antiviral compounds for the treatment of paramyxoviridae infections and pharmaceutical compositions

专利摘要:
The present invention relates to methods for treating infections by Paramyxoviridae virus by administering ribosides, riboside phosphates and prodrugs thereof, of Formula (I): wherein the 1 'position of the nucleoside sugar is substituted. The compounds, compositions and methods provided are particularly useful for the treatment of infections by human parainfluenza virus and respiratory syncytial.
公开号:BR112013001553B1
申请号:R112013001553-5
申请日:2011-07-22
公开日:2021-01-12
发明作者:Jay P. Parrish；Adrian S. Ray；Dorothy Agnes Theodore；Richard L. Mackman
申请人:Gilead Sciences, Inc.；
IPC主号:

专利说明:

[0001] [001] The present invention relates in general to methods and compounds for the treatment of Paramyxoviridae virus infections, in particular, methods and nucleosides for the treatment of respiratory syncytial virus infections and parainfluenza virus infections. BACKGROUND OF THE INVENTION
[0002] [002] Paramyxoviruses of the Paramyxoviridae family are single-stranded and negative sense RNA viruses, which are responsible for many predominant human and animal diseases. These viruses comprise at least two main subfamilies, Paramyxovirinae and Pneumovirinae. The Paramyxovirina subfamily includes human parainfluenza viruses (HPIV), measles viruses and mumps viruses. Although vaccines are available to prevent measles and mumps infections, these infections caused 745,000 deaths in 2001, so additional treatments would be desirable for susceptible populations. PIVH are the second most common cause of lower respiratory tract infection in younger children and together account for about 75% of cases of Croup (http://www.cdc.gov/ncidod/dvrd/revb/respiratory/hpivfeat.htm ). PIVHs can cause repeated infections throughout life, including disease of the upper respiratory tract and even serious diseases of the lower respiratory tract (eg, pneumonia, bronchitis, and bronchiolitis), the latter being especially of caution among the elderly, and among patients with compromised immune systems (Sable, Infect. 1995, 9, 987-1003). Currently, there are no vaccines available to prevent HPIV infections. Therefore, there is a need for anti-Paramyxovirine therapies.
[0003] [003] The Pneumovirinae subfamily includes human respiratory syncytial virus (HRSV). Almost all children will have had an HRSV infection on their second birthday. HRSV is the leading cause of respiratory infections in infancy and childhood with 0.5 to 2% of those infected who require hospitalization. The elderly and adults with chronic heart disease, lung disease or those who are immunocompromised are also at a high risk of developing severe HRSV disease (http://www.cdc.gov/rsv/index.html). No vaccine to prevent HRSV infection is currently available. The monoclonal antibody palivizumab is available for high-risk infants, for example, premature children or those with any heart or lung disease, but the cost for general use is often prohibitive. Ribavirin has also been used to treat HRSV infections, but it has limited effectiveness. Therefore, there is a need for anti-Pneumovirinae therapies and anti-Paramyxoviridae therapies in general.
[0004] [004] Pyrrole nucleobase [1,2-f] [1,2,4] triazine ribosides, imidazo [1,5-f] [1,2,4] triazine, imidazo [1,2-f] [1 , 2.4] triazine, and [1,2,4] triazole [4,3-f] [1,2,4] triazine were disclosed in Carbohydrate Research 2001, 331 (1), 77-82; Nucleosides & Nucleotides (1996), 15 (1-3), 793-807; Tetrahedron Letters (1994), 35 (30), 5339-42; Heterocycles (1992), 34 (3), 569-74; J. Chem. Soc. Perkin Trans. 1985, 3, 621-30; J. Chem. Soc. Perkin Trans. 1 1984, 2, 229-38; WO 2000056734; Organic Letters (2001), 3 (6), 839-842; J. Chem. Soc. Perkin Trans. 1999, 20, 2929-2936; and J. Med. Chem. 1986, 29 (11), 2231-5. Pyrrole [1,2-f] [1,2,4] triazine nucleobase ribosides with antiviral, anti-HCV, and anti-RdRp activity have been described by Babu (WO2008 / 089105 and WO2008 / 141079) and Francom (WO2010 / 002877 ).
[0005] [005] Butler, et al., WO2009132135, discloses 1 'substituted ribosides and prodrugs comprising pyrrole [1,2-f] [1,2,4] triazine nucleobases that have anti-HCV and anti-RdRp activity. However, no method of treating Paramyxoviridae infections with these compounds has been described. SUMMARY OF THE INVENTION
[0006] [006] Methods and compounds are provided for the treatment of infections caused by the Paramyxoviridae virus family.
[0007] [007] A method for treating a Paramyxoviridae infection in a mammal in need thereof is provided comprising administering a therapeutically effective amount of a Formula I compound:
[0008] [008] In another embodiment, the method comprises administering a therapeutically effective amount of a racemate, enantiomer, diastereoisomer, tautomer, polymorph, pseudopolymorph, amorphous form, hydrate or solvate of a compound of Formula I or a pharmaceutically acceptable salt or ester thereof. to a mammal in need of it.
[0009] [009] In another embodiment, the method comprises treating a Paramyxovirin infection in a mammal in need of it by administering a therapeutically effective amount of a Formula I compound or a pharmaceutically acceptable salt or ester thereof.
[0010] [0010] In another embodiment, the method comprises treating an infection by parainfluenza virus, measles or mumps in a mammal in need of it by administering a therapeutically effective amount of a Formula I compound or a pharmaceutically acceptable salt or ester thereof.
[0011] [0011] In another embodiment, the method comprises treating a parainfluenza virus infection in a mammal in need of it by administering a therapeutically effective amount of a Formula I compound or a pharmaceutically acceptable salt or ester thereof.
[0012] [0012] In another embodiment, the method comprises treating a Pneumovirinae infection in a mammal in need of it by administering a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt or ester thereof.
[0013] [0013] In another embodiment, the method comprises treating a respiratory syncytial virus infection in a mammal in need of it by administering a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt or ester thereof.
[0014] In another embodiment, the method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising an effective amount of the compound of Formula I, or a pharmaceutically acceptable salt or ester thereof, in combination with a pharmaceutically acceptable diluent or carrier.
[0015] In another embodiment, the method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt or ester thereof, in combination with at least one additional therapeutic agent.
[0016] a) uma primeira composição farmacêutica compreendendo um composto de Fórmula I; ou um sal, solvato ou éster farmaceuticamente aceitável do mesmo; e b) uma segunda composição farmacêutica compreendendo pelo menos um agente terapêutico adicional ativo contra vírus infecciosos Paramyxoviridae. [0016] In another embodiment, the method comprises administering a therapeutically effective amount of a combination of pharmaceutical agent 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 Paramyxoviridae viruses.
[0017] [0017] In another embodiment, the present application provides a method for inhibiting a Paramyxoviridae RNA-dependent RNA polymerase, comprising contacting a Paramyxoviridae virus-infected cell with an effective amount of a Formula I compound; or a pharmaceutically acceptable salt, solvate and / or ester thereof.
[0018] [0018] In another embodiment, the use of a Formula I compound or a pharmaceutically acceptable salt, solvate and / or ester thereof is provided to treat a viral infection caused by the Paramyxoviridae virus.
[0019] [0019] In another aspect, the invention further provides processes and new intermediates described here that are useful for preparing compounds of Formula I of the invention.
[0020] [0020] In other respects, new methods for synthesis, analysis, separation, isolation, purification, characterization and testing of the compounds of this invention are provided. DETAILED DESCRIPTION OF EXEMPLARY MODALITIES
[0021] [0021] The reference will now be made in detail to certain modalities of the invention, examples that are illustrated in the description, structures and formulas that accompany it. While the invention will be described in conjunction with the listed modalities, it will be understood that they are not intended to limit the invention to those modalities. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents, which may be included within the scope of the present invention.
[0022] [0022] In another embodiment, a method for treating a Paramyxoviridae infection in a mammal in need thereof is provided comprising administering a therapeutically effective amount of a Formula I compound represented by Formula II:
[0023] [0023] In one embodiment of the method for treating an infection by Paramyxoviridae by administering a compound of Formula II, R1 of Formula II is H. In another aspect of this modality R6 of Formula II is N3, CN, halogen, (C1-C8) alkyl , (C1-C8) substituted alkyl, (C2-C8) alkenyl, (C2-C8) substituted alkenyl, (C2-C8) alkynyl, or (C2-C8) substituted alkynyl. In another aspect of this modality, R6 of Formula II is CN, methyl, ethylene or ethynyl. In another aspect of this modality, R6 of Formula II is CN. In another aspect of this modality, R6 of Formula II is methyl. In another aspect of this modality, R5 of Formula II is H. In another aspect of this modality, R2 of Formula II is ORa. In another aspect of this modality, R2 of Formula II is OH. In another aspect of this modality, R2 of Formula II is F. In another aspect of this modality, R3 of Formula II is ORa. In another aspect of this modality, R3 of Formula II is OH, -OC (= O) R11, or -OC (= O) OR11. In another aspect of this modality, R3 of Formula II is OH. In another aspect of this modality, R8 of Formula II is NR11R 12. In another aspect of this modality, R8 of Formula II is NH2. In another aspect of this modality, R8 of Formula II is OR11. In another aspect of this modality, R8 of Formula II is OH. In another aspect of this modality, R9 of Formula II is H. In another aspect of this modality, R9 of Formula II is NR11R 12. In another aspect of this modality, R9 of Formula II is NH2. In another aspect of this modality, R7 of Formula II is H, -C (= O) R11, -C (= O) OR11 or
[0024] [0024] In another embodiment of the method of treating a Paramyxoviridae infection comprising administering a Formula II compound, the Paramyxoviridae infection is caused by a Paramyxovirine virus. In another aspect of this modality, the Paramyxovirina virus is a parainfluenza, measles or mumps virus. In another aspect of this modality, the Paramyxovirina virus is a Respirovirus virus. In another aspect of this modality, the Paramyxovirina virus is a human parainfluenza virus type 1 or 3.
[0025] [0025] In another embodiment of the method of treating a Paramyxoviridae infection comprising administering a Formula II compound, the Paramyxoviridae infection is caused by a Pneumovirinae virus. In another aspect of this modality, the Pneumovirinae virus is a respiratory syncytial virus. In another aspect of this modality, the Pneumovirinae virus is a human respiratory syncytial virus.
[0026] [0026] In another embodiment, a method for treating a Paramyxoviridae infection in a mammal in need thereof is provided comprising administering a therapeutically effective amount of a Formula I compound represented by Formula III:
[0027] [0027] In one embodiment of the method of treating a Paramyxoviridae infection comprising administering a Formula III compound, Formula III R6 is N3, CN, halogen, (C1-C8) alkyl, (C1-C8) substituted alkyl, (C2 -C8) alkenyl, (C2-C8) substituted alkenyl, (C2-C8) alkynyl or (C2-C8) substituted alkynyl. In another aspect of this modality, R6 of Formula III is CN, methyl, ethylene or ethynyl. In another aspect of this modality, R6 of Formula III is CN. In another aspect of this modality, R6 of Formula III is methyl. In another aspect of this modality, R2 of Formula III is ORa. In another aspect of this modality, R2 of Formula III is OH. In another aspect of this modality, R2 of Formula III is F. In another aspect of this modality, R3 of Formula III is OH, -OC (= O) R11 or -OC (= O) OR11. In another aspect of this modality, Formula III R3 is OH. In another aspect of this modality, R8 of Formula III is NR11R 12. In another aspect of this modality, R8 of Formula III is NH2. In another aspect of this modality, R8 of Formula III is OR11. In another aspect of this modality, R8 of Formula III is OH. In another aspect of this modality, R9 of Formula III is H. In another aspect of this modality, R9 of Formula III is NR11R 12. In another aspect of this modality, R9 of Formula III is NH2. In another aspect of this modality, R7 of Formula III is H, -C (= O) R11, - C (= O) OR11 or
[0028] [0028] In another embodiment of the method of treating a Paramyxoviridae infection comprising administering a Formula III compound, Formula III R6 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 R8 is NH2. In another aspect of this modality, R6 of Formula III is CN, methyl, ethylene or ethynyl. In another aspect of this modality, R6 of Formula III is CN. In another aspect of this modality, R6 of Formula III is methyl. In another aspect of this modality, R2 of Formula III is ORa. In another aspect of this modality, R2 of Formula III is OH, -OC (= O) R11 or -OC (= O) OR11. In another aspect of this modality, R2 of Formula III is OH. In another aspect of this modality, R2 of Formula III is F. In another aspect of this modality, R3 of Formula III is OH, -OC (= O) R11 or -OC (= O) OR11. In another aspect of this modality, Formula III R3 is OH. In another aspect of this modality, R9 of Formula III is H. In another aspect of this modality, R9 of Formula III is NR11R 12. In another aspect of this modality, R9 of Formula III is NH2. In another aspect of this modality, R7 of Formula III is H, -C (= O) R11, - C (= O) OR11 or
[0029] [0029] In another embodiment of the method of treating a Paramyxoviridae infection comprising administering a Formula III compound, Formula III R6 is CN, methyl, ethylene or ethynyl, R8 is NH2, and R9 is H. In another aspect of this modality, Formula III R6 is CN. In another aspect of this modality, R6 of Formula III is methyl. In another aspect of this modality, R2 of Formula III is ORa. In another aspect of this modality, R2 of Formula III is OH, -OC (= O) R11 or -OC (= O) OR11. In another aspect of this modality, R2 of Formula III is OH. In another aspect of this modality, R2 of Formula III is F. In another aspect of this modality, R3 of Formula III is OH, -OC (= O) R11 or - OC (= O) OR11. In another aspect of this modality, Formula III R3 is OH. In another aspect of this modality, R7 of Formula III is H, -C (= O) R11, -C (= O) OR11 or
[0030] [0030] In another embodiment of the method of treating a Paramyxoviridae infection comprising administering a Formula III compound, the Paramyxoviridae infection is caused by a Paramyxovirine virus. In another aspect of this modality, the Paramyxovirina virus is a parainfluenza, measles or mumps virus. In another aspect of this modality, the Paramyxovirina virus is a Respirovirus virus. In another aspect of this modality, the Paramyxovirina virus is a Human parainfluenza virus type 1 or 3.
[0031] [0031] In another embodiment of the method of treating a Paramyxoviridae infection comprising administering a Formula III compound, the Paramyxoviridae infection is caused by a Pneumovirinae virus. In another aspect of this modality, the Pneumovirinae virus is a respiratory syncytial virus. In another aspect of this modality, the Pneumovirinae virus is a human respiratory syncytial virus.
[0032] [0032] In one embodiment, a compound of Formula IV is provided:
[0033] [0033] In an embodiment of the compound of Formula IV, R6 is N3, CN, halogen, (C1-C8) alkyl, (C1-C8) substituted alkyl, (C2-C8) alkenyl, (C2-C8) substituted alkenyl, (C2-C8) alkynyl or (C2-C8) substituted alkynyl. In another aspect of this modality, R6 is CN, methyl, ethylene or ethynyl. In another aspect of this modality, R6 is CN. In another aspect of this modality, R6 is methyl. In another aspect of this modality, R1 is H. In another aspect of this modality, R3 is OH, -OC (= O) R11 or -OC (= O) OR11. In another aspect of this modality, R3 is OH. In another aspect of this modality, R8 is NR11R 12. In another aspect of this modality, R8 is NH2. In another aspect of this modality, R 8 is OR11. In another aspect of this modality, R8 is OH. In another aspect of this modality, R9 is H. In another aspect of this modality, R9 is NR11R 12. In another aspect of this modality, R9 is NH2. In another aspect of this modality, R7 is H, -C (= O) R11, -C (= O) OR11 or
[0034] [0034] In another embodiment of a compound of Formula IV, R6 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 R8 is NH2. In another aspect of this modality, R6 is CN, methyl, ethylene or ethynyl. In another aspect of this modality, R6 is CN. In another aspect of this modality, R6 is methyl. In another aspect of this modality, R1 is H. In another aspect of this modality, R3 is OH, -OC (= O) R11 or -OC (= O) OR11. In another aspect of this modality, R3 is OH. In another aspect of this modality, R9 is H. In another aspect of this modality, R9 is NR11R 12. In another aspect of this modality, R9 is NH2. In another aspect of this modality, R7 is H, -C (= O) R11, - C (= O) OR11 or
[0035] [0035] In another aspect of this modality, R7 is H. In another aspect of this modality, R7 is
[0036] [0036] In another embodiment of the compound of Formula IV, R6 is CN, methyl, ethylene or ethynyl, R8 is NH2, and R9 is H. In another aspect of this modality, R1 is H. In another aspect of this modality, R6 is CN . In another aspect of this modality, R6 is methyl. In another aspect of this modality, R3 is OH, - OC (= O) R11 or -OC (= O) OR11. In another aspect of this modality, R3 is OH. In another aspect of this modality, R7 is H, -C (= O) R11, -C (= O) OR11 or
[0037] [0037] In another aspect of this modality, R7 is H. In another aspect of this modality, R7 is
[0038] [0038] In another embodiment, a method for treating a Paramyxoviridae infection in a mammal in need thereof is provided comprising administering a therapeutically effective amount of a compound of Formulas I-IV, wherein R11 or R 12 is independently H, ( C1- C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C4-C8) carbocyclylalkyl, optionally substituted aryl, optionally substituted heteroaryl, -C (= O) (C1- C8) alkyl, -S (O) n (C1-C8) alkyl or aryl (C1-C8) alkyl. In another embodiment, R11 and R 12 taken together with a nitrogen to which they are both attached, form a 3- to 7-membered heterocyclic ring to which a carbon atom of said heterocyclic ring can optionally be replaced by -O-, -S- or -NRa -. Therefore, by way of example and not limitation, the fraction – NR11R 12 can be represented by heterocycles:
[0039] [0039] In another embodiment, a method is provided for treating a Paramyxoviridae infection in a mammal in need thereof comprising administering a therapeutically effective amount of a compound of Formula I-IV, wherein each R3, R4, R5, R6, R 11 or R 12 is, independently, (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl or aryl (C1- C8) alkyl, wherein said (C1-C8) alkyl, (C2 -C8) alkenyl, (C2-C8) alkynyl or aryl (C1- C8) alkyl are independently optionally substituted by one or more halo, hydroxy, CN, N3, N (Ra) 2 or ORa. Therefore, by way of example and not limitation, R3, R4, R5, R6, R 11 or R 12 could represent fractions such as - CH (NH2) CH3, -CH (OH) CH2CH3, -CH (NH2) CH (CH3) 2, -CH2CF3, - (CH2) 2CH (N3) CH3, - (CH2) 6NH2 and the like.
[0040] [0040] In another embodiment, a method is provided for treating a Paramyxoviridae infection in a mammal in need thereof comprising administering a therapeutically effective amount of a Formula I-IV compound, wherein R3, R4, R5, R6, R 11 or R 12 is (C1-C8) alkyl in which one or more of the non-terminal carbon atoms of each said (C1- C8) alkyl can be optionally substituted by -O-, -S- or -NRa -. Therefore, by way of example and not limitation, R3, R4, R5, R6, R 11 or R 12 could represent fractions such as -CH2OCH3, -CH2OCH2CH3, -CH2OCH (CH3) 2, -CH2SCH3, - (CH2) 6OCH3, - (CH2) 6N (CH3) 2 and the like.
[0041] [0041] In another embodiment, a method is provided to treat a Paramyxoviridae infection in a sample comprising administering an effective amount of a compound of Formula I selected from the group consisting of:
[0042] [0042] In another embodiment, a compound of Formula IV is provided which is
[0043] [0043] In another embodiment, a compound of Formula I is provided which is
[0044] [0044] Unless stated otherwise, the following terms and phrases as used here are intended to have the following meanings:
[0045] [0045] When trade names are used here, applicants independently intend to include the product brand and the product brand active ingredients.
[0046] [0046] As used herein, "a compound of the invention" or "a compound of Formula I" means a compound of Formula I or a pharmaceutically acceptable salt thereof. Similarly, with respect to isolable intermediates, the phrase "a compound of Formula (number)" means a compound of that Formula and pharmaceutically acceptable salts thereof.
[0047] [0047] "Alkyl" is hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms. For example, an alkyl group can have 1 to 20 carbon atoms (that is, C1-C20 alkyl), 1 to 8 carbon atoms (that is, C1-C8 alkyl) or 1 to 6 carbon atoms (that is, 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, i-propyl, -CH (CH3) 2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, - CH2CH (CH3) 2 ), 2-butyl (s-Bu, s-butyl, -CH (CH3) CH2CH3), 2-methyl-2-propyl (t-Bu, t-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 (-CH2CH2CH (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 (CH3) 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).
[0048] [0048] "Aloxy" means a group containing the Formula -O-alkyl, in which an 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 to 20 carbon atoms (ie, C1-C20 alkoxy), 1 to 12 carbon atoms (ie, C1-C12 alkoxy) or 1 to 6 carbon atoms (ie , C1-C6 alkoxy). Examples of suitable alkoxy groups include, but are not limited to, methoxy (-O-CH3 or -OMe), ethoxy (-OCH2CH3 or -OEt), t-butoxy (-O-C (CH3) 3 or -OtBu) and the like.
