.31 mmol), 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 about 15 minutes, and N,N-diisopropylethylamine (8.67 mL, 49.78 mmol) was added. After about 4 hours, the reactants were diluted with ethyl acetate (100 mL), cooled to about 0 °C, and combined with aqueous citric acid solution (5 wt%, 100 mL). The organic phase was washed with aqueous citric acid solution (5 wt%, 100 mL), saturated ammonium chloride solution (40 mL), potassium carbonate solution (10 wt%, 2 × 100 mL), and saturated salt solution (100 mL). The organic phase was dried over sodium sulfate and concentrated under reduced pressure to give 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].

[1076] (S)-2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)- 2-Ethylbutyl 5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propionate Preparation of (Compound 32)

[1077]

[1078] Crude acetone (12.85 g) was combined with tetrahydrofuran (50 mL) and concentrated under reduced pressure. The residue was dissolved in tetrahydrofuran (100 mL), cooled to approximately 0 °C, and concentrated HCl (20 mL) was slowly added. The mixture was then heated to ambient temperature. After the initial acetone, as indicated by HPLC analysis, was consumed, water (100 mL) was added, followed by a saturated aqueous sodium bicarbonate solution (200 mL). The mixture was extracted with ethyl acetate (100 mL), the organic phase was washed with a saturated brine solution (50 mL), dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of methanol and ethyl acetate (0 to 20%). The fraction containing the product was concentrated under reduced pressure to provide the product.

[1079] B. Antiviral activity

[1080] Another aspect of the invention relates to a method for inhibiting viral infection, including the step of treating a sample or individual suspected of requiring such inhibition with the composition of the invention.

[1081] Within the scope of this invention, samples suspected of containing viruses include natural or artificial materials, such as living organisms; tissue or cell cultures; biological samples such as biomaterial 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 synthesizing desired glycoproteins; and so on. 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. Samples include organisms such as humans, as well as artificial materials such as cell cultures.

[1082] If desired, the antiviral activity of the compounds of the present invention can be observed by any method after application of the composition, including direct and indirect methods for detecting such activity. Quantitative, qualitative, and semi-quantitative methods for determining such activity are all contemplated. One of the screening methods described above is typically applied; however, any other method, such as observing the physiological characteristics of living organisms, is also applicable.

[1083] The antiviral activity of the compounds of this invention can be measured using known standard screening protocols. For example, the antiviral activity of the compounds can be measured using the following general protocols:

[1084]

[1085] HCS: High Content Imaging

[1086] HeLa: HeLa epithelial cells (cervical cancer)

[1087] Example 36. Determination of antiviral activity and cytotoxicity of Lassa virus and Junin virusThe antiviral activities of compounds 1, 9, and 32 against Lassa virus (LASV) and Junin virus (JUNV) were measured. All studies using wild-type viruses were conducted at Biosafety Level 4 (BSL-4) at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). Antiviral assays using attenuated JUNV strains were conducted at a BSL-2 laboratory at Utah State University. Lassa virus antiviral assays were performed in HeLa cells. Junin virus antiviral assays were performed in Vero and HeLa cells.

[1088] Antiviral assays were performed using a high-content imaging system in 384- or 96-well plates protected with BSL-4 to quantify viral antigen yield as a measure of viral replication. Each plate included a "virus-free" control (column 2) and a "1% DMSO" control (column 3) to determine 0% and 100% viral replication signals, respectively. Primary antibodies used for viral antigen detection 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 antibodies were 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 antibodies were removed, and cells were washed three times with 1×PBS. The secondary antibodies were 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 DR05500) diluted in 1×PBS. Cell images were acquired using a Perkin Elmer Opera confocal microscope (Perkin Elmer, Waltham, MA) with 10× air objectives, with 5 images collected per well. Virus-specific antigens were quantified by measuring fluorescence emission at 488 nm, and nuclei were quantified by measuring fluorescence emission at 640 nm. Z' values ​​for all antiviral assays were >0.3.

[1089] The inhibition percentage of each test concentration relative to the 0% and 100% inhibition controls was calculated, and the EC50 of each compound was determined by nonlinear regression. 50 The value represents the effective concentration of a compound that inhibits viral replication by 50%.