[0049] [0049] "Haloalkyl" is an alkyl group, as defined above, in which one or more hydrogen atoms in the alkyl group is replaced by a halogen atom. The alkyl portion of a haloalkyl group can have 1 to 20 carbon atoms (ie, C1-C20 haloalkyl), 1 to 12 carbon atoms (ie, C1-C12 haloalkyl) or 1 to 6 carbon atoms (ie , C1-C6 alkyl). Examples of suitable haloalkyl groups include, but are not limited to, -CF3, -CHF2, -CFH2, -CH2CF3 and the like.
[0050] [0050] "Alkenyl" is a hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms with at least one unsaturation site, that is, a carbon-carbon double bond, sp2. For example, an alkenyl group can have 2 to 20 carbon atoms (that is, C2-C20 alkenyl), 2 to 8 carbon atoms (that is, C2-C8 alkenyl) or 2 to 6 carbon atoms (that is, 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).
[0051] [0051] "Alquinyl" is a hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms with at least one unsaturation site, that is, a carbon-carbon triple bond, sp. For example, an alkynyl group can have 2 to 20 carbon atoms (that is, C2-C20 alkynyl), 2 to 8 carbon atoms (that is, C2-C8 alkyl) or 2 to 6 carbon atoms (that is, C2-C6 alkynyl). Examples of suitable alkynyl groups include, but are not limited to, acetylene (-CCH), propargyl (-CH2C-CH) and the like.
[0052] [0052] "Alkylene" refers to a saturated, branched or linear chain or cyclic hydrocarbon radical containing two centers of monovalent radical derived from the removal of two hydrogen atoms from the same or two different carbon atoms from a parent alkane. For example, an alkylene group can have 1 to 20 carbon atoms, 1 to 10 carbon atoms or 1 to 6 carbon atoms. Typical alkylene radicals include, but are not limited to, methylene (-CH2-), 1,1-ethyl (-CH (CH3) -), 1,2-ethyl (-CH2CH2-), 1,1-propyl (-CH (CH2CH3) ) -), 1,2-propyl (-CH2CH (CH3) -), 1,3-propyl (-CH2CH2CH2-), 1,4-butyl (-CH2CH2CH2CH2-) and the like.
[0053] [0053] "Alkenylene" refers to an unsaturated, branched or linear chain or cyclic hydrocarbon radical containing two centers of monovalent radical derived from the removal of two hydrogen atoms from the same or two different carbon atoms from an alkene parent. For example, an alkenylene group can have 1 to 20 carbon atoms, 1 to 10 carbon atoms or 1 to 6 carbon atoms. Typical alkenylene radicals include, but are not limited to, 1,2-ethylene (-CHCH-).
[0054] [0054] "Alquinylene" refers to an unsaturated, branched or linear chain or cyclic hydrocarbon radical containing two monovalent radical centers derived from the removal of two hydrogen atoms from the same or two different carbon atoms from a parent alkaline. For example, an alkynylene group can have 1 to 20 carbon atoms, 1 to 10 carbon atoms or 1 to 6 carbon atoms. Typical alkylene radicals include, but are not limited to, acetylene (-CC-), propargyl (-CH2CC-) and 4-pentynyl (-CH2CH2CH2CC-).
[0055] [0055] “Amino” generally refers to a nitrogen radical that can be considered an ammonia derivative containing the Formula –N (X) 2, where each "X" is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted carbocyclyl substituted, substituted or unsubstituted heterocyclyl, etc. The hybridization of nitrogen is approximately sp3. Non-limiting types of amino include -NH2, -N (alkyl) 2, -NH (alkyl), -N (carbocyclyl) 2, -NH (carbocyclyl), - N (heterocyclyl) 2, -NH (heterocyclyl), -N (aryl) 2, -NH (aryl), -N (alkyl) (aryl), - N (alkyl) (heterocyclyl), -N (carbocyclyl) (heterocyclyl), -N (aryl) (heteroaryl), - N ( alkyl) (heteroaryl) etc. The term "alkylamino" refers to an amino group substituted by 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 generally refers to alkylamino groups, as defined above, in which at least one substituted alkyl, as defined herein, is attached to the amino nitrogen atom. Non-limiting examples of substituted alkylamino 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.
[0056] [0056] "Arila" means an aromatic hydrocarbon radical derived from the removal of a hydrogen atom from a simple carbon atom from a parental aromatic ring system. For example, an aryl group can have 6 to 20 carbon atoms, 6 to 14 carbon atoms or 6 to 10 carbon atoms. Typical aryl groups include, but are not limited to, radicals derived from benzene (e.g., phenyl), substituted benzene, naphthalene, anthracene, biphenyl and the like.
[0057] [0057] "Arylalkyl" refers to an acyclic alkyl radical in which one of the hydrogen atoms attached to a carbon atom, typically a terminal carbon atom or sp3, is replaced by an aryl radical. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and the like. The arylalkyl group can comprise 7 to 20 carbon atoms, for example, the alkyl fraction is 1 to 6 carbon atoms and the aryl fraction is 6 to 14 carbon atoms.
[0058] [0058] "Arylalkenyl" refers to an acyclic alkenyl radical in which one of the hydrogen atoms attached to a carbon atom, typically a terminal carbon atom or sp3, but still an sp2 carbon atom, is replaced by an aryl radical . The aryl portion of the arylalkenyl may include, for example, any of the aryl groups described herein, and the alkenyl portion of the arylalkenyl may include, for example, any of the alkenyl groups described herein. The arylalkenyl group can comprise 8 to 20 carbon atoms, for example, the alkenyl fraction is 2 to 6 carbon atoms and the aryl fraction is 6 to 14 carbon atoms.
[0059] [0059] "Arylalkynyl" refers to an acyclic alkynyl radical in which one of the hydrogen atoms attached to a carbon atom, typically a terminal carbon atom or sp3, but still a sp carbon atom, is replaced by an aryl radical . The aryl portion of the arylalkynyl may include, for example, any of the aryl groups described herein, and the alkynyl portion of the arylalkynyl may include, for example, any of the alkynyl groups described herein. The arylalkynyl group can comprise 8 to 20 carbon atoms, for example, the alkynyl fraction is 2 to 6 carbon atoms and the aryl fraction is 6 to 14 carbon atoms.
[0060] [0060] The term "substituted" in reference to alkyl, alkylene, aryl, arylalkyl, alkoxy, heterocyclyl, heteroaryl, carbocyclyl, etc., for example, "substituted alkyl", "substituted alkylene", "substituted aryl", "substituted arylalkyl "," Substituted heterocyclyl "and" substituted carbocyclyl "means alkyl, alkylene, aryl, arylalkyl, heterocyclyl, carbocyclyl respectively, where one or more hydrogen atoms are each independently replaced by a non-hydrogen substituent. Typical substituents include, but are not limited to, -X, -Rb, -O-, = O, -ORb, -SRb, -S-, -NRb 2, -N + R b 3, = NRb, -CX3, -CN, -OCN, -SCN, -N = C = O, -NCS, -NO, -NO2, = N2, -N3, -NHC (= O) Rb, -OC (= O) Rb, -NHC (= O) NRb 2, -S (= O) 2-, -S (= O) 2OH, -S (= O) 2R b, -OS (= O) 2ORb, -S (= O) 2NRb 2, -S (= O) Rb, -OP (= O) (ORb) 2, -P (= O) (ORb) 2, -P (= O) (O-) 2, -P (= O) (OH) 2, - P (O) (ORb) (O-), -C (= O) Rb, -C (= O) X, -C (S) Rb, -C (O) ORb, -C (O) O-, -C (S) ORb, -C (O) SRb, -C (S) SRb, -C (O) NRb 2, -C (S) NRb 2, -C (= NRb) NRb 2, where each X is independently a halogen: F, Cl, Br or I; and each Rb is independently H, alkyl, aryl, arylalkyl, a heterocycle or a protecting group or prodrug moiety. Alkylene, alkenylene, and alkynylene groups can also be similarly substituted. Unless otherwise indicated, when the term "substituted" is used in conjunction with groups such as arylalkyl, which have two or more fractions capable of substitution, the substituents may be attached to the aryl fraction, the alkyl fraction or both.
[0061] [0061] The term “prodrug” as used here refers to any compound that when administered to a biological system generates the substance of the drug, ie active ingredient, as a result of spontaneous chemical reactions, chemical reactions catalyzed by enzyme , photolysis and / or metabolic chemical reactions. A prodrug is then covalently modified or latent analogue form of a therapeutically active compound.
[0062] [0062] One skilled in the art will recognize that the substituents and other fractions of the compounds of Formula I-IV must be selected to provide a compound that is sufficiently stable to provide a pharmaceutically useful compound that can be formulated into an acceptably stable composition. Compounds of Formula I-IV that have said stability are contemplated to be within the scope of the present invention.
[0063] [0063] "Heteroalkyl" refers to an alkyl group where one or more carbon atoms have been replaced with a hetero atom, such as, O, N or S. For example, if the carbon atom of the alkyl group that is attached to the parent molecule is replaced by a heteroatom (for example, O, N or S) the resulting heteroalkyl groups are, respectively, an alkoxy group (for example, -OCH3etc.), an amine (for example, -NHCH3, -N (CH3) 2 , etc.) or a thioalkyl group (for example, -SCH3). If a non-terminal carbon atom of the alkyl group that is not attached to the parent molecule is replaced by a heteroatom (for example, O, N or S) the resulting heteroalkyl groups are, respectively, an alkyl ether (for example, -CH2CH2- O-CH3, etc.), an alkyl amine (for example, -CH2NHCH3, -CH2N (CH3) 2, etc.) or a thioalkyl ether (for example, -CH2-S-CH3). If the terminal carbon atom of the alkyl group is replaced by a heteroatom (for example, O, N or S), the resulting heteroalkyl groups are, respectively, a hydroxyalkyl group (for example, -CH2CH2-OH), an aminoalkyl group ( for example, -CH2NH2) or an alkyl thiol group (for example, -CH2CH2-SH). A heteroalkyl group can have, for example, 1 to 20 carbon atoms, 1 to 10 carbon atoms or 1 to 6 carbon atoms. A C1-C6 heteroalkyl group means a heteroalkyl group containing 1 to 6 carbon atoms.
[0064] [0064] "Heterocycle" or "heterocyclyl" as used here includes by way of example and not limiting those heterocycles described in Paquette, Leo A .; Principles of Modern Heterocyclic Chemistry (WA Benjamin, New York, 1968), particularly chapters 1, 3, 4, 6, 7, and 9; The Chemistry of Heterocyclic Compounds, A Series of Monographs "(John Wiley & Sons, New York, 1950 to present), in Private Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960 ) 82: 5566. In a specific embodiment of the invention "heterocycle" includes a "carbocycle" as defined here, in which one or more (for example 1, 2, 3 or 4) carbon atoms have been replaced with a hetero atom (for example The terms "heterocycle" or "heterocyclyl" include saturated rings, partially unsaturated rings, and aromatic rings (i.e., heteroaromatic rings), substituted heterocyclyl include, for example, heterocyclic rings substituted with any of the substituents described here including carbonyl groups. A non-limiting example of a substituted heterocyclyl carbonyl is:
[0065] [0065] Examples of heterocycles include, by way of example and without limitation, pyridyl, dihydropyridyl, tetrahydropyridyl (piperidyl), thiazolyl, tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrrolyl, pyrrolyl, pyrrolyl, pyrrolyl, pyrrole, pyrazole , tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, tetrahydroquinolinyl, trinhydroquinoline, tetrahydroquinoline, tetrahydroquinoline, tetrahydroquinoline , 6H-1,2,5-thiadiazinyl, 2H, 6H-1,5,2-dithiazinyl, thienyl, thiantrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxatinil, 2H-pyrrolyl, isothiazolil, isoxazolil, pyrazinyl, pyridazinyl, pyridazinyl, pyridazinyl, pyridine , isoindolyl, 3H-indolyl, 1H-indazoli, purinyl, 4Hquinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, β-carbolinyl, phenantridinyl, phenantridinyl, pyridine anthrolinyl, phenazinyl, phenothiazinyl, furazanil, phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl, benzazolinyl, benzazolinyl, benzazolinyl, benzazolinyl
[0066] [0066] By way of example and without limitation, carbon-linked heterocycles are bonded at position 2, 3, 4, 5 or 6 of a pyridine, position 3, 4, 5 or 6 of a pyridazine, position 2, 4, 5 or 6 of a pyrimidine, position 2, 3, 5 or 6 of a pyrazine, position 2, 3, 4 or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole, position 2, 4 or 5 of an oxazole, imidazole or thiazole, position 3, 4 or 5 of an isoxazole, pyrazole or isothiazole, position 2 or 3 of an aziridine, position 2, 3 or 4 of an azetidine, position 2, 3, 4, 5, 6 , 7 or 8 of a quinoline or position 1, 3, 4, 5, 6, 7 or 8 of an isoquinoline. Even more typically, carbon attached to heterocycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4 -pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl or 5-thiazolyl.
[0067] [0067] By way of example, and not limitation, nitrogen attached to heterocycles are attached at position 1 of an aziridine, azetidine, pyrrolidine, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline , pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of an isoindole, or isoindoline, position 4 of a morpholine, and position 9 of a carbazole or β-carboline . Even more typically, nitrogen attached to heterocycles includes 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl and 1-piperidinyl.
[0068] [0068] "Heterocyclylalkyl" refers to an acyclic alkyl radical in which one of the hydrogen atoms attached to a carbon atom, typically a terminal carbon atom or sp3, is replaced by a heterocyclyl radical (that is, a heterocyclyl fraction). alkylene). Typical heterocyclyl alkyl groups include, but are not limited to, heterocyclyl-CH2-, 2- (heterocyclyl) ethan-1-yl, and the like, wherein the "heterocyclyl" moiety includes any of the heterocyclyl groups described above, including those described in Principles of Modern Heterocyclic Chemistry. One skilled in the art will understand that the heterocyclyl group can be attached to the alkyl portion of the heterocyclyl alkyl via a carbon-carbon bond or a carbon-heteroatom bond, with the proviso that the resulting group is chemically stable. The heterocyclyl alkyl group comprises 3 to 20 carbon atoms, for example, the alkyl portion of the arylalkyl group is 1 to 6 carbon atoms and the heterocyclyl fraction is 2 to 14 carbon atoms. Examples of heterocyclylalkyls include, by way of example and without limitation, 5-membered heterocycles containing sulfur, oxygen, and / or nitrogen containing heterocycles such as thiazolylmethyl, 2-thiazolylethan-1-yl, imidazolylmethyl, oxazolylmethyl, thiadiazolylmethylc., 6-membered heterocycles sulfur, oxygen and / or nitrogen such as piperidinylmethyl, piperazinylmethyl, morpholinylmethyl, pyridinylmethyl, pyridizylmethyl, pyrimidylmethyl, pyrazinylmethylc.
[0069] [0069] "Heterocyclylalkenyl" refers to an acyclic alkenyl radical in which one of the hydrogen atoms attached to a carbon atom, typically a terminal carbon atom or sp3, but still a sp2 carbon atom, is replaced by a heterocyclyl radical (that is, a heterocyclylalkenylene fraction). The heterocyclyl portion of the alkenyl heterocyclyl group includes any of the heterocyclyl groups described herein, including those described in Principles of Modern Heterocyclic Chemistry, and the alkenyl portion of the alkenyl heterocyclyl group includes any of the alkenyl groups described herein. One skilled in the art will understand that the heterocyclyl group can be attached to the alkenyl portion of the alkenyl heterocyclyl via a carbon-carbon bond or a carbon-heteroatom bond, with the proviso that the resulting group is chemically stable. The alkenyl heterocyclyl group comprises 4 to 20 carbon atoms, for example, the alkenyl portion of the alkenyl heterocyclyl group is 2 to 6 carbon atoms and the heterocyclyl fraction is 2 to 14 carbon atoms.
[0070] [0070] "Heterocyclylalkynyl" refers to an acyclic alkynyl radical in which one of the hydrogen atoms attached to a carbon atom, typically a terminal carbon atom or sp3, but still a sp carbon atom, is replaced by a heterocyclyl radical (that is, a heterocyclylalkynylene fraction). The heterocyclyl portion of the heterocyclyl alkynyl group includes any of the heterocyclyl groups described herein, including those described in Principles of Modern Heterocyclic Chemistry, and the alkynyl portion of the heterocyclyl alkynyl group includes any of the alkynyl groups described herein. One skilled in the art will understand that the heterocyclyl group may be attached to the alkynyl portion of the heterocyclyl alkynyl via a carbonocarbon bond or a carbon-heteroatom bond, with the proviso that the resulting group is chemically stable. The heterocyclyl alkynyl group comprises 4 to 20 carbon atoms, for example, the alkynyl portion of the heterocyclyl alkynyl group is 2 to 6 carbon atoms and the heterocyclyl fraction is 2 to 14 carbon atoms.
[0071] [0071] "Heteroaryl" refers to an aromatic heterocyclyl containing at least one heteroatom in the ring. Non-limiting examples of suitable heteroatoms that can be included in the aromatic ring include oxygen, sulfur and nitrogen. Non-limiting examples of heteroaryl rings include all of the aromatic rings listed in the definition of "heterocyclyl", including pyridinyl, pyrrolyl, oxazolyl, indolyl, isoindolyl, purinyl, furanyl, thienyl, benzofuranyl, benzothiophenyl, carbazolyl, imidazolyl, thiazolyl, pyrolazole, isoxazol, pyridine isothiazolyl, quinolyl, isoquinolyl, pyridazyl, pyrimidyl, pyrazyl, etc.
[0072] [0072] "Carbocycle" or "carbocyclyl" refers to a saturated aromatic ring (i.e., cycloalkyl), partially unsaturated (for example, cycloalkenyl, cycloalcadienylc.) Or aromatic ring containing 3 to 7 carbon atoms as a monocycle, 7 to 12 carbon atoms as a bicycle, and up to about 20 carbon atoms as a bicycle. Monocyclic carbocycles have 3 to 7 ring atoms, even more typically 5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, for example, arranged as a bicycle system [4.5], [5.5], [5.6] or [6.6], or 9 or 10 ring atoms arranged as a bicycle system [5.6] or [6.6], or spiro-fused rings. Non-limiting examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl , 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, and phenyl. Non-limiting examples of bicycle bicycles include naphthyl, tetrahydronaphthalene and decalin.
[0073] [0073] "Carbocyclylalkyl" refers to an acyclic alkyl radical in which one of the hydrogen atoms attached to a carbon atom is replaced by a carbocyclyl radical as described here. Typical, but not limiting, examples of carbocyclylalkyl groups include cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclopentylmethyl and cyclohexylmethyl.
[0074] [0074] "Arylheteroalkyl" refers to a heteroalkyl as defined here, in which a hydrogen atom (which may be attached to a carbon atom a heteroatom) has been replaced by an aryl group as defined here. The aryl groups can be attached to a carbon atom of the heteroalkyl group, or to a heteroatom of the heteroalkyl group, as long as the resulting arylheteroalkyl group provides a chemically stable fraction. For example, an arylheteroalkyl group can have the general formula -alkyleneO-aryl, -alkylene-O-alkylene-aryl, -alkylene-NH-aryl, -alkylene-NH-alkylene-aryl, -alkylene-S-aryl, -alkylene-S-alkylene-aryl, etc. In addition, any of the alkylene fractions in the general formulas above can be substituted with any of the substituents defined or exemplified here.
[0075] [0075] "Heteroarylalkyl" refers to an alkyl group, as defined here, in which a hydrogen atom has been replaced by a heteroaryl group as defined here. Non-limiting examples of heteroaryl alkyl include -CH2-pyridinyl, -CH2-pyrrolyl, -CH2-oxazolyl, -CH2-indolyl, -CH2-isoindolyl, -CH2-purinyl, -CH2-furanyl, -CH2-thienyl, -CH2- benzofuranil, -CH2-benzothiophenyl, -CH2-carbazolyl, -CH2-imidazolyl, -CH2-thiazolyl, -CH2-isoxazolyl, -CH2-pyrazolyl, -CH2-isothiazolyl, -CH2-quinolyl, -CH2-isoquinolyl, -CH2 pyridazyl, -CH2-pyrimidyl, -CH2-pyrazyl, -CH (CH3) -pyridinyl, -CH (CH3) -pyrrolyl, -CH (CH3) -oxazolyl, -CH (CH3) -indolyl, -CH (CH3) - isoindolyl, -CH (CH3) -purinyl, -CH (CH3) -furanyl, -CH (CH3) -thienyl, -CH (CH3) -benzofuranyl, -CH (CH3) -benzothiophenyl, -CH (CH3) -carbazolil, -CH (CH3) -imidazolyl, -CH (CH3) -thiazolyl, -CH (CH3) -isoxazolyl, -CH (CH3) -pyrazolyl, -CH (CH3) -isothiazolyl, -CH (CH3) -quinolyl, -CH (CH3) -isoquinolyl, -CH (CH3) -pyridazyl, -CH (CH3) -pyrimidyl, -CH (CH3) -pyrazyl, etc.
[0076] [0076] The term "optionally substituted" with reference to a particular fraction of the compound of Formula I-III (for example, an optionally substituted aryl group) refers to a fraction in which all the substituents are hydrogen or in which one or more of the hydrogens of the fraction can be replaced by substituents such as those listed under the definition of "substituted".
[0077] [0077] The term "optionally substituted" in reference to a particular fraction of the compound of Formula I-III (for example, the carbon atoms of said (C1-C8) alkyl can be optionally substituted by -O-, -S- or –NRa -) means that one or more of the methylene groups of (C1-C8) alkyl can be replaced by 0, 1, 2 or more of specified groups (for example, –O-, -Sor –NRa -).
[0078] [0078] The term "non-terminal carbon atoms" in reference to an alkyl, alkenyl, alkynyl, alkylene, alkenylene, or alkynylene fraction refers to the carbon atoms in the fraction that intervene between the first carbon atom of the fraction and the last carbon atom in the fraction. Therefore, by way of example and not limitation, in the alkyl -CH2 (C *) H2 (C *) H2CH3 fraction or alkylene -CH2 (C *) H2 (C *) H2CH2 fraction - the C * atoms could be considered to be the non-terminal carbon atoms.
[0079] [0079] Alternative waxes Y and Y1 are nitrogen oxides such as + N (O) (R) or + N (O) (OR). These nitrogen oxides, as shown here attached to a carbon atom, can still be represented by separate charge groups as
[0080] [0080] "Ligand" or "bond" means a chemical fraction comprising a covalent bond or a chain of atoms. Binders include repeated units of alkyloxy (eg, polyethylene-oxide, PEG, polymethylene-oxide) and alkylamino (eg, polyethyleneamino, Jeffamine®); and diacid ester and amides including succinate, succinamide, diglycolate, malonate and caproamide.
[0081] [0081] The terms like “linked to oxygen”, “linked to nitrogen”, “linked to carbon”, “linked to sulfur” or “linked to phosphorus” mean that if a link between two fractions can be formed by using more than a type of atom in a fraction, then the bond formed between the fractions is through the specified atom. For example, a nitrogen-bound amino acid could be linked to a nitrogen atom of the amino acid rather than through an oxygen or carbon atom of the amino acid.
[0082] [0082] In some embodiments of the compounds of Formula I-IV, one or more of W1 or W2 are independently a radical of a naturally occurring α-amino acid ester bound to nitrogen. 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. The esters of these amino acids comprise any of those described for the substituent R, particularly those in which R is optionally substituted (C 1 -C 8) alkyl.
[0083] [0083] The base term "purine" or "pyrimidine" includes, among others, adenine, N6 -alkylpurines, N6 -acylpurines (where acyl is C (O) (alkyl, aryl, alkylaryl, or arylalkyl), N6-benzylpurine , N6 -halopurine, N6 -vinylpurine, N6 - acetylenic purine, N6 -acyl purine, N6 -hydroxyalkyl purine, N6 -alylaminopurine, N6 -thioalkyl purine, N2 -alkylpurines, N2 -alkyl-6-thiopurines, thymine, cytosine, 5-fluorocytosine, 5-methylcytosine, 6-azapyrimidine, including 6-azacytosine, 2- and / or 4-mercaptopyrimidine, uracil, 5-halouracil, including 5-fluorouracil, C5-alkylpyrimidines, C5-benzylpyrimidines, C5-halopyrimidines, -vinylpyrimidine, C5 - acetylenic pyrimidine, C5 -acyl pyrimidine, C5 -hydroxyalkyl purine, C5 - amidopyrimidine, C5 -cyanopyrimidine, C5 -5-iodopyrimidine, C6 -iodine-pyrimidine, C 5 -Br-vinyl-pyrimine vinyl pyrimidine, C5 -nitropyrimidine, C5 -aminopyrimidine, N2 -alkylpurines, N2 -alkyl-6-thiopurines, 5-azacitidinyl, 5-azauracilyl, triazolopyridinyl, imide zolopyridinyl, pyrrolopyrimidinyl, and pyrazolopyrimidinyl. Purine bases include, but are not limited to, guanine, adenine, hypoxanthine, 2,6-diaminopurine, and 6-chloropurine. The Formula I-III purine and pyrimidine bases are linked to the ribose sugar, or emsmo analog, through a nitrogen atom in the base. The functional oxygen and nitrogen groups in the base can be protected as needed or desired. Suitable protecting groups are well known to those skilled in the art, and include include trimethylsilyl, dimethyl-hexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl groups, and acyl groups such as acetyl and propionyl, methanesulfonyl, and ptoluenesulfonyl.
[0084] [0084] Unless otherwise specified, the carbon atoms of the compounds of Formula I-IV are intended to have a valence of four. In some representations of chemical structure where carbon atoms do not have a sufficient number of linked variables to produce a valence of four, the remaining carbon substituents needed to provide a valence of four cases assumed to be hydrogen. For example,
[0085] [0085] "Protection group" refers to a fraction of a compound that masks or alters the properties of a functional group or the properties of the compound as a whole. The chemical structure of a protection group varies widely. A function of a protection group is to serve as an intermediary in the synthesis of the parent drug substance. Chemical protection 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. Protection groups are generally used to mask the reactivity of certain functional groups, to aid in the efficiency of reactions desired chemicals, for example, creating and breaking chemical bonds in an orderly and planned way.The protection of functional groups of a compound alters other physical properties in addition to the reactivity of the protected functional group, such as polarity, lipophilicity (hydrophobicity), and other properties which can be measured by common analytical tools. Chemically protected intermediates can themselves be biologically active or inactive.
[0086] [0086] Protected compounds can also present altered properties, and in some cases, optimized in vitro and in vivo, such as passage through cell membranes and resistance to enzymatic degradation or sequestration. In this role, compounds protected with intended therapeutic effects can be referred to as prodrugs. Another function of a protection group is to convert the parent drug into a prodrug, whereby the parent drug is released upon conversion of the prodrug in vivo. Because the active prodrugs are absorbed more effectively than the parent drug, the prodrugs can have greater potency in vivo than the parent drug. The protecting groups are removed in vitro, in the case of chemical intermediates, or in vivo, in the case of prodrugs. With chemical intermediates, it is not particularly important that the resulting products after deprotection, for example, alcohols, are physiologically acceptable, although in general it is more desirable if the products are pharmacologically harmless.
[0087] [0087] “Prodrug fraction” means a labile functional group that separates from the active inhibitory compound during metabolism, systemically, within a cell, by hydrolysis, enzymatic cleavage, or by some other processes (Bundgaard, Hans, “Design and Application of Prodrugs ”in Textbook of Drug Design and Development (1991), P. Krogsgaard-Larsen and H. Bundgaard, Eds. Harwood Academic Publishers, pp. 113-191). Enzymes that are capable of an enzymatic activation mechanism with the prodrug phosphonate compounds of the invention include, among others, amidases, esterases, microbial enzymes, phospholipases, cholinesterases and phosphases. Prodrug fractions can serve to increase solubility, absorption and lipophilicity to optimize drug release, bioavailability and efficacy.
[0088] [0088] A prodrug fraction can include an active metabolite or the drug itself.
[0089] [0089] Exemplary prodrug fractions include the hydrolytically sensitive or labile acyloxymethyl esters -CH2OC (= O) R30 and acyloxymethyl carbonates -CH2OC (= O) OR30 where R30 is C1-C6 alkyl, C1-C6 substituted alkyl, C6 -C20 aryl or C6-C20 substituted aryl. The acyloxyalkyl ester was used as a prodrug strategy for carboxylic acids and then applied to phosphates and phosphonates by Farquhar et al (1983) J. Pharm. Sci. 72: 324; still US patent 4816570, 4968788, 5663159 and 5792756. In certain compounds of the invention, a prodrug fraction is part of a phosphate group. The acyloxyalkyl ester can be used to release phosphoric acids through cell membranes and to increase oral bioavailability. A close variant of the acyloxyalkyl ester, the alkoxycarbonyloxyalkyl (carbonate) ester, can further improve oral bioavailability as a pro-drug fraction in the compounds of the inventive combinations. An exemplary acyloxymethyl ester is pivaloyloxymethoxy, (POM) -CH2OC (= O) C (CH3) 3. A fraction of acyloxymethyl carbonate prodrug is pivaloyloxymethylcarbonate (POC) -CH2OC (= O) OC (CH3) 3.
[0090] [0090] The phosphate group can be a fraction of phosphate prodrug. The prodrug fraction can be sensitive to hydrolysis, such as, among others, those comprising a pivaloyloxymethyl carbonate (POC) or POM group. Alternatively, the prodrug fraction may be sensitive to enzyme-enhanced cleavage, such as a lactate ester or a phosphonamidate ester group.
[0091] [0091] Aryl esters of phosphorous groups, especially phenyl esters, are reported to have improved oral bioavailability (DeLambert et al (1994) J. Med. Chem. 37: 498). Phenyl esters containing a phosphate-ortho carboxylic ester have also been described (Khamnei and Torrence, (1996) J. Med. Chem. 39: 4109-4115). Benzyl esters are reported to generate stop phosphonic acid. In some cases, substituents in the ortho or para position can accelerate hydrolysis. Benzyl analogs with an acylated phenol or an alkylated phenol can generate the phenolic compound through the action of enzymes, for example, esterases, oxidases etc., which in turn cleaves on the benzyl CO bond to generate the phosphoric acid and the intermediate quinone . Examples of this class of prodrugs are described by Mitchell et al (1992) J. Chem. Soc. Perkin Trans. I 2345; Brook et al WO 91/19721. Still other benzyl prodrugs have been described containing a carboxylic ester group attached to benzyl methylene (Glazier et al WO 91/19721). Uncle-containing prodrugs are reported to be useful for the intracellular release of phosphonate drugs. These pro-esters contain an ethylthio group in which the thiol group is esterified with an acyl group or combined with another thiol group to form a disulfide. The deesterification or reduction of the disulfide generates the intermediate thio free which subsequently breaks down to phosphoric acid and episulfide (Puech et al (1993) Antiviral Res., 22: 155-174; Benzaria et al (1996) J. Med. Chem. 39: 4958). Cyclic phosphonate esters have also been described as prodrugs of compounds containing phosphorus (Erion et al, US Patent 6312662).
[0092] [0092] It should be noted that all enantiomers, diastereoisomers, and racemic mixtures, tautomers, polymorphs, pseudopolymorphs of compounds within the scope of Formula I-IV and pharmaceutically acceptable salts thereof are included by the present invention. All mixtures of said enantiomers and diastereoisomers are within the scope of the present invention.
[0093] [0093] A compound of Formula I-IV and its pharmaceutically acceptable salts can exist as different polymorphs or pseudopolymorphs. As used here, crystalline polymorphism means the ability of a crystalline compound to exist in different crystal structures. Crystalline polymorphism can result from differences in crystal packaging (packaging polymorphism) or differences between different conformers in the same molecule (conformational polymorphism). As used here, crystalline pseudopolymorphism means the ability of a hydrate or solvate of a compound to exist in different crystal structures. The pseudopolymorphs of the present invention may exist due to differences in crystal packaging (pseudopolymorphism packaging) or due to differences in packaging between different conformers of the same molecule (conformational pseudopolymorphism). The present invention comprises all polymorphs and pseudopolymorphs of the compounds of Formula I-III and their pharmaceutically acceptable salts.
[0094] [0094] A compound of Formula I-IV and its pharmaceutically acceptable salts may still exist as an amorphous solid. As used here, an amorphous solid is a solid in which there is no large range order of the positions of the atoms in the solid. This definition applies as such when the size of the crystal is two nanometers or less. Additives, including solvents, can also be used to create the amorphous forms of the present invention. The present invention comprises all amorphous forms of the compounds of Formula I-IV and their pharmaceutically acceptable salts.
[0095] [0095] Selected substituents comprising the compounds of Formula I-IV are present in a recursive degree. In this context, "recursive substituent" means that a substituent can recite another case of the same. Due to the recursive nature of said substituents, theoretically, a large number of compounds can be present in any said modality. For example, Rx comprises a substituent Ry. Ry can be R. R can be W3. W3 can be W4 and W4 can be R or comprise substituents comprising Ry. A person skilled in the art of medicinal chemistry understands that the total number of said substituents is reasonably limited by the desired properties of the intended compound. Said properties include, by way of example, and not limitation, physical properties such as molecular weight, solubility or P log, application properties as activity against the intended target, and practical properties such as ease of synthesis.
[0096] [0096] By way of example, and not limitation, W3 and Ry are recursive substituents in certain modalities. Typically, each recursive substituent can 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 in a certain modality. More typically, each recursive substituent can independently occur 12 or less times in a certain embodiment. Even more typically, each recursive substituent can independently occur 3 or less times in a certain embodiment. For example, W3 will occur 0 to 8 times, Ry will occur 0 to 6 times in a certain mode. Even more typically, W3 will occur 0 to 6 times and Ry will occur 0 to 4 times in a certain mode.
[0097] [0097] Recursive substituents are an intended aspect of the invention. A specialist in the medical chemistry technique understands the versatility of said substituents. To the degree that recursive substituents are present in an embodiment of the invention, the total number will be determined as set out above.
[0098] [0098] The modifier “about” used in connection with the quantity is inclusive of the declared value and has the meaning dictated by the context (for example, it includes the degree of error associated with the measurement of the particular quantity).
[0099] [0099] The term “treat”, as used here, unless otherwise indicated, means to reverse, alleviate, inhibit the progress of, or prevent the disorder or condition to which that term applies, or one or more symptoms of said disorder or condition. The term "treatment", as used here, refers to the act of treating, as "treating" is defined immediately above.
[0100] [00100] The term "therapeutically effective amount", as used here, is the amount of Formula I-IV compound present in a composition described here that is required to provide a desired level of the drug in the secretions and tissues of the airways and lungs , or alternatively, into the bloodstream of a subject to be treated to generate an anticipated physiological response or desired biological effect when said composition is administered by the chosen route of administration. The precise amount will depend on several factors, for example, the particular compound of Formula I-IV, the specific activity of the composition, the delivery device employed, the physical characteristics of the composition, its intended use, as well as patient considerations such as severity condition status, patient cooperation, etc., and can be readily determined by a person skilled in the art based on the information provided here.
[0101] [00101] The term "normal saline" means a water solution containing 0.9% (w / v) NaCl.
[0102] [00102] The term "hypertonic saline" means a water solution containing more than 0.9% (w / v) NaCl. For example, 3% hypertonic saline could contain 3% (w / v) NaCl.
[0103] [00103] The compounds of Formula I-IV can comprise a phosphate group such as R7, which can be a pro-drug fraction
[0104] [00104] A W5 heterocycle can be a monocycle containing 3 to 7 members in the ring (2 to 6 carbon atoms and 1 to 3 hetero atoms selected from N, O, P and S) or a bicycle containing 7 to 10 members in the ring (4 to 9 carbon atoms and 1 to 3 hetero atoms selected from N, O, P and S). W5 heterocyclic monocycles can have 3 to 6 ring atoms (2 to 5 carbon atoms and 1 to 2 hetero atoms selected from N, O and S); or 5 or 6 ring atoms (3 to 5 carbon atoms and 1 to 2 hetero atoms selected from N and S). W5 heterocyclic bicycles have 7 to 10 ring atoms (6 to 9 carbon atoms and 1 to 2 hetero atoms selected from N, O, and S) arranged as a bicycle system [4.5], [5.5], [5.6], or [6.6]; or 9 to 10 ring atoms (8 to 9 carbon atoms and 1 to 2 hetero atoms selected from N and S) arranged as a bicycle system [5.6] or [6.6]. The heterocycle W5 can be linked to Y2 via a carbon, nitrogen, sulfur or other atom by a stable covalent bond.
[0105] [00105] W5 heterocycles include, for example, pyridyl, dihydropyridyl, piperidine, pyridazinyl, pyrimidinyl, pyrazinyl, s-triazinyl, oxazolyl, imidazolyl, thiazolyl, isoxazolil, pyrazolyl, isothiazolyl, thyranilyl, furanilil, thyranilyl, furanilil, furanililil, furanililil, furanililil, furanililil, furanililil. W5 also includes, among others, examples such as:
[0106] [00106] W5 carbocycles and heterocycles can be independently substituted with 0 to 3 R groups, as defined above. For example, W5 carbocycles replaced include:
[0107] [00107] Examples of substituted phenyl carbocycles include:
[0108] [00225] Modalities of
[0109] [00226] where each Y2b is, independently, O or N (R). In another aspect of this modality, each Y2b is O and each Rx is independently:
[0110] [00227] where M12c is 1, 2 or 3 and each Y2 is independently a bond, O, CR2, or S. In another aspect of this embodiment, one Y2b-Rx is NH (R) and the other Y2b-Rx is O -Rx where Rx is:
[0111] [00228] where M12c is 2. In another aspect of this modality, each Y2b is O and each Rx is independently:
[0112] [00229] where M12c is 2. In another aspect of this modality, each Y2b is O and each Rx is independently:
[0113] [00230] where M12c is 1 and Y2 is a bond, O or CR2
[0114] [00231] Other modalities of
[0115] [00232] where each Y3 is, independently, O or N (R). In another aspect of this modality, each Y3 is O. In another aspect of this modality, the substructure is:
[0116] [00233] where Ry is W5 as defined here
[0117] [00234] Another form of Formula I-IV includes the substructures:
[0118] [00235] where each Y2c is, independently, O, N (Ry) or S.
[0119] [00236] Another modality of
[0120] [00237] In another aspect of the Formula Ib modality, each Y and Y3 is O. In another aspect of the Formula Ib modality, W1 or W2 is Y2b-Rx; each Y, Y3 and Y 2b is O and Rx is:
[0121] [00238] where M12c is 1, 2 or 3 and each Y2 is independently a bond, O, CR2, or S. In another aspect of Formula Ib, W1 or W2 is Y2b-Rx; each Y, Y3 and Y2b is O and Rx is:
[0122] [00239] where M12c is 2. In another aspect of Formula Ib, W1 or W2 is Y2b-Rx; each Y, Y3 and Y2b is O and Rx is:
[0123] [00240] where M12c is 1 and Y2 is a bond, O or CR2.
[0124] [00241] Another modality of
[0125] [00242] of compounds of Formula I-IV include a substructure:
[0126] [00243] wherein W5 is a carbocycle such as phenyl or substituted phenyl. In another aspect of this modality, the substructure is:
[0127] [00244] where Y2b is O or N (R) and the phenyl carbocycle is replaced by 0 to 3 R groups. In another aspect of this modality of the substructure, Rx is:
[0128] [00245] where M12c is 1, 2 or 3 and each Y2 is independently a bond, O, CR2 or S.
[0129] [00246] Another modality of
[0130] [00247] of Formula I-IV includes substructures:
[0131] [00248] The chiral carbon of the amino acid and lactate fractions can be the R or S configuration or the racemic mixture.
[0132] [00249] Another modality of
[0133] [00250] Another modality of
[0134] [00251] In one aspect of this embodiment, each Rx is, independently, (C1-C8) alkyl. In another aspect of this embodiment, each R x is, independently, C6-C20 aryl or C6-C20 substituted aryl.
[0135] [00252] In a preferred mode,
[0136] [00253] Another modality of
[0137] [00254] The variables used in Tables 20. 1 to 20. 37 have the following definitions: each R21 is independently H or (C1-C8) alkyl; each R22 is independently H, R21, R23 or R24 where each R24 is independently replaced by 0 to 3 R23; each R23 is independently R23a, R23b, R23c or R23d, since when R23 is attached to a heteroatom, then R23 is R23c or R23d; each R23a is independently F, Cl, Br, I, -CN, N3 or -NO2; each R23b is independently Y21; each R23c is independently -R2x, -N (R2x) (R2x), -SR2x, -S (O) R2x, -S (O) 2R2x, -S (O) (OR2x), -S (O) 2 (OR2x ), -OC (= Y21) R2x, -OC (= Y21) OR2x, -OC (= Y21) (N (R2x) (R2x)), -SC (= Y21) R2x, -SC (= Y21) OR2x, -SC (= Y21) (N (R2x) (R2x)), -N (R2x) C (= Y21) R2x, - N (R2x) C (= Y21) OR2x or -N (R2x) C (= Y21) (N (R2x) (R2x)); each R23d is independently -C (= Y21) R2x, -C (= Y21) OR2x or -C (= Y21) (N (R2x) (R2x)); each R2x is independently H, (C1-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, aryl, heteroaryl; or two R2x taken together with a nitrogen to which they are both attached form a 3- to 7-membered heterocyclic ring to which a carbon atom of said heterocyclic ring can optionally be replaced by -O-, -S- or -NR21-; and wherein one or more of the non-terminal carbon atoms of each said (C1-C8) alkyl can be optionally substituted by -O-, -S- or -NR21-; each R24 is independently (C1-C8) alkyl, (C2-C8) alkenyl, or (C2-C8) alkynyl; each R25 is independently R24 where each R24 is replaced by 0 to 3 groups R23; each R25a is independently (C1-C8) alkylene, (C2-C8) alkenylene, or (C2-C8) alkylene any of which is said (C1-C8) alkylene, (C2-C8) alkenylene, or (C2-C8) alkylene is replaced by 0-3 R23 groups; each W23 is independently W24 or W25; each W24 is independently R25, -C (= Y21) R25, -C (= Y21) W25, -SO2R25 or -SO2W25; each W25 is independently carbocycle or heterocycle where W25 is independently substituted by 0 to 3 groups R22; and each Y21 is independently O or S.