[1090] Example 37. Huning virus assay - Vero

[1091] Vero or Vero E6 cells were seeded at 20,000 cells / well in 100 μL MEM + 2% FBS in 96-well plates. The compound diluted in DMSO was mixed with 120 μL MEM + 2% FBS. 100 μL of each test compound was transferred to two wells of the 96-well plate. 20 μL of virus solution in MEM + 20% FBS was added to make the final test concentrations 47, 4.7, 0.47, and 0.047 μM, and the multiplicity of infection (MII) 0.003 pfu / cell. The test plates were incubated until the untreated virus control approached maximum cytopathic effect (CPE) (5–7 days). The plates were then stained with neutral red dye for 2 hours, eluted in citrate / ethanol buffer, and read on a spectrophotometer at 540 nm. EC50 was calculated by regression analysis. 50 The value is the concentration of the test compound required to reduce virus-induced CPE by 50%, as measured by neutral red staining.

[1092] Example 38. Huning virus assay - HeLa

[1093] HeLa cells were seeded at 2000 cells per well in 384-well plates, and the compound was added to the assay plate as described in section 3.2.1. The assay plate was transferred to a BSL-4 kit and infected with 0.3 pfu / cell JUNV, resulting in ~50% cell expression of viral antigen within 48 hours. The assay plate was incubated for 48 hours, and viral replication was quantified by immunostaining using antibodies that recognize viral glycoproteins.

[1094] Example 39. Lassa virus assay

[1095] HeLa cells were seeded at 2000 cells per well in 384-well plates, and the compound was added to the assay plate as described in section 3.2.1. The assay plate was transferred to a BSL-4 kit and infected with 0.1 pfu / cell LASV, resulting in >60% cell expression of viral antigen within 48 hours. The assay plate was incubated for 48 hours, and viral replication was quantified by immunostaining using antibodies that recognize viral glycoproteins.

[1096] Table 2: Antiviral assays for Lassa virus and Junin virus

[1097] Table 2: In vitro antiviral activity of compounds 1, 9 and 32 against arena virus

[1098]

[1099] ND not detected

[1100] JUNV = Junin virus, LASV = Lassa virus

[1101] Example 40. Determination of antiviral activity and cytotoxicity of MERS-CoV and SARS-CoV

[1102] The antiviral activities of compounds 9 and 32 against MERS virus (MERS-CoV) and SARS virus (SARS-CoV) were measured.

[1103] Antiviral assays were conducted at USAMRIID and the University of North Carolina at Chapel Hill.

[1104] Example 41. MERS-CoV Antiviral Assay (USAMRIID)

[1105] Vero E6 cells were seeded in 384-well plates, and serial dilutions of compound 32 or compound 9 were added to the assay plates via direct titration using an HP D300 digital dispenser (Hewlett-Packard, PalOAlto, CA). Plates were transferred to BSL-4 kits and infected with MERS-CoV (strain Jordan N3) at a multiplicity of infection of 0.5 plaque-forming units (pfu) per cell. Infected cultures were incubated for 48 hours. Viral replication levels in compound-treated and control-vector-treated cultures were determined by quantifying virus-specific antigen levels after immunostaining with an antibody against MERS-CoV Spike(S) protein. The primary antibody (40069-RP02 rb-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 plates were incubated at room temperature for 60 minutes. The primary antibody was removed, and cells were washed three 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 with Draq5 (Biostatus, Shepshed Leicestershire, UK, Cat#DR05500) diluted in 1×PBS. Cells were reverse stained with CellMask Deep Red (Thermo Fisher Scientific, Waltham, MA, catalog number C10046) to enhance the detection of cytoplasmic compartments. Cell images were acquired using a Perkin Elmer Opera confocal microscope (Perkin Elmer, Waltham, MA) with a 10× air objective, collecting 5 images per well. Virus-specific antigens were quantified by measuring fluorescence emission at 488 nm, and nuclei were quantified by measuring fluorescence emission at 640 nm. High-content image analysis was performed to quantify the percentage of infected cells and cell viability. Dose-response analysis using the Levenberg-Marquardt algorithm as a curve-fitting strategy was conducted in GeneData Screener software to determine EC50 values.