[0138] [00255] Rx modalities include esters, carbamates, carbonates, thioesters, amides, thioamides and urea groups:
[0139] Any reference to the compounds of the invention described herein still includes a reference to a physiologically acceptable salt thereof. Examples of physiologically acceptable salts of the compounds of the invention include salts derived from an appropriate base, such as an alkali metal or alkaline earth metal (for example, Na +, Li +, K +, Ca + 2 and Mg + 2), ammonium and NR4 + (in that R is defined here). Physiologically acceptable salts of a nitrogen atom or an amino group include (a) acid addition salts formed with inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acids, phosphoric acid, nitric acid and the like; (b) salts formed with organic acids, such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, isethionic acid, acid lactobionic, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, ptoluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, malonic acid, sulfosalicylic acid, glycolic acid, 2-hydroxy-3-na , salicylic acid, stearic acid, phthalic acid, mandelic acid, lactic acid, ethanesulfonic acid, lysine, arginine, glutamic acid, glycine, serine, threonine, alanine, isoleucine, leucine and the like; and (c) salts formed from elementary anions for example, chlorine, bromine, and iodine. Physiologically acceptable salts of a hydroxy group compound include the anion of said compound in combination with an appropriate cation such as Na + and NR4 +.
[0140] [00257] For therapeutic use, salts of active ingredients of the compounds of the invention will be physiologically acceptable, that is, they will be salts derived from a physiologically acceptable acid or base. However, salts of acids or bases that are not physiologically acceptable can still be used, for example, in the preparation or purification of a physiologically acceptable compound. All salts, whether or not derived from a physiologically acceptable acid or base, are within the scope of the present invention.
[0141] [00258] Finally, it should be understood that the compositions here comprise compounds of the invention in their non-ionized form, as well as the zwitterionic form, and combinations with the stoichiometric amounts of water as in hydrates.
[0142] [00259] The compounds of the invention, exemplified by Formula I-IV can have chiral centers, for example, chiral carbon or phosphorus atoms. The compounds of the invention thus include racemic mixtures of all stereoisomers, including enantiomers, diastereoisomers and atropisomers. In addition, the compounds of the invention include optical isomers enriched or resolved into any of the asymmetric and chiral atoms. In other words, the apparent chiral centers of the descriptions are provided as chiral isomers or racemic mixtures. Both racemic and diastereoisomeric mixtures, as well as individual isolated or synthesized optical isomers, substantially free of their enantiomeric partners or diastereoisomeric partners, are all within the scope of the invention. The racemic mixtures are separated into their individual optically substantially pure isomers by well-known techniques such as, for example, the separation of diastereoisomeric salts formed with optically active adjuncts, for example, acids or bases followed by conversion back to optically active substances. In most cases, the desired optical isomer is synthesized by means of stereospecific reactions, starting with the appropriate stereoisomer of the desired starting material.
[0143] [00260] The term "chiral" refers to molecules that have the non-superimposed property of the specular image partner, while the term "achiral" refers to molecules that are superimposed on their specular image partner.
[0144] [00261] The term "stereoisomers" refers to compounds that have identical chemical constitution, but differ with respect to the arrangement of atoms or groups in space.
[0145] [00262] "Diastereoisomer" refers to a stereoisomer with two or more chirality centers and whose molecules are not mirror images of one another. Diastereoisomers have different physical properties, for example, melting points, boiling points, spectral properties, reactivities and biological properties. For example, compounds of Formula I-IV may have a chiral phosphorus atom when R7 is:
[0146] [00263] and W1 and W2 are different. When at least one of W1 or W2 still has a chiral center, for example, with W1 or W2 is a naturally occurring nitrogen-bound αamino acid ester, then the Formula I-IV compound will exist as diastereoisomers because there are two centers of chirality in the molecule. All said diastereoisomers and their uses described herein are included by the present invention. Mixtures of diastereoisomers can be separated under high resolution analytical procedures such as electrophoresis, crystallization and / or chromatography. Diastereoisomers may have different physical attributes such as, among others, solubility, chemical stability and crystallinity and may also have different biological properties such as, among others, enzymatic stability, absorption and metabolic stability.
[0147] [00264] "Enantiomers" refers to two stereoisomers of a compound that are not specular images not superimposed on one another.
[0148] [00265] The stereochemical definitions and conventions used here generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active forms, that is, they have the ability to rotate the plane of polarized light. In the description of the optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule around its chiral centers. The prefixes del, D and L, or (+) and (-) are used to designate the rotation signal of the light plane polarized by the compound, with S, (-), or 1 meaning that the compound is levorotatory while a compound prefixed with R, (+), or d is dextrorotatory. For a certain chemical structure, these stereoisomers are identical except that they are mirror images of one another. A specific stereoisomer can also be referred to as an enantiomer, and a mixture of said isomers is generally called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which can occur where there is no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.
[0149] [00266] Whenever a compound described here is replaced by more than one of the same group designated, for example, "R" or "R1", then it will be understood that the groups can be the same or different, that is, each group is independently selected. The wavy lines, ﹋, indicate the site of covalent bonding unions of the contiguous substructures, groups, fractions or atoms.
[0150] [00267] The compounds of the invention may still exist as tautomeric isomers in certain cases. Although only a delocalized resonance structure can be described, all of these forms are contemplated within the scope of the invention. For example, eno-amine tautomers may exist for purine, pyrimidine, imidazole, guanidine, amidine, and tetrazole systems and all of their possible tautomeric forms are within the scope of the invention. Methods of inhibiting a Paramyxoviridae polymerase
[0151] [00268] Another aspect of the invention relates to methods of inhibiting the activity of Paramyxoviridae polymerase comprising the step of treating a sample suspected to contain Paramyxoviridae with a composition of the invention.
[0152] [00269] The compositions of the invention can act as inhibitors of Paramyxoviridae polymerase, as intermediates for said inhibitors or have other uses as described below. The inhibitors will bind to sites on the surface or in a Paramyxoviridae polymerase cavity containing a unique geometry to Paramyxoviridae polymerase. Paramyxoviridae polymerase binding compositions can bind with varying degrees of reversibility. Those compounds binding substantially irreversibly are ideal candidates for use in this method of the invention. Once labeled, compositions that bind substantially irreversibly are useful as probes for the detection of Paramyxoviridae polymerase. Thus, the invention relates to methods of detecting Paramyxoviridae polymerase in a sample suspected to contain Paramyxoviridae polymerase comprising the steps of: treating a sample suspected to contain Paramyxoviridae polymerase with a composition comprising a compound of the invention attached to a marker; and observe the effect of the sample on the marker activity. Appropriate markers are well known in the field of diagnosis and include stable free radicals, fluorophores, radioisotopes, enzymes, chemiluminescent groups and chromogens. The compounds here are labeled in a conventional manner using functional groups such as hydroxyl, carboxyl, sulfhydryl or amino.
[0153] [00270] Within the context of the invention, samples suspected of containing Paramyxoviridae polymerase include natural or man-made materials as living organisms; tissue or cell cultures; biological samples as samples of biological material (blood, serum, urine, cerebrospinal fluid, tears, sputum, saliva, tissue samples, and the like); laboratory samples; food, water, or air samples; samples of bioproducts such as cell extracts, particularly recombinant cells synthesizing a desired glycoprotein; and the like. Typically the sample will be suspected to contain an organism that produces Paramyxoviridae polymerase, often a pathogenic organism like a Paramyxoviridae virus. Samples can be contained in any medium including water and organic solvent / water mixtures. The samples include living organisms such as humans, and man-made materials such as cell cultures.
[0154] [00271] The treatment step of the invention comprises adding the composition of the invention to the sample or it comprises adding a precursor of the composition to the sample. The addition step comprises any method of administration as described above.
[0155] [00272] If desired, the activity of Paramyxoviridae polymerase after application of the composition can be observed by any method including direct and indirect methods of detecting the activity of Paramyxoviridae polymerase. Quantitative, qualitative, and semi-quantitative methods of determining the activity of Paramyxoviridae polymerase are all included. Typically one of the screening methods described above is applied, however, any other method such as observing the physiological properties of a living organism is still applicable.
[0156] [00273] Organisms that contain Paramyxoviridae polymerase include the Paramyxoviridae viruses. The compounds of this invention are useful in the treatment or prophylaxis of Paramyxoviridae infections in animals or men.
[0157] [00274] However, in screening compounds capable of inhibiting human Paramyxoviridae viruses, it should be kept in mind that the results of the enzyme assays may not correlate with cell culture assays. Thus, a cell-based assay should be the primary screening tool. Screenings for Paramyxoviridae polymerase inhibitors.
[0158] [00275] The compositions of the invention are screened for inhibitory activity against Paramyxoviridae polymerase by any of the conventional techniques for evaluating the activity of the enzyme. Within the context of the invention, typically compositions are first screened for inhibition of Paramyxoviridae polymerase in vitro and compositions showing inhibitory activity are then iterated for in vivo activity. Compositions containing Ki in vitro (inhibitory constants) of less than about 5 X 10-6 M and preferably less than about 1 X 10-7 M are preferred for in vivo use.
[0159] [00276] Useful in vitro screenings have been described in detail and will not be elaborated here. However, the examples describe appropriate in vitro assays. Pharmaceutical formulations
[0160] [00277] The compounds of this invention are formulated with conventional vehicles and excipients, which will be selected according to ordinary practice. The tablets will contain excipients, glidants, fillers, binders and the like. Aqueous formulations are prepared in a sterile form, and when intended for delivery by another than oral administration they will generally be isotonic. All formulations will optionally contain excipients such as those set out in "Handbook of Pharmaceutical Excipients" (1986). Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextran, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and the like. The pH of a formulation varies from about 3 to about 11, but it is usually about 7 to 10.
[0161] [00278] Although it is possible for the active ingredients to be administered alone it may be preferable to present them as pharmaceutical formulations. A formulation, for both veterinary and human use, of the invention comprises at least one active ingredient, as defined above, together with one or more acceptable carriers thereof and optionally other therapeutic ingredients, particularly the additional therapeutic ingredients as discussed here. The vehicles must be "acceptable" in the sense of being compatible with other ingredients of a formulation and physiologically innocuous of the recipient.
[0162] [00279] Formulations those suitable for the previous routes of administration. The formulations can conveniently be presented in unit dosage form and can be prepared by any of the methods known in the pharmacy art. Techniques and formulations are generally found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). Said methods include the step of bringing in association the active ingredient with the vehicle that constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing the active ingredient in association with liquid vehicles or finely divided solid vehicles or both and then, if necessary, shaping the product.
[0163] [00280] The formulations of the present invention suitable for oral administration can be presented as capsules as discrete units, capsules or tablets, each containing a predetermined amount of the active ingredient; as a granule or powder; as a solution or suspension in an aqueous or non-aqueous liquid; or as a liquid oil-in-water emulsion or a liquid water-in-oil emulsion. The active ingredient can also be administered as a bolus, eletuary or paste.
[0164] [00281] A tablet is made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compression, in a suitable machine, the active ingredient in the form of free flow as a powder or granules, optionally mixed with a binder, inert diluent, lubricant, preservative, active surface or dispersing agent. Molded tablets can be prepared by molding in an appropriate machine, a mixture of the powdered active ingredient moistened with an inert liquid diluent. The tablets can optionally be coated or labeled and are optionally formulated to provide slow or controlled release of the active ingredient thereof.
[0165] [00282] For infections of the eye or other external tissues, for example, mouth and skin, the formulations are preferably applied as a topical ointment or cream containing active ingredients in an amount of, for example, 0.075 to 20% w / w (including active ingredients in a range between 0.1% and 20% in increments of 0.1% w / w such as 0.6% w / w, 0.7% w / w etc.), preferably 0.2 to 15% w / w and more preferably 0.5 to 10% w / w. When formulated in an ointment, the active ingredients can be used with a paraffinic or water-miscible ointment base. Alternatively, the active ingredients can be formulated into a cream with a base of oil cream in water.
[0166] [00283] If desired, the aqueous phase of the cream base may include, for example, at least 30% w / w of a polyhydric alcohol, that is, an alcohol containing two or more hydroxyl groups such as propylene glycol, butane 1,3- diol, mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400) and mixtures thereof. Topical formulations may desirably include a compound that enhances the absorption or penetration of the active ingredient through the skin or other affected areas. Examples of said skin penetration enhancers include dimethyl sulfoxide and related analogs.
[0167] [00284] The oil phase of the emulsions of this invention can consist of known ingredients in a known way. While the phase may include only one emulsifier (also known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included along with a lipophilic emulsifier, which acts as a stabilizer. It is also preferred to include an oil and a fat. Together, emulsifiers with or without stabilizers make up the so-called emulsifying wax and the wax together with the oil and fat make up the so-called emulsifying ointment base that forms the oily dispersed phase of cream formulations.
[0168] [00285] Emulsifiers and emulsion stabilizers, suitable for use in a formulation of the invention include Tween® 60, Span® 80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.
[0169] [00286] The choice of suitable fats or oils for a formulation is based on achieving the desired cosmetic properties. The cream should preferably be a non-greasy product, which does not stain and washable with the appropriate consistency to prevent the escape of tubes or other containers. Straight or branched chain dibasic or monobasic alkyl esters such as diisoadipate, isocetyl stearate, coconut fatty acid diester, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a mixture of Known branched esters known as Crodamol CAP can be used, the latter three being preferred esters. These can be used alone or in combination, depending on the properties required. Alternatively, high-melting lipids such as white soft paraffin and / or liquid paraffin or other mineral oils are used.
[0170] [00287] The pharmaceutical formulations according to the present invention comprise a combination according to the invention together with one or more pharmaceutically acceptable vehicles or excipients and optionally other therapeutic agents. Pharmaceutical formulations containing the active ingredient can be any form suitable for the intended method of administration. When used for oral use, for example, tablets, troches, lozenges, aqueous or oily suspensions, granules or dispersible powders, emulsions, hard or soft capsules, syrups or elixirs can be prepared. Compositions intended for oral use can be prepared according to any method known in the art of making pharmaceutical compositions and said compositions may contain one or more agents including sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable preparation. Tablets containing the active ingredient in the mixture with a non-toxic pharmaceutically acceptable excipient that are suitable for making tablets are acceptable. These excipients can be, for example, inert diluents, such as calcium or sodium carbonate, lactose, calcium or sodium phosphate; granulating and disintegrating agents, such as corn starch, or alginic acid; binding agents, such as starch, acacia or gelatin; and lubricating agents, such as magnesium stearate, stearic acid or talc. The tablets can be uncoated or coated by techniques known as microencapsulation to delay disintegration and absorption in the gastrointestinal tract and thus provide a sustained action for a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax can be employed.
[0171] [00288] Formulations for oral use can also be presented as hard gelatin capsules, where the active ingredient is mixed with an inert solid diluent, for example, calcium phosphate or kaolin, or as soft gelatin capsules, in which the ingredient The active ingredient is mixed with water or an oily medium, such as peanut oil, liquid paraffin or olive oil.
[0172] [00289] The aqueous suspensions of the invention contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Said excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginates, polyvinylpyrrolidone, tragacanth and acacia gum, and dispersing or wetting agents such as a naturally occurring phosphatide (eg, lecithin), a product condensation of an alkylene oxide with a fatty acid (for example, polyoxyethylene stearate), a condensation product of ethylene oxide with a long-chain aliphatic alcohol (for example, heptadecaethylene-oxymethanol), an oxide condensation product ethylene with a partial ester derived from a fatty acid and a hexitol anhydride (for example, polyoxyethylene sorbitan monooleate). The aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl phydroxy-benzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents such as sucrose or saccharin.
[0173] [00290] Oil suspensions can be formulated by suspending the active ingredient in a vegetable oil, arachis oil, olive oil, coconut oil or sesame oil, or in a mineral oil such as liquid paraffin. Oral suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set out above, and flavoring agents can be added to provide a palatable oral preparation. These compositions can be preserved by the addition of an antioxidant such as ascorbic acid.
[0174] [00291] Dispersible powders and granules of the invention suitable for the preparation of an aqueous suspension by the addition of water provide the active ingredient in the mixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those described above. Additional excipients, for example, sweeteners, flavors and coloring agents, may be present.
[0175] [00292] The pharmaceutical compositions of the invention can be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil, such as olive oil, arachis oil, a mineral oil, such as liquid paraffin, or a mixture of these. Suitable emulsifying agents include naturally occurring gums, such as gum tragacanth and acacia gum, naturally occurring phosphatides, such as soy lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion can also contain sweetening and flavoring agents. Syrups and elixirs can be formulated with sweetening agents such as glycerol, sorbitol or sucrose. Said formulations can contain an emollient, a preservative, a flavoring agent or a dye.
[0176] [00293] The pharmaceutical compositions of the invention may be in the form of a sterile injectable preparation, such as a sterile injectable oil or aqueous suspension. This suspension can be formulated according to the known technique using those suitable dispersing or wetting agents that have been mentioned above. The sterile injectable preparation can also be a suspension or sterile injectable solution in a parenterally non-toxic diluent or solvent, such as a solution in 1,3-butane diol or prepared as a lyophilized powder. Among the appropriate vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, conventionally sterile fixed oils can be used as a solvent or suspending medium. For this purpose, any soft-fixed oil can be used including synthetic di- or monoglycerides. In addition, fatty acids, such as oleic acid, likewise, can be used in the preparation of injectables.
[0177] [00294] The amount of active ingredient that can be combined with the carrier material to produce a single dosage form will vary depending on the treated host and the particular mode of administration. For example, a gradual release formulation for oral administration to humans can contain approximately 1 to 1000 mg of active material, combined with an adequate and convenient amount of support material that can vary from about 5 to about 95% of the total compositions (weight: weight). The pharmaceutical composition can be prepared to provide the easily measurable amount for administration. For example, an aqueous solution for intravenous infusion may contain about 3 to 500 g of the active ingredient per milliliter of solution solution in the order that infusion of an appropriate volume at a rate of approximately 30 mL / hr can occur.
[0178] [00295] Formulations suitable for topical administration to the eye also include eye drops in which the active ingredient is dissolved or suspended in an appropriate vehicle, especially an aqueous solvent for the active ingredient. The active ingredient is preferably present in said formulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10%, and particularly about 1.5% w / w.
[0179] [00296] Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored base, usually sucrose and acacia or tragacanth; lozenges comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in an appropriate liquid carrier.
[0180] [00297] Formulations for rectal administration can be presented as a suppository with an appropriate base comprising, for example, cocoa butter or a salicylate.
[0181] [00298] Formulations suitable for intrapulmonary or nasal administration have a particle size, for example, in the range of 0.1 to 500 microns, such as 0.5, 1, 30, 35 etc., which is administered by rapid inhalation through nasal passage or by inhalation through the mouth to reach the alveolar sacs. Appropriate formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for the administration of aerosol or dry powder can be prepared according to conventional methods and can be released with other compound therapeutic agents as compounds hitherto used in the treatment or prophylaxis of Paramyxoviridae infections as described below.
[0182] [00299] In another aspect, the invention is a new composition, effective, safe, non-irritating and physiologically compatible comprising a compound of Formula I-IV, or a pharmaceutically acceptable salt thereof, suitable for the treatment of potentially paramyxoviridae infections and bronchiolitis associated. Preferred pharmaceutically acceptable salts are salts of inorganic acids including hydrochloride, hydrobromide, sulfate or phosphate salts as they may cause with less pulmonary irritation. Preferably, the inhalable formulation is released into the endobronchial space in an aerosol comprising particles with a median mass aerodynamic diameter (MMAD) between about 1 and about 5 µm. Preferably, the Formula I-IV compound is formulated for aerosol delivery using a nebulizer, pressurized metered dose inhaler (pMDI), or dry powder inhaler (DPI).
[0183] [00300] Using non-limiting nebulizers include nebulizers by atomization, jet, ultrasonic, pressurized, vibrating porous plate, or equivalents including those nebulizers using adaptive aerosol delivery technology (Denyer, J. Aerosol medicine pulmonary drug delivery 2010, 23 supp 1, S1-S10). A jet nebulizer uses air pressure to break a liquid solution into aerosol droplets. An ultrasonic nebulizer works by a piezoelectric crystal that shears a liquid into small aerosol droplets. A pressurized nebulization system forces the solution under pressure through small pores to generate aerosol droplets. A porous vibrating plate device uses rapid vibration to shear a flow of liquid into appropriate droplet sizes.
[0184] [00301] In a preferred embodiment, the nebulizer formulation is released into the endobronchial space in an aerosol comprising particles with a MMAD predominantly between about 1 µm to about 5 µm using a nebulizer capable of aerosolizing a formulation of the Formula I-IV compound in MMAD particles required. To be optimally therapeutically effective and to avoid upper and systemic respiratory side effects, most aerosol particles should not have an MMAD greater than about 5 µm. If an aerosol contains a large number of particles with an MMAD greater than 5 µm, the particles are deposited in the upper airways, decreasing the amount of drug released to the site of inflammation and bronchoconstriction in the lower respiratory tract. If the aerosol MMAD is less than about 1 µm, the particles have a tendency to remain suspended in the inhaled air and are subsequently exhaled during exhalation.
[0185] [00302] When formulated and released according to the method of the invention, the aerosol formulation for nebulization offers an effective therapeutic dose of the compound of Formula I-IV to the site of the Paramyxoviridae infection sufficient to treat the Paramyxoviridae infection The amount of drug administered should be adjusted to reflect the efficiency of delivering an effective therapeutic dose of the Formula I-IV compound. In a preferred embodiment, a combination of the aqueous aerosol formulation with the spray nebulizer, jet, pressurized, vibrating porous plate, or ultrasonic, allows, depending on the nebulizer, about at least 20 to about 90%, usually about 70% release of the administered dose of the compound of Formula I-IV into the airways. In a preferred embodiment, at least about 30 to about 50% of the active compound is released. More preferably, about 70 to about 90% of the active compound is released.
[0186] [00303] In another embodiment of the present invention, a compound of Formula I-IV or a pharmaceutically acceptable salt thereof, is released as an inhalable dry powder. The compounds of the invention are administered endobronchially as a dry powder formulation to release fine fine particles of the compound into the endobronchial space using dry powder or metered dose inhalers. For DPI release, the Formula I-IV compound is processed into particles with, predominantly, MMAD between about 1 µm to about 5 µm by spray drying milling, critical fluid processing, or solution precipitation. Media grinding, jet grinding and spray drying devices and procedures capable of producing particle sizes with an MMAD between about 1 µm to about 5 µm are well known in the art. In one embodiment, excipients are added to the Formula I-IV compound before processing into particles of the required sizes. In another embodiment, excipients are mixed with particles of the required size to assist in the dispersion of drug particles, for example, using lactose as an excipient.
[0187] [00304] Particle size determinations are made using devices known in the art. For example, a multi-stage Anderson cascade impactor or another method such as those specifically cited in US Pharmacopoeia Chapter 601 as devices featuring aerosols within dry powder and metered dose inhalers.
[0188] [00305] In another preferred embodiment, a Formula I-IV compound is released as a dry powder, using a device such as a dry powder inhaler from other dry powder dispersion devices. Non-limiting examples of dry powder inhalers and devices include those described in US 5,458,135; US 5,740,794; US 5775320; US 5,785,049; US 3,906,950; US 4,013,075; US 4,069,819; US 4,995,385; US 5,522,385; US 4,668,218; US 4,667,668; US 4,805,811 and US 5,388,572. There are two main designs of dry powder inhalers. A design is a measuring device in which a reservoir for the drug is placed inside the device and the patient adds a dose of the drug to the inhalation chamber. The second design is a device measured at the factory in which each individual dose was made in a separate container. Both systems depend on a drug formulation in small 1µm MMAD particles and about 5 µm often involve co-formulation with larger excipient particles such as, among others, lactose. The powdered drug is placed in the inhalation chamber (either by measuring device or by breaking a dose measured at the factory) and the patient's inspiratory flow accelerates the powder out of the device and into the oral cavity. The non-laminar flow characteristics of the powder path cause the excipient-drug aggregates to decompose, and the mass of the large excipient particles causes their impaction at the back of the throat, while the smallest drug particles are deposited deeply in the lungs. In preferred embodiments, a Formula I-IV compound, or a pharmaceutically acceptable salt thereof, is released as a dry powder using the type of dry powder inhaler as described here, wherein the dry powder MMAD, exclusive of any excipient, it is predominantly in the range of 1 µm to about 5 µm.
[0189] [00306] In another preferred embodiment, a compound of Formula I-IV is supplied as a dry powder using a metered dose inhaler. Non-limiting examples of metered dose inhalers and devices include those described in US 5,261,538; US 5,544,647; US 5,622,163; US 4,955,371; US 3,565,070; US 3,361,306 and US 6,116,234. In preferred embodiments, a Formula I-IV compound, or a pharmaceutically acceptable salt thereof, is released as a dry powder using a type of dry powder inhaler in which the dry powder MMAD, exclusive of any excipient, is predominantly in the range of about 1-5 µm.
[0190] [00307] Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient of said vehicles as they are known in the art to be suitable.
[0191] [00308] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostats and solutes that process an isotonic formulation with the intended recipient's blood; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickeners.
[0192] [00309] The formulations are presented in multiple dose or unit dose containers, for example, sealed ampoules and vials and can be stored in a lyophilized (lyophilized) condition, requiring only the addition of the liquid sterile vehicle, for example, water for injection , immediately before use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the type described above. Preferred unit dosage formulations are those that contain a daily unit dose or daily sub-dose, as recited above, or an appropriate fraction of the active ingredient.
[0193] [00310] It should be understood that in addition to particularly the ingredients mentioned above as formulations of this invention, it may include other conventional agents of the art, taking into account the type of formulation in question, for example, those suitable for oral administration may include flavoring agents.
[0194] [00311] The invention further provides veterinary compositions comprising at least one active ingredient, as defined above together with a veterinary vehicle thereof.
[0195] [00312] Veterinary vehicles are useful for the purpose of administering the composition and can be solid, liquid or gaseous materials that are otherwise inert or acceptable in the veterinary technique and are compatible with the active ingredient. These veterinary compositions can be administered orally, parenterally or any other desired route.
[0196] [00313] The compounds of the invention are used to provide controlled-release pharmaceutical formulations containing as active ingredient one or more compounds of the invention ("controlled-release formulations") in which the release of the active ingredient is controlled and regulated to allow less dosage of frequency or to improve the pharmacokinetic or toxicity profile of a certain active ingredient.
[0197] [00314] The effective dose of the active ingredient depends at least on the nature of the condition being treated, toxicity, whether the compound is being used prophylactically (lower doses) or against an active viral infection, the method of release and pharmaceutical formulation and will be determined by the doctor using conventional dose escalation studies. It can be expected to be about 0.0001 to about 100 mg / kg body weight per day; typically from about 0.01 to about 10 mg / kg body weight per day; more typically, from about, 01 to about 5 mg / kg body weight per day; more typically, from about 0.5 to about 0.5 mg / kg body weight per day. For example, the daily candidate dose for an adult human of approximately 70 kg of body weight will vary from 1 mg to 1000 mg, preferably between 5 mg and 500 mg, and can take the form of single or multiple doses. Routes of administration
[0198] [00315] One or more compounds of the invention (referred to herein as the active ingredients) are administered by any route appropriate to the condition being treated. Suitable routes include oral, rectal, nasal, pulmonary, topical (including sublingual and buccal), vaginal and parenteral (including intrathecal, subcutaneous, intramuscular, intravenous, intradermal and epidural), and the like. It will be appreciated that the preferred route may vary, for example, with the condition of the beneficiary. An advantage of the compounds of the present invention is that orally they are bioavailable and can be dosed orally. Combination Therapy
[0199] [00316] The compositions of the invention are still used in combination with other active ingredients. For the treatment of Paramyxoviridae virus infections, preferably the other active therapeutic agent is active against Paramyxoviridae virus infections, particularly respiratory syncytial virus infections and / or parainfluenza virus infections. Non-limiting examples of these other active therapeutic agents are ribavirin, palivizumab, motavizumab, RSV-IGIV (RespiGam®), MEDI-557, A-60444, MDT637, BMS-433771, and mixtures thereof.
[0200] [00317] Many of the Paramyxoviridae virus infections are respiratory infections. Therefore, additional active therapies used to treat respiratory symptoms and sequelae of infection can be used in combination with the compounds of Formula I-IV. Additional agents are preferably administered orally or by direct inhalation. For example, other additional preferred therapeutic agents in combination with the compounds of Formula I-IV, for the treatment of viral respiratory infections include, but are not limited to, bronchodilators and corticosteroids.
[0201] [00318] Glucocorticoids, which were first introduced as an asthma therapy in 1950 (Carryer, Jornal of Allergy, 21, 282-287, 1950), remain the most potent and consistently effective therapy for this disease, although its mechanism of action is not yet fully understood (Morris, J. Allergy Clin. Immunol., 75 (1 Pt) 1-13, 1985). Unfortunately, oral glucocorticoid therapies are associated with profound undesirable side effects such as trunk obesity, hypertension, glaucoma, glucose intolerance, accelerated cataract formation, bone mineral loss, and psychological effects, all of which limit their use as therapeutic agents. long-term (Goodman and Gilman, 10th edition, 2001). One solution to systemic side effects is to release steroid drugs directly to the site of inflammation. Inhaled corticosteroids (ICS) were developed to mitigate the serious adverse effects of oral steroids. Non-limiting examples of corticosteroids that can be used in combinations with the compounds of Formula I-IV are dexamethasone, dexamethasone sodium phosphate, fluorometolone, fluorometolone acetate, loteprednol, loteprednol etabonate, hydrocortisone, prednisolone, fludrocortone, triamcinolone, triamcinolone, triamcinolone, triamcinolone, triamcinolone, triamcinolone, beclomethasone diproprionate, methylprednisolone, fluocinolone, fluocinolone acetonide, flunisolide, fluocortin-21-butylate, flumetasone, flumetasone pivalate, budesonide, halobetasol propionate, mometasone furoate, fluticasone propionate, ciclesonide; or pharmaceutically acceptable salts thereof.
[0202] [00319] Other anti-inflammatory agents, working through cascading anti-inflammatory mechanisms are also useful as additional therapeutic agents in combination with the compounds of Formula I-IV, for the treatment of viral respiratory infections. Applying "anti-inflammatory signal transduction modulators" (referred to in this text as AISTM), as phosphodiesterase inhibitors (for example, specific PDE-4, PDE-5 or PDE-7), transcription factor inhibitors (for example, blocking NFκB by inhibiting IKK), or kinase inhibitors (for example, blocking P38 MAP, JNK, PI3K, EGFR or Syk) is a logical approach to shutting down inflammation as these small molecules target a limited number of common intracellular pathways - those signal transduction pathways that are critical points for anti-inflammatory therapeutic intervention (see review by PJ Barnes, 2006). These additional non-limiting therapeutic agents include: 5- (2,4-difluoro-phenoxy) -1-isobutyl-1H-indazol-6-carboxylic acid (2-dimethylamino-ethyl) -amide (P38 Map kinase inhibitor ARRY797); 3-cyclopropylmethoxy-N- (3,5-dichloro-pyridin-4-yl) -4-difluoromethoxy-benzamide (PDE-4 inhibitor Roflumilast); 4- [2- (3-cyclopentyloxy-4-methoxyphenyl) -2-phenylethyl] -pyridine (PDE-4 inhibitor CDP-840); N- (3,5-dichloro-4-pyridinyl) -4- (difluoromethoxy) -8 - [(methylsulfonyl) amino] -1-dibenzofurancarboxamide (PDE-4 Oglemilast inhibitor); N- (3,5-dichloro-pyridin-4-yl) -2- [1- (4-fluorbenzyl) -5-hydroxy1H-indol-3-yl] -2-oxo-acetamide (PDE-4 AWD inhibitor 12-281); 8-methoxy-2-trifluormethyl-quinoline-5-carboxylic acid (3,5-dichloro-1-oxy-pyridin-4-yl) -amide (PDE-4 Sch 351591 inhibitor); 4- [5- (4-fluorophenyl) -2- (4-methanesulfinylphenyl) -1H-imidazol-4-yl] -pyridine (P38 SB-203850 inhibitor); 4- [4- (4-fluorophenyl) -1- (3-phenyl-propyl) -5-pyridin-4-yl-1H-imidazol-2-yl] -but-3-in-1-ol (inhibitor of P38 RWJ-67657); 2-diethylamino-ethyl ester of 4-cyano-4- (3-cyclopentyloxy-4-methoxy-phenyl) -cyclohexanecarboxylic acid (Cilomilast 2-diethyl-ethyl ester prodrug, PDE-4 inhibitor); (3-chloro-4-fluorophenyl) - [7-methoxy-6- (3-morpholin-4-yl-propoxy) -quinazolin-4-yl] -amine (Gefitinib, EGFR inhibitor); and 4- (4-methyl-piperazin-1-ylmethyl) -N- [4-methyl-3- (4-pyridin-3-yl-pyrimidin-2-ylamino) -phenyl] -benzamide (Imatinib, EGFR inhibitor ).
[0203] [00320] Combinations comprising inhaled β2-adrenoreceptor agonist bronchodilators such as formoterol, albuterol or salmeterol with the compounds of Formula I-IV are also suitable combinations, among others, useful for the treatment of viral respiratory infections.
[0204] [00321] Combinations of inhaled β2-adrenoreceptor agonist bronchodilators such as formoterol or salmeterol with ICS are also used to treat bronchoconstriction and inflammation (Symbicort® and Advair®, respectively). Combinations comprising these combinations of ICS and β2-adrenoreceptor agonist together with the compounds of Formula I-IV are also suitable, but, among others, combinations useful for the treatment of viral respiratory infections.
[0205] [00322] For the treatment or prophylaxis of pulmonary bronchoconstriction, anticholinergics are of potential use and therefore useful as additional therapeutic agents in combination with the compounds of Formula I-IV, for the treatment of viral respiratory infections. These anticholinergics include, among others, antagonists of the muscarinic receptor (particularly of the M3 subtype) that have demonstrated therapeutic efficacy in men for the control of cholinergic tone in COPD (Witek, 1999); 1- {4-hydroxy-1- [3,3,3-tris- (4-fluorophenyl) -propionyl] -pyrrolidine-2-carbonyl} -pyrrolidine-2-carboxylic acid (1-methyl-piperidin-4-ylmethyl ) -amide; 3- [3- (2-diethylamino-acetoxy) -2-phenyl-propionyloxy] -8-isopropyl-8-methyl-8-azonia-bicyclo [3.2.1] octane (IpratropiumN, N-diethylglycinate); 1-aza-bicyclo [2.2.2] oct-3-yl ester of 1-cyclohexyl-3,4-dihydro-1H-isoquinoline-2-carboxylic acid (Solifenacin); 1-aza-bicyclo [2.2.2] oct-3-yl ester of 2-hydroxymethyl-4-methanesulfinyl-2-phenyl-butyric acid (Revatropate); 2- {1- [2- (2,3-dihydro-benzofuran-5-yl) -ethyl] -pyrrolidin-3-yl} -2,2-diphenyl-acetamide (Darifenacin); 4-azepan-1-yl-2,2-diphenyl-butyramide (Buzepide); 7- [3- (2-diethylamino-acetoxy) -2-phenyl-propionyloxy] -9-ethyl-9-methyl-3-oxa-9-azoniatricycle [3.3.1.02,4] nonane (Oxitrope-N, N- diethylglycinate); 7- [2- (2-diethylaminoacetoxy) -2,2-di-thiophen-2-yl-acetoxy] -9,9-dimethyl-3-oxa-9-azoniatricycle [3.3.1.02,4] nonane (Tiotropium- N, N-diethylglycinate); 2- (3-diisopropylamino-1-phenyl-propyl) -4-methyl-phenyl ester of dimethylamino-acetic acid (Tolterodine-N, N-dimethylglycinate); 3- [4,4-bis- (4-fluorophenyl) -2-oxo-imidazolidin1-yl] -1-methyl-1- (2-oxo-2-pyridin-2-yl-ethyl) -pyrrolidinium; 1- [1- (3-fluoro-benzyl) -piperidin-4-yl] -4,4-bis- (4-fluorophenyl) -imidazolidin-2-one; 1-cyclooctyl-3- (3-methoxy-1-aza-bicyclo [2.2.2] oct-3-yl) -1-phenyl-prop-2-in-1-ol; 3- [2- (2-diethylaminoacetoxy) -2,2-di-thiophen-2-yl-acetoxy] -1- (3-phenoxy-propyl) -1-azoniabicyclo [2.2.2] octane (Aclidinio-N, N-diethylglycinate); or 1-methyl-1- (2-phenoxy-ethyl) -piperidin-4-yl ester of (2-diethylamino-acetoxy) -di-thiophen-2-yl-acetic acid.
[0206] [00323] The compounds of Formula I-IV can also be combined with mucolytic agents to treat the infection and the symptoms of respiratory infections. A non-limiting example of a mucolytic agent is ambroxol. Similarly, compounds of Formula I-IV can be combined with expectorant agents to treat infection and the symptoms of respiratory infections. A non-limiting example of an expectorant is guaifenesin.
[0207] [00324] Nebulized hypertonic saline is used to improve the immediate and long-term clearance of small airways in patients with lung diseases (Kuzik, J. Pediatrics 2007, 266). The Formula I-IV compounds can also be combined with nebulized hypertonic saline particularly when the infection of Paramyxoviridae virus is complicated with bronchiolitis. The combination of the Formula I-IV compounds with hypertonic saline may also include any of the additional agents discussed above. In a preferred aspect, nebulized about 3% hypertonic saline is used.
[0208] [00325] It is also possible to combine any compound of the invention with one or more additional active therapeutic agents in a unit dosage form for simultaneous or sequential administration to the patient. Combination therapy can be administered as a sequential or simultaneous regimen. When administered sequentially, the combination can be administered in two or more administrations.
[0209] [00326] Co-administration of a compound of the invention with one or more other active therapeutic agents generally refers to the simultaneous or sequential administration of a compound of the invention and one or more other active therapeutic agents, so that the therapeutically effective amount of the compound of invention and one or more other active therapeutic agents are present in the patient's body.
[0210] [00327] Co-administration includes administration of unit dosages of the compounds of the invention before or after administration of unit dosages of one or more other active therapies, for example, administration of compounds of the invention within seconds, minutes or hours of administration of an or more other active therapeutic agents. For example, a unit dose of a compound of the invention can be administered first, followed by seconds or minutes by administering a unit dose of one or more other active therapeutic agents. Alternatively, a unit dose of one or more other therapeutic agents can be administered first, followed by administration of a unit dose of a compound of the invention within seconds or minutes. In some cases, it may be desirable to administer a unit dose of a compound of the invention first, followed, after a period of hours (e.g., 1-12 hours), by administration of a unit dose of one or more other therapeutic agents . In other cases, it may be desirable to administer a unit dose of one or more other active therapeutic agents first, followed, after a period of hours (eg 1-12 hours), by administration of a unit dose of a compound of invention.
[0211] [00328] The combination therapy can provide "synergy" and "synergistic" ie the effect obtained when the active ingredients used together is greater than the sum of the effects that result from the use of compounds separately. A synergistic effect can be achieved when the active ingredients are: (1) co-formulated and administered or released simultaneously in a combined formulation; (2) released by alternation or in parallel as separate formulations; or (3) by some other regime. When released in alternation therapy, a synergistic effect can be achieved when the compounds are administered or released sequentially, for example, in separate tablets, capsules or pills, or by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, that is, serially, whereas in combination therapy, effective doses of two or more active ingredients are administered together. An antiviral synergistic effect denotes an antiviral effect that is greater than the purely expected additive effects of the individual compounds in the combination.
[0212] [00329] In yet another embodiment, the present application provides methods of inhibiting Paramyxoviridae polymerase in a cell, comprising: contacting an HCV-infected cell with an effective amount of a Formula I-IV compound, or a salt, solvate and / or pharmaceutically acceptable ester thereof, through which Paramyxoviridae polymerase is inhibited.
[0213] [00330] In yet another embodiment, the present application provides methods of inhibiting Paramyxoviridae polymerase in a cell, comprising: contacting an HCV-infected cell with an effective amount of a compound of Formula I-IV, or a salt, solvate and / or pharmaceutically acceptable ester thereof, and at least one additional therapeutic agent by which Paramyxoviridae polymerase is inhibited.
[0214] [00331] In yet another embodiment, the present application provides methods of inhibiting Paramyxoviridae polymerase in a cell, comprising: contacting a cell infected with Paramyxoviridae virus with an effective amount of a compound of Formula I-IV, or a salt, solvate and / or pharmaceutically acceptable ester thereof, and at least one additional therapeutic agent selected.
[0215] [00332] In yet another embodiment, the present application provides methods for treating a Paramyxoviridae virus infection in a patient, comprising: administering to the patient a therapeutically effective amount of a Formula I-IV compound, or a salt, solvate and / or pharmaceutically acceptable ester even.
[0216] [00333] In yet another embodiment, the present application provides methods for treating a Paramyxoviridae virus infection in a patient, comprising: administering to the patient a therapeutically effective amount of a compound of Formula I-IV, or a salt, solvate and / or pharmaceutically acceptable ester itself, and at least one additional therapeutic agent, by means of which the Paramyxoviridae polymerase is inhibited.