[1106] Example 42. Antiviral assays for MERS-CoV and SARS-CoV

[1107] HAE cell cultures isolated from lung tissue were cultured at the air-liquid interface for 6 weeks to promote differentiation. The top surfaces of HAE cultures were washed 24 hours and 1 hour before infection with 1×PBS, followed by infection at 37°C for >1 hour with 1×PBS. Differentiated HAE cultures were infected with recombinant MERS-CoV expressing red fluorescent protein (MERS-CoV RFP) and recombinant SARS-CoV expressing green fluorescent protein (SARS-CoV GFP) at a multiplicity of infection (MIU) of 0.1 pfu / cell. To infect HAE cultures, the top washing solution was removed, viral inoculum was added, and the inoculated cultures were incubated at 37°C for 2.5 hours. After removing the inoculum, the top surfaces of HAE cultures were washed three times with 500 μL of 1×PBS to remove residual virus. Triple-diluted 3-fold dilutions of five compounds 9 were prepared starting at 10 μM and added to the outer side of the culture substrate in HAELI medium approximately 30 minutes before infection. Viral replication was assessed by fluorescence imaging of the cell cultures after 48 hours of incubation. In addition, viral replication was quantified by measuring the yield of infectious virus in the HAE top wash on Vero cell monolayers via plaque assay and by quantifying the viral RNA yield from total cellular RNA via real-time PCR.

[1108] Table 3: MERS antiviral assay

[1109] Table 3: In vitro antiviral activity of compound 32 against coronavirus

[1110] <![CDATA[EC 50 (μM)]]> Measurement Virus MERS-CoV cell lines Vero Compound 9 0.46 Compound 32 0.58

[1111] Example 43. Real-time PCR assay for MERS-CoV and SARS-CoV

[1112] Forty-eight hours post-infection, primary HAE cultures from the antiviral assays described above were harvested in 500 μL of 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 by incubation at 55°C using SuperScript III (Life Technologies, Grand Island, NY, USA). After the first-strand cDNA was generated, ORF1 (genomic RNA) and ORF8 or ORF9 (subgenomic RNA of MERS-CoV and SARS-CoV, 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'). Readings 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 were expressed as a log10-fold change in the copy number of viral ORF1 and ORF8 encoding RNA (MERS-CoV) and / and ORF9 encoding RNA (SARS-CoV) in treated and untreated cells using the ΔΔCt method {10431}.

[1113] Example 44. In Calu-3 In vitro efficacy in 2B4 cells

[1114] 48 hours before infection, Calu-3 2B4 cells were injected at a dose of 5 × 10⁻⁶. 4Cells / well were seeded in 96-well black-walled clear plates. The medium was changed 24 hours prior to infection. A 20 mM stock solution of compound 32 was serially diluted 3-fold in 100% DMSO to...

Claims

1. Use of a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of an Arenaviridae infection caused by a Lassa virus or a Junin virus, wherein the compound is: , or .

2. The use of claim 1, wherein the compound is: or ; or a pharmaceutically acceptable salt thereof.

3. The use of claim 1, wherein the compound is: or a pharmaceutically acceptable salt thereof.

4. The use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable carrier or excipient.

5. The use of claim 1, wherein the medicament is further administered with a therapeutically effective amount of at least one other therapeutic agent or a combination thereof selected from the group consisting of a corticosteroid, an anti-inflammatory signal transduction modulator, a beta 2-adrenergic receptor agonist bronchodilator, an anticholinergic, a mucolytic, a hypertonic saline, and other drugs for the treatment of Arenaviridae viral infections; or mixtures thereof.

6. The use of claim 5, wherein the at least one other therapeutic agent is selected from the group consisting of ribavirin, favipiravir (also known as T-705 or Avigan), T-705 monophosphate, T-705 diphosphate, T-705 triphosphate, ST-193, and mixtures thereof.

7. The use of claim 1, wherein the Arenaviridae infection is caused by a Lassa virus.

8. The use of claim 1, wherein the Arenaviridae infection is caused by a Junin virus.

9. The use of claim 1, wherein the Arenaviridae infection is caused by a Lassa virus strain selected from Josiah, NL, z148, Macenta, AV, and CSF.

10. The use of claim 1, wherein the Arenaviridae polymerase is inhibited.

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