[0217] [00334] In yet another embodiment, the present application provides methods for treating a Paramyxoviridae virus infection in a patient, comprising: administering to the patient a therapeutically effective amount of a compound of Formula I-IV, or a salt, solvate and / or pharmaceutically acceptable ester, and at least one additional therapeutic agent. Metabolites of the compounds of the invention
[0218] [00335] Also included within the scope of this invention are the in vivo metabolic products of the compounds described here, insofar as said products are new and not obvious about the prior art. Said products can result, for example, from the oxidation, reduction, hydrolysis, amidation, esterification and the like of the administered compound, mainly due to enzymatic processes. Thus, the invention includes new and non-obvious compounds produced by a process comprising contacting a compound of this invention with a mammal for a period of time sufficient to produce a metabolic product therefrom. Said products are typically identified by the preparation of a radiolabeling compound (for example, 14C or 3H) of the invention, administering this parenteral route at a detectable dose (for example, more than about 0.5 mg / kg) to an animal such as rat, mouse, guinea pig, monkey, or a human being, leaving enough time for metabolism to occur (typically about 30 seconds to 30 hours) and isolation of their conversion products from urine, blood or other biological samples. These products are easily isolated, once they are labeled (others are isolated by the use of antibodies capable of binding to epitopes surviving in the metabolite). The metabolite structures are determined in a conventional manner, for example, by MS or NMR analysis. In general, the analysis of metabolites is done in the same way as studies of metabolism of conventional drugs known to those skilled in the art. Conversion products, provided they are not otherwise found in vivo, are useful in diagnostic assays for the therapeutic dosage of the compounds of the invention, even if they have no HCV polymerase inhibitory activity alone.
[0219] [00336] Recipes and methods for determining the stability of compounds in gastrointestinal secretions of substitutes are known. The compounds are defined here as stable in the gastrointestinal tract where less than about 50 mol percent of protected groups are deprotected in intestinal substitute or gastric juice after incubation for 1 hour at 37 ° C. Just because the compounds are stable in the gastrointestinal tract does not mean that they cannot be hydrolyzed in vivo. The prodrugs of the invention will typically be stable in the digestive system, but can be substantially hydrolyzed to the parent drug in the digestive lumen, liver or other metabolic organs, or within cells in general. Examples
[0220] [00337] Certain abbreviations and acronyms are used to describe experimental details. Although most of these would be understood by a person skilled in the art, Table 1 contains a list of many of these abbreviations and acronyms.
[0221] [00338] Ethyl alanine ester hydrochloride salt (1.69 g, 11 mmol) was dissolved in anhydrous CH2Cl2 (10 mL) and the mixture stirred with cooling to 0 ° C under N2 (g). Phenyl dichlorophosphate (1.49 mL, 10 mmol) was added followed by addition under a drop of Et3N for 10 min. The reaction mixture was then slowly heated to RT and stirred for 12 h. Anhydrous Et2O (50 mL) was added and the mixture stirred for 30 min. The solid that formed was removed by filtration, and the filtrate concentrated under reduced pressure. The residue was subjected to silica gel chromatography eluting with 0-50% EtOAc in hexanes to generate intermediate A (1.13 g, 39%).
[0222] [00339] 1H NMR (300 MHz, CDCl3) δ 7.39-7.27 (m, 5H), 4.27 (m, 3H), 1.52 (m, 3H), 1.32 (m, 3H ).
[0223] [00340] 31P NMR (121.4 MHz, CDCl3) δ 8.2, 7.8.
[0224] [00341] The 2-ethylbutyl alanine ester chlorophosphoramidate B was prepared using the same procedure as chloridate A except for the replacement of 2-ethylbutyl alanine ester with ethyl alanine ester. The material is used crude in the next reaction. Treatment with methanol or ethanol forms the displaced product with the LCMS signal requirement.
[0225] [00342] The isopropyl alanine chlorophosphoramidate C ester was prepared using the same procedure as chloridate A except for the replacement of isopropyl alanine ester with ethyl alanine ester. The material is used crude in the next reaction. Treatment with methanol or ethanol forms the displaced product with the LCMS signal requirement.
[0226] [00343] The commercially available lactol (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 stirred at RT for 48 h. The reaction mixture was poured into ice-cold H2O (500 ml) and the mixture stirred for 20 min. The mixture was extracted with EtOAc (3 x 200 ml) and the combined organic extracts were then washed with H2O (3 x 200 ml). The organic extract was dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The residue was dissolved in CH2Cl2 and subjected to silica gel chromatography eluting with 25% EtOAc in hexanes to generate the lactone (9.55 g, 96%).
[0227] [00344] 1H NMR (400 MHz, DMSO) δ 7.30-7.34 (m, 13H), 7.19-7.21 (m, 2H), 4.55-4.72 (m, 6H) , 4.47 (s, 2H), 4.28 (d, J = 3.9 Hz, 1H), 3.66 (m, 2H).
[0228] [00345] LCMS m / z 436.1 [M + H2O], 435.2 [M + OH] - Tr = 2.82 min
[0229] [00346] HPLC Tr = 4.59 [2-98% ACN in H2) for 5 min @ 2 ml / min flow.
[0230] [00347] 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 for 20 min. in RT and then cooled to -78ºC after which time a solution of n-BuLi (6 mL, 1.6 N in hexanes, 9.6 mmol) was added slowly. The reaction mixture was stirred for 10 min. at -78 ° C and then 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 dissolved in a mixture of CH2Cl2 and H2O (100 ml, 1: 1). The organic layer was separated and washed with H2O (50 ml). The organic layer was then dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The residue was subjected to silica gel chromatography eluting with 0-50% EtOAc in hexanes to provide the product as a 1: 1 mixture of anomers (345 mg, 26% yield).
[0231] [00348] LCMS m / z 553 [M + H].
[0232] [00349] The hydroxy nucleoside (1.1 g, 2.0 mmol) was dissolved in anhydrous CH2Cl2 (40 mL) and the solution cooled with stirring at 0 ° C under N2 (g). TMSCN (0.931 mL, 7 mmol) was added and the mixture stirred for an additional 10 min. TMSOTf (1.63 mL, 9.0 mmol) was slowly added to the reaction and the mixture stirred for 1 h. The reaction mixture was then diluted with CH2Cl2 (120 mL) and aqueous NaHCO3 (120 mL) was added to quench the reaction. The reaction mixture was stirred for another 10 min and the organic layer separated. The organic layer was extracted with CH2Cl2 (150 ml) and the combined organic extracts dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The residue was dissolved in a minimum amount of CH2Cl2 and subjected to silica gel chromatography eluting with a gradient of 0-75% EtOAc and hexanes to generate the cyclic nucleoside tribenzyl as a mixture of anomers. (0.9 g, 80%).
[0233] [00350] 1H NMR (300 MHz, CD3CN) δ 7.94 (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), 6.41 (bs, 2H), 5.10 (d , J = 3.9 Hz, 0.5H), 4.96 (d, J = 5.1 Hz, 0.5H), 4.31 - 4.85 (m, 7H), 4.09-4, 18 (m, 2H), 3.61-3.90 (m, 2H).
[0234] [00351] LCMS m / z 562 [M + H].
[0235] [00352] Tribenzil cyano nucleoside (70 mg, 0.124 mmol) was dissolved in anhydrous CH2Cl2 (2 mL) and cooled to -78 ° C under N2 (g). A solution of BCl3 (1N in CH2Cl2, 0.50 mL, 0.50 mmol) was added and the reaction mixture stirred for 1 h. at -78ºC. When the reaction was complete by LC / MS, MeOH was added to quench the reaction. The reaction mixture was allowed to warm to RT and the solvent removed under reduced pressure. The residue was subjected to HPLC reverse phase C18, eluting for 5 min with H2O (0.1% TFA), followed by a gradient of 0-70% MeCN in H2O (0.1% TFA) for 35 min, to elute the αanomer (20 mg, 37%), and β-anomer 1 (20 mg, 37%). (α-anomer)
[0236] [00353] 1H NMR (300 MHz, D2O) δ 7.96 (s, 1H), 7.20 (d, J = 4.8 Hz, 1H), 6.91 (d, J = 4.8 Hz, 1H), 4.97 (d, J = 4.4 Hz, 1H), 4.56-4.62 (m, 1H), 4.08-4.14 (m, 1H), 3.90 (dd , J = 12.9, 2.4 Hz, 1H), 3.70 (dd, J = 13.2, 4.5 Hz, 1H). (β-anomer)
[0237] [00354] 1H NMR (400 MHz, DMSO) δ 7.91 (s, 1H), 7.80-8.00 (br s, 2H), 6.85-6.89 (m, 2H), 6, 07 (d, J = 6.0 Hz, 1H), 5.17 (br s, 1H), 4.90 (br s, 1H), 4.63 (t, J = 3.9 Hz, 1H), 4.02-4.06 (m, 1H), 3.94 (br s, 1H), 3.48-3.64 (m, 2H).
[0238] [00355] LCMS m / z 292.2 [M + H], 290.0 [M-H]. Tr = 0.35 min.
[0239] [00356] 13C NMR (400 MHZ, 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
[0240] [00357] HPLC Tr = 1.32 min (2R, 3R, 4R, 5R) -2- (4-aminopyrrolo [1,2-f] [1,2,4] triazin-7-yl) -3-fluor-4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-carbonitrile (Compound 2)
[0241] [00358] 2-Deoxy-2-fluor-4,5-O, O-dibenzyl-D-arabinose. 1'-Methoxy-2-deoxy2-fluor-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 is stirred for 5 h. 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 column Combiflash HP Gold) eluting with 0–100% EtOAc in hexanes to generate 2-deoxy-2-fluor-4,5-O, O-dibenzyl-Darabinose (695 mg, 72%) as a white solid: Rf = 0.52 (25% EtOAc in hexanes);
[0242] [00359] 1H NMR (300 MHz, CDCl3) δ 7.30 (m, 10H), 5.35 (m, 1H), 4.68–4.29 (m, 7H), 3.70 (d, J = 10.5 Hz, 1H), 3.50 (d, J = 10.5 Hz, 2H).
[0243] [00360] 19F NMR (282.2 MHz, CDCl3) δ –207 (m), –211 (m).
[0244] [00361] LCMS m / z 350 [M + H2O].
[0245] [00362] (3R, 4R, 5R) -4- (benzyloxy) -5- (benzyloxymethyl) -3-fluordihydrofuran2 (3H) -one. 2-Deoxy-2-fluor-4,5-O, O-dibenzyl-D-arabinose (4.3 g, 12.8 mmol) was dissolved in CH2Cl2 (85 mL) was treated with 4 Å MS (10 g) and dichromate pyridinium (14.4 g, 38.3 mmol). The resulting mixture was stirred for 24 h and then filtered through a pad of Celite. The eluent was concentrated under reduced pressure and the residue subjected to silica gel chromatography (120 g SiO2 column HP Gold Combiflash) eluting with 0–100% EtOAc in hexanes to generate (3R, 4R, 5R) -4- (benzyloxy) - 5- (benzyloxymethyl) -3-fluordihydrofuran-2 (3H) -one as a clear oil (3.5 g, 83%): Rf = 0.25 (25% EtOAc in hexanes).
[0246] [00363] 1H NMR (300 MHz, CDCl3) δ 7.37 (m, 10H), 5.45 (dd, J = 49, 5.7, Hz, 1H), 4.85 (d, J = 11, 7 Hz, 1H), 4.52 (m, 4 H), 4.29 (d, J = 5.4 Hz, 1H), 2.08 (dd, J = 15.3, 10.2 Hz, 2H ).
[0247] [00364] 19F NMR (282.2 MHz, CDCl3) δ –216.
[0248] [00365] LCMS m / z 348 [M + H2O].
[0249] [00366] HPLC (6–98% MeCN – H2O gradient, 0.05% TFA modifier) tR = 5.29 min. Phenomenex Synergi 4 m Hidro-RP 80 A, 50 × 4.60 mm, 4 micron; 2 mL / min flow
[0250] [00367] (3R, 4R, 5R) -2- (4-aminopyrrolo [1,2-f] [1,2,4] triazin-7-yl) -4- (benzyloxy) -5- (benzyloxymethyl) - 3-fluortetrahydrofuran-2-ol. 7-Bromopyrrolo [1,2- f] [1,2,4] -triazin-4-amine (68 mg, 0.319 mmol) in THF (1.4 mL) was treated with TMSCl (89 µL, 0.703 mmol) and the mixture stirred for 2 h. The mixture was then cooled to –78 ° C and treated with nBuLi (1.0 M in hexanes, 1.09 mL, 1.09 mmol). The solution was stirred for 30 min and then treated with (3R, 4R, 5R) -4- (benzyloxy) - 5- (benzyloxymethyl) -3-fluordihydrofuran-2 (3H) -one (106 mg, 0.319 mmol) under drip in THF (1.4 mL). The resulting mixture was stirred for 30 min and then AcOH (83 µL, 1.44 mmol) in THF (1.0 mL) was added to quench the reaction. The mixture was heated to RT and then concentrated under reduced pressure. The residue was diluted with EtOAc (100 ml) and washed with saturated NaCl solution (50 ml). The organic layer was dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The residue was subjected to silica gel chromatography (40 g SiO2 HP Gold Combiflash column) eluting with 0–100% EtOAc in hexanes followed by a 0–100% gradient (20% MeOH in EtOAc) in EtOAc to generate (3R, 4R, 5R) -2- (4-aminopyrrolo [1,2- f] [1,2,4] triazin-7-yl) -4- (benzyloxy) -5- (benzyloxymethyl) -3-fluortetrahydrofuran- 2-ol as a white solid (68 mg, 44%, 60/40 mixture of α / β isomers). Rf = 0.32 (EtOAc).
[0251] [00368] 1H NMR (300 MHz, CDCl3) δ 8.05 (s, 1H), 7.86 (s, 1H), 7.81 (s, 1H), 7.64 (s, 1H), 7, 26 (m, 10H), 6.95 (m, 1H), 6.71 (m, 1H), 6.08 (m, 1H), 5.34 (m, 1H), 4.65 (m, 6H ), 4.71 (m, 2H).
[0252] [00369] 19F NMR (282.2 MHz, CDCl3) δ –211 (m).
[0253] [00370] LCMS m / z 465 [M + H].
[0254] [00371] HPLC (6–98% MeCN – H2O gradient, 0.05% TFA modifier) tR = 4.37 min. (α-isomer), 4.54 min. (β-isomer).
[0255] [00372] (3R, 4R, 5R) -2- (4-aminopyrrolo [1,2-f] [1,2,4] triazin-7-yl) -4- (benzyloxy) -5- (benzyloxymethyl) - 3-fluortetrahydrofuran-2-carbonitrile: (3R, 4R, 5R) -2- (4-aminopyrrolo [1,2-f] [1,2,4] triazin-7-yl) -4- (benzyloxy) -5- (benzyloxymethyl) -3- fluortetrahydrofuran-2-ol (195 mg, 0.42 mmol) was dissolved 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 h and then cooled to 0 ° C. The mixture was treated with saturated NaHCO3 solution (20 drops) then heated to RT 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 SiO2 HP Gold Combiflash column) eluting with 0–100% EtOAc in hexanes to generate (3R, 4R, 5R) -2- (4-aminopyrrole [1,2-f] [1,2,4] triazin-7-yl) -4- (benzyloxy) -5- (benzyloxymethyl) -3-fluortetrahydrofuran-2-carbonitrile as a white solid (110 mg, 55%, 60/40 mixture α / β isomers). Data for both isomers: Rf = 0.53 (EtOAc).
[0256] [00373] 1H NMR (300 MHz, CDCl3) δ 8.01 (s, 1H), 7.94 (s, 1H), 7.30 (m, 10H), 7.00 (d, J = 4.5 Hz, 1H), 6.93 (d, J = 4.8 Hz, 1H), 6.87 (d, J = 5.4 Hz, 1H), 6.70 (d, J = 4.8 Hz, 1H), 5.85 (dd, J = 52, 3.3 Hz, 1H), 5.55 (dd, J = 53, 4.5 Hz, 1H), 4.71 (m, 7H), 3, 87 (m, 2H), 3.72 (m, 2H).
[0257] [00374] 19F NMR (282.2 MHz, CDCl3) δ –196 (m), –203 (m).
[0258] [00375] LCMS m / z 474 [M + H].
[0259] [00376] HPLC (6–98% MeCN – H2O gradient, 0.05% modifying TFA) tR = 4.98 min.
[0260] [00377] (2R, 3R, 4R, 5R) -2- (4-aminopyrrolo [1,2-f] [1,2,4] triazin-7-yl) -3-fluor-4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-carbonitrile (2) (3R, 4R, 5R) -2- (4-aminopyrrolo [1,2-f] [1,2,4] triazin-7-yl) -4- (benzyloxy) -5- (benzyloxymethyl) -3-fluortetrahydrofuran-2-carbonitrile (110 mg, 0.23 mmol) was dissolved in CH2Cl2 (1.5 mL) and cooled to 0 ° C. The reaction mixture was treated with BCl3 (1.0 M in CH2Cl2, 766 µL, 0.77 mmol) and stirred for 2 h. The mixture was then cooled to –78 ° C and treated with Et3N (340 µL, 2.44 mmol) followed by MeOH (2 mL) before allowing to warm to RT. The reaction was concentrated under reduced pressure and then co-evaporated with MeOH (3 x 5 ml). The residue was then suspended in H2O (5 ml) and treated with NaHCO3 (1 g). The solution was stirred for 10 min and then concentrated under reduced pressure. The residue was filtered and washed with MeOH (3 × 10 mL) in a glass funnel (coarse) and the eluent concentrated under reduced pressure. The residue was subjected to reverse phase HPLC (6–98% MeCN in H2O gradient with 0.05% TFA modifier) to generate (2R, 3R, 4R, 5R) -2- (4-aminopyrrole [1,2-f ] [1,2,4] triazin-7-yl) -3-fluor-4-hydroxy5- (hydroxymethyl) tetrahydrofuran-2-carbonitrile 2 as a white solid (16.8 mg, 25%) and α -isomer.
[0261] [00378] Data for the α-isomer: Rf = 0.13 (10% MeOH in EtOAc).
[0262] [00379] 1H NMR (300 MHz, CD3OD) δ 8.09 (s, 1H), 7.28 (d, J = 5.1 Hz, 1H), 7.17 (d, J = 5.1 Hz, 1H), 5.42 (dd, J = 53, 3.3 Hz, 1H), 4.20 (m, 2H), 3.99 (d, J = 3.6 Hz, 1H), 3.77 ( d, J = 3.6 Hz, 1H).
[0263] [00380] 19F NMR (282.2 MHz, CDCl3) δ –197 (m).
[0264] [00381] LCMS m / z 294 [M + H].
[0265] [00382] HPLC (2–98% MeCN – H2O gradient, 0.05% modifying TFA) tR = 1.49 min.
[0266] [00383] 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 cooled to 0 ° C with stirring under N2 (g). A solution of methyl magnesium chloride (2 mL, 6 mmol) (3N in THF) was added and the resulting mixture stirred overnight. Acetic acid (7 mmol) was added to quench the reaction and then the solvents were removed by rotating under reduced pressure. The residue was redissolved in CH2Cl2 and the solution subjected to a plug of silica gel to isolate the product (0.355 g) as a crude mixture. LC / MS (m / z: 480, M + 1). The crude material was dissolved in anhydrous CH2Cl2 (20 ml) and placed under N2 (g). The solution was stirred and treated with methanesulfonic acid (0.2 ml, 2.74 mmol). The reaction mixture was stirred for 12 h at RT and then quenched by the addition of Et3N (3.5 mmol). The mixture was concentrated under reduced pressure and the residue subjected to silica gel chromatography to provide the substituted methyl nucleoside (0.174 g, 0.377 mmol, 44% yield) as a 4: 1 mixture of beta- and alpha-anomers respectively.
[0267] [00384] 1H NMR (300 MHz, CD3CN) major anomer δ 7.87 (s, 1H), 7.27- 7.40 (m, 10 H), 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 ).
[0268] [00385] 19F NMR (282.2 MHz, CD3CN) main anomer δ -207 (m, 1F)
[0269] [00386] LCMS m / z 463 [M + H].
[0270] [00387] The benzylated nucleoside material (0.134 g, 0.290 mmol), Degussa catalyst (0.268 g) and AcOH (30 mL) were mixed together. The reaction atmosphere was charged with H2 (g) and the reaction stirred for 2 h. The catalyst was removed by filtration and the mixture concentrated under reduced pressure. The residue was dissolved in a minimum amount of H2O and subjected to reverse phase HPLC (C18 hydro RP column) to isolate β-anomer 3 (0.086 g, 0.217 mmol, 57% yield).
[0271] [00388] 1H NMR (300 MHz, D2O) δ7.87 (s, 1H), 7.22 (d, J = 4.8 Hz, 1H), 6.87 (d, J = 4.8 Hz, 1H ), 5.35 (dd, J = 54, 3.6 Hz, 1H), 3.97-4.10 (m, 2H), 3.81 (dd, J = 12.6, 2.1 Hz, 1H), 3.64 (dd, J = 12.6, 4.8 Hz, 1H), 1.65 (d, J = 4.2 Hz, 3H).
[0272] [00389] 19F NMR (282.2 MHz, CD3CN) δ -207 (m, 1F).
[0273] [00390] A small amount of alpha anomer was characterized as follows.
[0274] [00391] 1H NMR (300 MHz, D2O) δ7.86 (s, 1H), 7.26 (d, J = 4.8 Hz, 1H), 6.85 (d, J = 4.8 Hz, 1H ), 5.31 (dd, J = 54, 3.9 Hz, 1H), 4.39 (ddd, J = 26.1, 9.9, 3.6 Hz, 2H), 4.00 - 4, 05 (m, 1H), 3.90 (dd, J = 12.3, 2.1 Hz, 1H), 3.66 (dd, J = 12.6, 4.8, 1H), 1.56 ( s, 3H).
[0275] [00392] 19F NMR (282.2 MHz, CD3CN) δ-198 (dd, J = 54, 26 Hz, 1F).
[0276] [00393] Nucleoside 3 (0.011 g, 0.04 mmol) was dissolved in trimethylphosphate (2 mL) and cooled to 0 ° C. The mixture was stirred under an atmosphere of N2 (g) and 1-Methylimidazole (0.320 ml, 5 mmol) followed by alaninyl monoisopropyl, monophenol phosphorchloridate C (0.240 ml, 4.4 mmol) was added. The reaction mixture was stirred for 2 h. at 0oC and then allowed to warm slowly to RT. While monitoring via LC / MS. When completed by LCMS, 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 hexanes. The product fractions were collected and concentrated. The residue was subjected to prep HPLC to generate the prodrug alanine isopropyl monoamidate 4 as a mixture of isomers (4.7 mg, 0.003 mmol, 6%).
[0277] [00394] 1H NMR (300 MHz, CD3CN) δ 7.87 (s, 1H), 7.17-7.44 (m, 5 H), 6.71-6.83 (m, 2H), 6, 14 (br, s, 2H), 5.38 (dd, J = 56, 3.3 Hz, 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)
[0278] [00395] LCMS m / z 552 [M + H].
[0279] [00396] Nucleoside 3 (0.026 g, 0.092 mmol) was dissolved in trimethylphosphate (2 mL) and cooled to 0 ° C. The mixture was stirred under N2 (g) and 1-methylimidazole (0.062 ml, 0.763 mmol) followed by chloridate A (0.160 g, 0.552 mmol) was added. The reaction mixture was stirred for 2 h. at 0oC and then allowed to warm slowly to RT. H2O (5 mL) was added to quench the reaction and then the mixture concentrated under reduced pressure. The residue was dissolved in CH2Cl2 and subjected to silica gel chromatography eluting with 0-100% EtOAc in hexanes. The product fractions were collected and concentrated. The crude product was eluted using 0 to 100 percent EtOAc in hexanes. The crude product was collected and concentrated under reduced pressure. The residue was subjected to prep HPLC to generate 5 (2.0 mg, 4% yield).
[0280] [00397] LCMS m / z 538 [M + H].
[0281] [00398] Nucleoside 3 (0.022 g, 0.056 mmol) was dissolved in trimethylphosphate (1 ml) and stirred under N2 (g). Phosphorous oxychloride (0.067 mL, 0.73 mmol) was added and the mixture stirred for 2 h. Monitoring by analytical ion exchange column determined the time in which> 80 percent monophosphate was formed. 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 min and then quenched by the addition of 1N solution of triethylammonium bicarbonate in H2O (5 mL). The mixture was concentrated under reduced pressure and the residue re-dissolved in H2O. The solution was subjected to ion exchange chromatography to generate the title 6 product (1.7 mg, 6% yield).
[0282] [00399] LCMS m / z 521 [M-H]. Tr = 0.41
[0283] [00400] Ion exchange HPLC TR = 9.40 min
[0284] [00401] ((3αR, 5S, 6αR) -2,2-dimethyl-tetrahydrofuro [2,3-d] [1,3] dioxol-5-yl) methanol
[0285] [00402] The acetate material (1.2 g, 5.5 mmol) (J. Org. Chem. 1985, 50, 3547, De Bernardo et al) was dissolved in a 1: 1 MeOH and THF mixture (10 mL) . A 1N NaOH (aq) solution (10mL) was added until the pH was 13. The reaction mixture was stirred for 2h and then neutralized to pH 8-9 by the addition of AcOH. The mixture was extracted with EtOAc (10 x 30mL) and the combined organic extracts dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was subjected to silica gel chromatography eluting with 0-70% EtOAc in hexanes to generate the desired product (866 mg, 90%).
[0286] [00403] 1H NMR (300 MHz, CDCl3) δ 5.84 (d, J = 3.6 Hz, 1H), 4.78 (t, J = 4.5 Hz, 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).
[0287] [00404] (3αR, 5S, 6αR) -5- (benzyloxymethyl) -2,2-dimethyl-tetrahydrofuro [2,3-d] [1,3] dioxol. Sodium hydride (188 mg, 7.46 mmol) was dissolved in anhydrous THF (5 ml) and stirred under N2 (g) in RT. The 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. and then benzyl bromide (892 µL, 7.46 mmol) was added. The reaction was stirred for 2 h and then poured into a mixture of cold aqueous NaHCO3 and EtOAc (30mL). The organic layer was separated and then the aqueous layer 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 eluting with 0-40% EtOAc in hexanes to generate the benzyl ether product (912 mg, 69%).
[0288] [00405] 1H NMR (300 MHz, CDCl3) δ 7.35-7.27 (m, 5H), 5.86 (d, J = 3.6 Hz, 1H), 4.74 (t, J = 4 , 2 Hz, 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).
[0289] [00406] (3R, 5S) -5- (benzyloxymethyl) -tetrahydrofuran-2,3-diol. The benzyl ether (910 mg, 3.44 mmol) was dissolved in a mixture 1: 1 AcOH and H2O (20 mL) and stirred at 60oC for 7h. The mixture was concentrated under reduced pressure and the residue subjected to silica gel chromatography eluting with 0-70% EtOAc in hexanes to generate the diol product (705 mg, 91%).
[0290] [00407] 1H NMR (300 MHz, CDCl3) δ 7.36-7.27 (m, 5H), 5.40 (d, J = 3.9 Hz, 0.5H), 5.17 (s, 0 , 5H), 4.67-4.56 (m, 3H), 4.33 (m, 0.5H), 4.24 (d, J = 4.8 Hz, 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).
[0291] [00408] (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 celite carbonate (3.46 g, 6.28 mmol). The resulting mixture was stirred at 80 ° C under N2 (g) for 2h. The mixture was then cooled to RT, filtered and concentrated under reduced pressure. The residue was subjected to silica gel chromatography eluting with 0-70% EtOAc in hexanes to generate the lactone product (600 mg, 86%).
[0292] [00409] 1H NMR (300 MHz, CDCl3) δ 7.39-7.27 (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).
[0293] [00410] (3R, 5S) -3- (benzyloxy) -5- (benzyloxymethyl) -dihydrofuran-2 (3H) -one. The lactone (600 mg, 2.7 mmol) was dissolved in EtOAc (30mL) and treated with silver oxide (626 mg, 2.7 mmol) followed by benzyl bromide (387 µL, 3.24 mmol). The reaction mixture was then stirred at 50oC under N2 (g) for 8h. More silver oxide (300 mg) was then added and the resulting mixture stirred at 50oC for 16h. More benzyl bromide (50 µL) and silver oxide (150 mg) were added and the mixture stirred for another 8 hours. The reaction mixture was allowed to cool, filtered and then concentrated under reduced pressure. The residue was subjected to silica gel chromatography eluting with 0-20% EtOAc in hexanes to generate the title product (742 mg, 88%).
[0294] [00411] 1H NMR (300 MHz, CDCl3) δ 7.39-7.27 (m, 10H), 4.99 (d, J = 11.4 Hz, 1H), 4.72 (m, 2H), 4.56 (m, 2H), 4.39 (t, J = 8.1 Hz, 1H), 3.72-3.51 (m, 2H), 2.42-2.25 (m, 2H) .
[0295] [00412] (3R, 5S) -2- (4-aminopyrrolo [1,2-f] [1,2,4] triazin-7-yl) -3- (benzyloxy) -5- (benzyloxymethyl) -tetra- hydrofuran-2-ol. The 7-bromopyrrolo [1,2-f] [1,2,4] triazin-4-amine (607 mg, 2.85 mmol) was dissolved in anhydrous THF (10 mL) and stirred under Ar (g) in RT . TMSCl (1.1 mL, 8.55 mmol) was added under a drip and the mixture stirred for 2h. The reaction was concentrated under reduced pressure and then dried under a high vacuum. The residue was suspended in THF (20 ml) and stirred under Ar (g) at -78 ° C. A 2.5M n-BuLi solution in hexane (2.28 mL, 5.7 mmol) was added under a drop for 10 min. and the resulting mixture stirred for 60 min. Lactone (742 mg, 2.37 mmol) dissolved in anhydrous THF (7 mL) was added to the above mixture for 20 min. The reaction mixture was stirred for 2 h. and then quenched with AcOH until pH 5-6. The mixture was allowed to warm 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 hexanes to generate the title product (250 mg, 24%).
[0296] [00413] LCMS m / z 447.2 [M + H], 445.1 [M – H].
[0297] [00414] (3R, 5S) -2- (4-aminopyrrolo [1,2-f] [1,2,4] triazin-7-yl) -3- (benzyloxy) -5- (benzyloxymethyl) -tetra- hydrofuran-2-carbonitrile. The alcohol (250 mg, 0.56 mmol) was dissolved in anhydrous CH2Cl2 (10 mL) and stirred under Ar (g) at -15 ° C. TMSCN (448 μL, 3.36 mmol) was added under a drip and the mixture stirred for 10 min. TMSOTf (466 μL, 2.58 mmol) was added under a drip for 10 min and the resulting mixture stirred for 90 min. at -15oC. More TMSCN (224 μL, 3 eq.) And TMSOTf (202 μL, 2 eq.) Was added and stirring continued for 5 h. Saturated aqueous NaHCO3 solution was added to quench the reaction and the mixture stirred for 10 min. The organic layer was separated and washed with saturated aqueous NaHCO3 solution, saturated NaCl solution, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was subjected to silica gel chromatography eluting with 0-70% EtOAc in hexanes to generate the title product (150 mg, 59%).
[0298] [00415] LCMS m / z 456.3 [M + H], 454.1 [M – H].
[0299] [00416] (2R, 3R, 5S) -2- (4-aminopyrrolo [1,2-f] [1,2,4] triazin-7-yl) -3-hydroxy-5- (hydroxymethyl) -tetra- hydrofuran-2-carbonitrile (7). The benzyl ether (150 mg, 0.329 mmol) was dissolved in anhydrous CH2Cl2 (2 mL) and the mixture stirred under Ar (g) at - 20oC. A 1M BCl3 solution in CH2Cl2 (724 μL, 0.724 mmol) was added under a drip and the resulting mixture stirred for 2 h. An additional 1M BCl3 in CH2Cl2 (724 μL, 0.724 mmol) was added and stirring continued for 2h. 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 and then treated with MeOH (10 ml). The reaction was allowed to warm to RT and then concentrated under reduced pressure. The residue was dissolved in MeOH and concentrated under reduced pressure. The residue was dissolved in MeOH again and treated with solid NaHCO3. The mixture was stirred for 5 min and then the solid removed by filtration. The solution was concentrated under reduced pressure and subjected to preparative HPLC to generate the desired product 7 (10 mg, 11%).
[0300] [00417] 1H NMR (300 MHz, D2O) δ7.71 (s, 1H), 6.75 (d, J = 4.5 Hz, 1H), 6.65 (d, J = 4.8 Hz, 1H ), 4.91 (t, J = 6.3 Hz, 1H), 4.57 (m, 1H), 3.67-3.47 (m, 2H), 2.18 (m, 2H).
[0301] [00418] LCMS m / z 276.1 [M + H], 274.0 [M – H]. 2 - (((((2R, 3S, 4R, 5R) -5- (4-aminopyrrolo [1,2-f] [1,2,4] triazin-7-yl) -5-cyano-3,4- dihydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) -phosphorylamino) (2S) -isopropyl propanoate (Compound 8)
[0302] [00419] Nucleoside 1 (45mg, 0.15mmol) was dissolved in anhydrous trimethyl phosphate (0.5ml) and the solution stirred under N2 (g) at 0oC. Methyl imidazole (36 μL, 0.45 mmol) was added to the solution. Chlorophosphoramidate 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 aqueous NaHCO3, saturated NaCl, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was subjected to silica gel chromatography eluting with 0-5% MeOH in CH2Cl2 followed by preparative HPLC to generate the product (20.9 mg, 25%).
[0303] [00420] 1H NMR (300 MHz, CD3OD) δ 7.95 (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);
[0304] [00421] 31P NMR (121.4 MHz, CD3OD) δ 3.8.
[0305] [00422] LCMS m / z 561.0 [M + H], 559.0 [MH]. 2 - (((((2R, 3S, 4R, 5R) -5- (4-aminopyrrolo [1,2-f] [1,2,4] triazin-7-yl) -5-cyano-3,4- (2S) -2-ethylbutyl dihydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphorylamino) propanoate (Compound 9)
[0306] [00423] Prepared from Compound 1 and chloridate B according to the same method for the preparation of compound 8.
[0307] [00424] 1H NMR (300 MHz, CD3OD) δ 7.87 (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).
[0308] [00425] 31P NMR (121.4 MHz, CD3OD) δ 3.7.
[0309] [00426] LCMS m / z 603.1 [M + H], 601.0 [MH].
[0310] [00427] Prepared from Compound 1 and chloridate A using the same method for the preparation of compound 8.
[0311] [00428] 1H NMR (300 MHz, CD3OD) δ 7.95 (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).
[0312] [00429] 31P NMR (121.4 MHz, CD3OD) δ 3.7.
[0313] [00430] LCMS m / z 547.0 [M + H], 545.0 [MH].
[0314] [00431] Compound 11 was prepared from Compound 2 and chloridate A using the same method as for the preparation of compound 8.
[0315] [00432] 1H NMR (300 MHz, CD3OD) δ 7.91 (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).
[0316] [00433] 31P NMR (121.4 MHz, CD3OD) δ 3.8.
[0317] [00434] LCMS m / z 549.0 [M + H], 547.1 [MH].
[0318] [00435] Nucleoside 1 (14.6 mg, 0.05 mmol) was dissolved in anhydrous trimethyl phosphate (0.5 ml) and stirred under N2 (g) in RT. POCl3 (9.2 μL, 0.1 mmol) was added and the mixture stirred for 60 min. Alanine ethyl ester hydrochloride (61 mg, 0.4 mmol) and then Et3N (70 μL, 0.5 mmol) was added. The resulting mixture was stirred for 15 min. and then more Et3N (70 μl, 0.5 mmol) was added to generate a pH solution of 9-10. The mixture was stirred for 2 h. and then diluted with EtOAc, washed with saturated aqueous NaHCO3 followed by saturated aqueous NaCl. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was subjected to preparative HPLC (column C18) to generate product 12 (5.5 mg, 16%).
[0319] [00436] 1H NMR (400 MHz, CD3OD) δ 8.13 (s, 1H), 7.41 (d, J = 4.8 Hz, 1H), 7.18 (d, J = 4.8 Hz, 1H), 4.78 (d, J = 5.6 Hz, 1H), 4.36 (m, 1H), 4.25-4.08 (m, 7H), 3.83 (m, 2H), 1.33-1.23 (m, 12H).
[0320] [00437] 31P NMR (121.4 MHz, CD3OD) δ 13.8.
[0321] [00438] LCMS m / z 570.0 [M + H], 568.0 [MH].
[0322] [00439] The nucleoside alcohol (0.6 g, 1.08 mmol) (prepared as described in the synthesis of Compound 1) was dissolved in anhydrous THF (8mL) and placed under N2 (g). The reaction mixture was stirred and cooled to 0 ° C and then treated with a 0.5N solution of ethinyl magnesium bromide in THF (17.2 mL, 17.2 mmol). The reaction mixture was stirred overnight at RT. AcOH (1.5 mL) was added to quench the reaction. The mixture was concentrated under reduced pressure and the residue redissolved in CH2Cl2. The solution is subjected to a plug of silica gel eluting with 0 to 80% EtOAc in hexanes to generate the title product as a crude mixture.
[0323] [00440] LCMS m / z 579 [M + H].
[0324] [00441] The crude ethinyl 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 for 12 h. in RT. When completed by LCMS, Et3N (0.56 mL) was added to quench the reaction. The reaction was concentrated under reduced pressure and the residue subjected to silica gel chromatography eluting with 0 to 75% EtOAc in hexanes to generate the ethynyl nucleoside as a mixture of anomers (0.200 g, 33% over 2 steps).
[0325] [00442] LCMS m / z 561 [M + H].
[0326] [00443] The tribenyl nucleoside (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 stirred for 1 h. at -78oC. A solution of MeOH (10 mL) and pyridine (2 mL) was added to quench the reaction and the mixture was allowed to rise to RT. The mixture was concentrated under reduced pressure and subjected to preparative HPLC to generate α-anomer (20 mg) and β-anomer 13 (110 mg).
[0327] [00444] (β-anomer) 1H NMR (300 MHz, DMSO) δ 7.81 (s, 1H), 7.76 (br s, 2H), 6.80-6.85 (m, 2H), 5 , 11 (d, J = 7.2 Hz, 1H), 4.90 (d, J = 6.0 Hz, 1H), 4.82 (dd, J = 7.2, 4.8 Hz, 1H) , 4.62 (t, J = 6.3 Hz, 1H), 3.95-3.99 (m, 1H), 3.85-3.91 (dd, J = 11.4, 5.7 Hz , 1H), 3.61-3.67 (m, 1H), 3.47-3.55 (m, 1H), 3.52 (d, J = 0.9 Hz, 1H).
[0328] [00445] (α -anomer) 1H NMR (300 MHz, DMSO) δ 7.80 (s, 1H), 7.59 (bs, 2H), 6.80 (d, J = 4.5 Hz, 1H) , 6.54 (d, J = 4.2 Hz, 1H), 5.00 (d, J = 7.2 Hz, 1H), 4.89 (d, J = 4.8 Hz, 1H), 4 , 74 (t, J = 5.7 Hz, 1H), 4.58 (t, J = 4.5 Hz, 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 = 0.6 Hz, 1H)
[0329] [00446] LCMS m / z 291 [M + H].
[0330] [00447] The tribenyl alcohol of the synthesis of Compound 1 (0.250 g, 0.453 mmol) was dissolved in anhydrous THF (25 ml) and stirred under N2 (g). The reaction mixture was cooled to 0 ° C and then a 3.0 N solution of methyl magnesium chloride in THF (1.2 mL, 3.62 mmol) was added. The reaction mixture was stirred overnight at RT. Acetic acid (1.5 mL) was added to quench the reaction and then the mixture was concentrated under reduced pressure. The residue was redissolved in CH2Cl2 and submitted to a plug of silica gel eluting with 0 to 80% EtOAc in hexanes. The crude product (0.452 g) was then used for the next reaction without further purification.
[0331] [00448] LCMS m / z 569 [M + H].
[0332] [00449] The crude methyl nucleoside (0.452 g, 0.796 mmol) was dissolved in anhydrous CH2Cl2 (20 mL) and stirred under N2 (g). Methanesulfonic acid (0.2 mL, 2.78 mmol) was added and the reaction stirred for 12 hr at RT. Et3N (0.56 mL) was added to quench the reaction and then the mixture concentrated under reduced pressure. The residue was subjected to silica gel chromatography eluting with 0 to 75% EtOAc in hexanes to generate the product as a mixture of anomers (0.20 g, 46% over 2 steps).
[0333] [00450] LCMS m / z 551 [M + H].
[0334] [00451] The tribenyl nucleoside (0.20 g, 0.364 mmol) was dissolved in AcOH (30 mL) and loaded with Pd / C (Degussa) (400 mg). The stirred mixture was washed with N2 (g) three times and then H2 (g) was introduced. The reaction was stirred under H2 (g) for 2 h. and then the catalyst removed by filtration. The solution was concentrated under reduced pressure and under the residue it was redissolved in H2O. The solution was subjected to preparative HPLC under neutral conditions to generate α-anomer and β-anomer 14 in 81% yield.
[0335] [00452] (α-anomer) 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) .
[0336] [00453] (β-anomer) 1H NMR (300 MHz, 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).
[0337] [00454] LCMS m / z 281 [M + H]. Bis (2,2-dimethylpropanothioate) of S, S'-2,2 '- ((((((2R, 3S, 4R, 5R) -5- (4-aminopyrrolo [1,2-f] [1,2, 4] triazin-7-yl) -5-cyano-3,4-dihydroxytetrahydro-furan-2-yl) methoxy) phosphoryl) bis (oxy) bis (ethane-2,1-di-yl) ( Compound 15)
[0338] [00455] Nucleoside 1 (0.028 g, 0.096 mmol) was dissolved in trimethylphosphate (1 ml). The reaction was stirred under N2 (g) and then treated with 1Htetrazole (0.021 g, 0.29 mmol). The reaction mixture was cooled to 0oC and the phosphate (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 h. and then quenched with 30% hydrogen peroxide (0.120 mL). The mixture was stirred for 30 min in RT and then treated with saturated aqueous sodium thiosulfate (1 ml). The mixture was stirred for 10 min. and then concentrated under reduced pressure. The residue was subjected to preparative HPLC to isolate the title product 15.
[0339] [00456] 1H NMR (300 MHz, CD3CN) δ 7.98 (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).
[0340] [00457] 31P NMR (121.4 MHz, CD3CN) δ -1.85 (s).
[0341] [00458] LCMS m / z 661 [M + H]. Bis (2,2-dimethylpropanothioate) of S, S'-2,2 '- ((((((2R, 3S, 4R, 5S) -5- (4-aminopyrrolo [1,2-f] [1,2, 4] triazin-7-yl) -5-ethynyl-3,4-dihydroxytetrahydrofuran-2-yl) methoxy) phosphoryl) bis (oxy) bis (ethane-2,1-di-yl) (Compound 16 )
[0342] [00459] Compound 16 was prepared using the same method as compound 15 except for substitution compound 13 as the starting nucleoside.
[0343] [00460] 1H NMR (300 MHz, CD3CN) δ 7.91 (s, 1H), 6.86 (d, J = 4.8 Hz, 1H), 6.76 (d, J = 4.5 Hz, 1H), 6.29 (bs, 2H), 4.69 (t, J = 2.7 Hz, 1H), 4.58 (d, J = 5.7 Hz, 1H), 4.14-4, 33 (m, 5H), 3.99-4.07 (m, 4H), 3.53 (d, J = 5.4 Hz, 1H), 3.11 (q, J = 5.7 Hz, 4H ), 1.22 (s, 18H).
[0344] [00461] LCMS m / z 658.9 [M +]. Tr = 2.31
[0345] [00462] Compound 17 was prepared from compound 1 using a procedure similar to the preparation of compound 6. The product was isolated as the sodium salt.
[0346] [00463] 1H NMR (400 MHz, D2O) δ 7.76 (s, 1H), 6.88 (d, J = 4.8 Hz, 1H), 6.73 (d, J = 4.4 Hz, 1H), 4.86 (d, J = 5.2 Hz, 1H), 4.43 (m, 1H), 4.39 (m, 1H), 4.05 (m, 1H), 3.94 ( m, 1H)
[0347] [00464] 31P NMR (121.4 MHz, D2O) δ -5.4 (d, 1P), -10.8 (d, 1P), -21.1 (t, 1P).
[0348] [00465] LCMS m / z 530 [M-H], 531.9 [M + H] Tr = 0.22 min
[0349] [00466] Ion exchange HPLC Tr = 9.95 min
[0350] [00467] Compound 18 was prepared from compound 13 using a procedure similar to the preparation of compound 6. The product was isolated as the TEA salt.
[0351] [00468] 1H NMR (300 MHz, D2O) δ 7.85 (s, 1H), 7.09 (d, J = 4.6 Hz, 1H), 6.95 (d, J = 4.7 Hz, 1H), 4.23 (m, 2H), 4.08 (m, 2H), 3.06 (q, J = 7.4 Hz, 20H), 1.14 (t, J = 7.3 Hz, 30H)
[0352] [00469] 31P NMR (121.4 MHz, D2O) δ -10.8 (d, 1P), -11.2 (d, 1P), -23.2 (t, 1P).
[0353] [00470] LCMS m / z 530.8 [M + H], Tr = 0.46
[0354] [00471] Ion exchange HPLC Tr = 9.40 min ((2R, 3S, 4R, 5S) -5- (4-aminopyrrolo [1,2- f] [1,2,4] triazin-7-yl) -3,4-dihydroxy tetrahydrogen triphosphate -5-methyltetrahydrofuran-2-yl) methyl (Compound 19)
[0355] [00472] Compound 19 was prepared from compound 14 using a procedure similar to the preparation of compound 6.
[0356] [00473] 1H NMR (400 MHz, D2O)  7.78 (s, 1H), 6.98 (m, 1H), 6.84 (m, 1H), 4.45 (m, 1H), 4, 04 (m, 4H), 1.54 (s, 3H).
[0357] [00474] 31P NMR (161 MHz, D2O) δ -10.6 (m), -23.0 (m).
[0358] [00475] LCMS m / z 521.0 [M + H].
[0359] [00476] ((2R, 3R, 4R, 5R) -5- (4-aminopyrrolo [1,2-f] [1,2,4] triazin-7-yl) -5-cyano- tetrahydrogen triphosphate 4-fluor-3-hydroxytetrahydrofuran2-yl) methyl (Compound 20)
[0360] [00477] Compound 20 was prepared from compound 2 using a procedure similar to the preparation of compound 6.
[0361] [00478] 1H NMR (400 MHz, D2O)  7.78 (s, 1H), 6.93 (d, J = 4.4 Hz, 1H), 6.78 (d, J = 4.8 Hz, 1H), 5.45 (dd, J = 53, 4.4 Hz, 1H), 4.38-4.50 (m, 2H), 4.13-4.20 (m, 2H).
[0362] [00479] 31P NMR (161 MHz, D2O) δ -5.7 (d, 1P), -11.0 (d, 1P), -21.5 (t, 1P).
[0363] [00480] LCMS m / z 533.9.0 [M + H], 532.0 [M-H] Tr = 1.25 min.
[0364] [00481] HPLC ion exchange Tr = 11.0 min Antiviral Activity
[0365] [00482] Another aspect of the invention relates to methods of inhibiting viral infections, comprising the step of treating a sample or a subject suspected of needing said inhibition with a composition of the invention.
[0366] [00483] Within the context of the invention, samples suspected of containing a virus include natural or man-made materials as living organisms; tissue or cell cultures; biological samples as samples of biological material (blood, serum, urine, cerebrospinal fluid, tears, sputum, saliva, tissue samples, and the like); laboratory samples; food, water, or air samples; samples of bioproducts such as cell extracts, particularly recombinant cells synthesizing a desired glycoprotein; and the like. Typically, the sample will be suspected of containing an organism that induces a viral infection, often a pathogenic organism such as a tumor virus. Samples can be contained in any medium including water and organic solvent / water mixtures. The samples include living organisms such as humans, and man-made materials such as cell cultures.
[0367] [00484] If desired, the antivirus activity of a compound of the invention after application of the composition can be observed by any method including direct and indirect methods of detecting said activity. Quantitative, qualitative, and semi-quantitative methods of determining this activity are all included. Typically one of the screening methods described above is applied, however, any other method such as observing the physiological properties of a living organism is still applicable.
[0368] [00485] The antiviral activity of a compound of the invention can be measured using standard screening protocols that are known. For example, a compound's antiviral activity can be measured using the following general protocols.
[0369] [00486] Assays of cytotoxicity and antiviral activity of respiratory syncytial virus (RSV) Anti-RSV activity
[0370] [00487] The antiviral activity against RSV is determined using an in vitro cytoprotection assay on Hep2 cells. In this assay, compounds inhibiting virus replication exhibit a cytoprotective effect against virus-induced cell death that can be quantified using a cell viability reagent. The method used is similar to the methods previously described in the published literature (Chapman et al., Antimicrob Agents Chemother. 2007, 51 (9): 3346-53.)
[0371] [00488] Hep2 cells are obtained from ATCC (Manassas, VI) and maintained in MEM medium supplemented with 10% fetal bovine serum and penicillin / streptomycin. The cells are passed twice a week, and maintained in confluent phases. Commercial stock of RSV strain A2 (Advanced Biotechnologies, Columbia, MD) is titrated before testing the compound to determine the appropriate dilution of the viral stock that generates a desirable cytopathic effect in Hep2 cells.
[0372] [00489] For antiviral tests, Hep2 cells were seeded in 96-well plates 24 hours before the assay at a density of 3000 cells / well. In a separate 96-well plate, the compounds to be tested are serially diluted in cell culture medium. Eight concentrations in three-fold serial dilution increments are prepared for each tested compound and 100 µl / well of each dilution is transferred in duplicate onto plates with seeded Hep2 cells. Subsequently, the appropriate dilution of virus stock previously determined by titration is prepared in cell culture medium and 100 µL / well is added to test plates containing the cells and compounds diluted in series. Each plate includes three wells of untreated infected cells and three wells of uninfected cells that served as controls for 0% and 100% virus inhibition, respectively. Following RSV infection, the test plates are incubated for 4 days in a tissue culture incubator. After incubation, RSV-induced cytopathic effect is determined using a TiterGlo reagent cell (Promega, Madison, WI) followed by a luminescence reading. Percent inhibition is calculated for each concentration tested against 0% and 100% inhibition controls and the EC50 value for each compound is determined by non-linear regression as a 50% RSV-induced cytopathic inhibition concentration. Ribavirin (acquired from Sigma, St. Louis, MO) is used as a positive control for antiviral activity. Cytotoxicity
[0373] [00490] The cytotoxicity of the tested compounds is determined in uninfected Hep2 cells in parallel with the antiviral activity using the cell viability reagent in a similar manner to that described above for other cell types (Cihlar et al., Antimicrob Agents Chemother. 2008 , 52 (2): 655-65.). The same protocol as for the determination of antiviral activity is used to measure the cytotoxicity of the compound except that the cells are not infected with RSV. Instead, fresh cell culture media (100 µL / well) without the virus is added to plates tested with prediluted cells and compounds. The cells are then incubated for 4 days followed by a cell viability test using the CellTiter Glo reagent and a luminescence reading. The untreated cell and cells treated with 50 µg / mL of puromycin (Sigma, St. Louis, MO) are used as controls for 100% and 0% cell viability, respectively. The percentage of cell viability is calculated for each concentration of the tested compound in relation to the 0% and 100% controls and the CC50 value is determined by non-linear regression as a concentration of compound reducing the cell viability by 50%.
[0374] [00491] RSV ribonucleoprotein (RNP) complexes were prepared using a modified method by Mason et al (1). HEp-2 cells were plated at a density of 7.1 x 104 cells / cm2 in MEM + 10% fetal bovine serum (FBS) and allowed to bind overnight at 37 ° C (5% CO2). After fixation, the cells were infected with RSV A2 (MOI = 5) in 35 mL of MEM + 2% FBS. At 20 hours after infection, the medium was replaced by MEM + 2% FBS supplemented with 2 µg / ml of actinomycin D and returned to 37 ° C for one hour. The cells were then washed once with PBS and treated with 35 ml of 250 µg / ml smooth-lecithin PBS for one minute, after which all the liquid was aspirated. The cells were harvested by demolishing them in 1.2 ml of buffer A [50 mM Tris acetate (pH 8.0), 100 mM potassium acetate, 1 mM DTT and 2 µg / ml actinomycin D] and lysed by passage repeated through an 18 gauge needle (10 times). The cell lysate was placed on ice for 10 minutes and then centrifuged at 2400g for 10 minutes at 4 ° C. The supernatant (S1) was removed and the pellet (P1) was interrupted in 600 µL of buffer B [10 mM Tris acetate (pH 8.0), 10 mM potassium acetate and 1.5 mM MgCl2] supplemented with 1% Triton X-100 by repeated passage through an 18 gauge needle (10 times). The resuspended pellet was placed on ice for 10 minutes and then centrifuged at 2400g for 10 minutes at 4 ° C. The supernatant (S2) was removed and the pellet (P2) was interrupted in 600 µL of buffer B supplemented with 0.5% deoxycholate and 0.1% Tween 40. The resuspended pellet was placed on ice for 10 minutes and then centrifuged. at 2400g for 10 minutes at 4 ° C. The supernatant fraction (S3), containing the enriched RSV RNP complexes, was collected and the protein concentration was determined by UV absorbance at 280 nm. Aliquoted S3 fractions of RSV RNP were stored at -80 ° C. RSV RNP Assay
[0375] [00492] Transcription reactions contained 25 µg of crude RSV RNP complexes in 30 µl of reaction buffer [50 mM TRIS-acetate (pH 8.0), 120 mM potassium acetate, 5% glycerol, 4.5 mM MgCl2, 3 mM DTT, 2 mM ethylene glycol-bis (2-aminoethylether) -tetraacetic (EGTA), 50 µg / mL BSA, 2.5 U RNasin (Promega), ATP, GTP, UTP, CTP and 1.5 uCi [α-32P] NTP (3000 Ci / mmol)]. The radiolabel nucleotide used in the transcription assay was selected to match the nucleotide analogue to be evaluated for inhibition of RSV RNP transcription. Cold competitive NTP was added to a final concentration of half its Km (ATP = 20 µM, GTP = 12.5 µM, UTP = 6 µM and CTP = 2 µM). The remaining three nucleotides were added in a final concentration of 100 µM.
[0376] 1) Mason, S., Lawetz, C., Gaudette, Y., Do, F., Scouten, E., Lagace, L., Simoneau, B. e Liuzzi, M. (2004) Poliadenilation-dependent screening assay for respiratory syncytial virus RNA transcriptase activity e identification of an inhibitor. Nucleic Acids Research, 32, 4758-4767.
[0377] [00494] The Parainfluenza Cytoprotection assay uses Vero cells and Parainfluenza 3 strain C 243. Briefly viruses and cells are mixed in the presence of test compound and incubated for 7 days. The virus is pre-titled so that control wells show 85 to 95% loss of cell viability due to virus replication. Therefore, the antiviral or cytoprotective effect is observed when compounds prevent the virus from replicating. Each assay plate contains cell control wells (cells only), virus control wells (cells plus virus), toxicity compound control wells (cells plus compound only), compound colorimetric control wells (compound only), as well as the experimental wells (composed of cells plus viruses). Compound cytoprotection and cytotoxicity are evaluated by MTS (CellTiter 96® reagent, Promega, Madison, WI) dye reduction. The% reduction in viral cytopathic effects (CPE) is determined and reported; IC50 (inhibitory concentration of virus replication by 50%), TC50 (concentration resulting in cell death of 50%) and a calculated TI (therapeutic index TC50 / IC50) are provided together with a graphical representation of the antiviral activity and cytotoxicity of the compound when the compounds are tested in dosage response. Each assay includes ribavirin as a positive control. Preparation of cells
[0378] [00495] Vero cells (kidney, African green monkey, Cercopithecus aetiops) were obtained from the American Type Culture Collection (ATCC, Rockville, Mariland) and are grown in Dulbecco medium modified by Eagle (DMEM) supplemented with 10% serum fetal bovine (FBS), 2.0 mM LGlutamine, 100 units / ml penicillin and 100 µg / ml streptomycin ("growth medium"). The cells are subcultured twice a week at a 1:10 split ratio using standard cell culture techniques. Determinations of total cell number and percentage of viability are performed using a hemocytometer and exclusion of blue trypan. The viability of the cells must be greater than 95% for the cells to be used in the assay. The cells are seeded in 96-well tissue culture plates the day before the assay at a concentration of 1 x 104 cells / well. Virus Preparation
[0379] [00496] The virus used for this assay is Parainfluenza 3 strain C 243. This virus was obtained from the American Type Culture Collection (ATCC) and was cultured in Vero cells to produce sets of stock viruses. For each assay, a pre-titrated virus aliquot is removed from the freezer (-80 ° C) and allowed to thaw slowly at room temperature in a biological safety chamber. The virus is resuspended and diluted in tissue culture medium so that the amount of virus added to each well is determined to generate between 85 and 95% of cell death in 6-7 days after infection. MTS staining for cell viability
[0380] [00497] In the termination test (7 days after infection), the test plates are stained with the soluble tetrazolium-based MTS dye (3- (4,5-dimethylthiazol-2-yl) -5- (3- carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium; CellTiter®96 reagent, Promega) to determine cell viability and quantify the toxicity of the compound. MTS is metabolized by the mitochondrial enzymes of metabolically active cells to generate a soluble formazan product, which allows rapid quantitative analysis of cell cytotoxicity and compound viability. This reagent is a single, stable solution that does not require preparation before use. At the end of the assay, 20-25 µL of MTS reagent is added per well and the microtiter plates are then incubated for 4-6 hours at 37 ° C, 5% CO2 to assess cell viability. Adhesive plate seals are used instead of caps, the sealed plate is inverted several times to mix the soluble formazan product and the plate is read spectrophotometrically at 490/650 nm with a Molecular Devices Vmax or SpectraMax Plus plate reader. Data analysis
[0381] [00498] Using an in-house computer program the% reduction of cytopathic effect (CPE),% of cell viability, IC25, IC50, IC95, TC25, TC50, and TC95 and other indices are calculated and the summary of the graphical result is introduced. The raw data for both antiviral activity and toxicity with a graphical representation of the data is provided in a printout summarizing the activity of the individual compound. The table below shows the activity of selected compounds against Parainfluenza 3 virus.
[0382] [00499] The specific pharmacological and biochemical responses observed in the described trials may vary according to and depending on the particular active compound selected or whether there are pharmaceutical vehicles present, as well as the type of formulation and mode of administration employed, and said expected variations or differences the results are contemplated according to the practice of the present invention.
[0383] [00500] All publications, patents and patent documents mentioned above are incorporated herein by reference, as if individually incorporated by reference.
[0384] [00501] The invention has been described with reference to several specific techniques and preferred and technical modalities. However, one skilled in the art will understand that many modifications and variations can be prepared while remaining within the spirit and scope of the invention.

权利要求:
Claims (19)
[0001]
Composed, characterized by the fact of presenting the structure:
[0002]
Compound according to claim 1, characterized by the fact that it has the structure:
[0003]
Compound according to claim 2, characterized by the fact that it has the structure:
[0004]
Compound according to claim 1, characterized by the fact that it has the structure:
[0005]
Compound according to claim 4, characterized by the fact that it has the structure:
[0006]
Compound according to any one of claims 1 to 5, characterized in that it is for use in the treatment of a Paramyxoviridae virus infection.
[0007]
. Compound according to claim 6, characterized by the fact that infection by Paramyxoviridae is caused by a Paramyxovirina virus.
[0008]
Compound according to claim 6, characterized by the fact that the infection by Paramyxoviridae is caused by a Respirovirus virus.
[0009]
Compound according to claim 6, characterized in that the infection by Paramyxoviridae is caused by a human parainfluenza virus type 1 or 3.
[0010]
Compound according to claim 6, characterized by the fact that the infection by Paramyxoviridae is caused by a Pneumovirinae virus.
[0011]
Compound according to claim 6, characterized by the fact that the infection by Paramyxoviridae is caused by a human respiratory syncytial virus.
[0012]
Pharmaceutical composition, characterized in that it comprises a compound defined in any one of claims 1 to 5 and a pharmaceutically acceptable carrier.
[0013]
Composition according to claim 12, characterized by the fact that it still comprises another agent selected from the group consisting of: (i) a corticosteroid selected from the group consisting of dexamethasone, dexamethasone sodium phosphate, fluormetolone, fluormetolone acetate, loteprednol, loteprednol etabonate, hydrocortisone, prednisolone, fludrocortisone, triamcinolone, triamcinone, triamcinone, acetone, triamcinolone, triamcinolone, triamcinone, acetonone fluocinolone acetonide, flunisolide, fluocortin-21-butylate, flumetasone, flumetasone pivalate, budesonide, halobetasol propionate, mometasone furoate, fluticasone propionate and ciclesonide, or a pharmaceutically acceptable salt thereof; (ii) an anti-inflammatory signal transduction modulator selected from the group consisting of 5- (2,4-difluoro-phenoxy) -1-isobutyl-1Hindazole-6-carboxylic acid (2-dimethylamino-ethyl) - amide (P38 Map kinase inhibitor ARRY-797); 3-cyclopropylmethoxy-N- (3,5-dichloro-pyridin-4-yl) -4-difluoromethoxybenzamide (PDE-4 inhibitor Roflumilast); 4- [2- (3-cyclopentyloxy-4-methoxyphenyl) - 2-phenyl-ethyl] -pyridine (PDE-4 inhibitor CDP-840); N- (3,5-dichloro-4-pyridinyl) -4- (difluoromethoxy) -8 - [(methylsulfonyl) amino] -1-dibenzofurancarboxamide (PDE-4 Oglemilast inhibitor); N- (3,5-dichloro-pyridin-4-yl) -2- [1- (4-fluorbenzyl) -5-hydroxy1H-indol-3-yl] -2-oxo-acetamide (PDE-4 AWD inhibitor 12-281); 8-methoxy2-trifluormethyl-quinoline-5-carboxylic acid (3,5-dichloro-1-oxy-pyridin-4-yl) -amide (PDE-4 Sch 351591 inhibitor); 4- [5- (4-fluorophenyl) -2- (4-methanesulfinyl-phenyl) - 1H-imidazol-4-yl] -pyridine (P38 SB-203850 inhibitor); 4- [4- (4-fluoro-phenyl) -1- (3-phenyl-propyl) -5-pyridin-4-yl-1H-imidazol-2-yl] -but-3-in-1-ol ( P38 inhibitor RWJ67657); 4-cyano-4- (3-cyclopentyloxy-4-methoxyphenyl) -cyclohexanecarboxylic acid 2-diethylamino-ethyl ester (Cilomilast 2-diethyl-ethyl ester prodrug, PDE-4 inhibitor); (3-chloro-4-fluorophenyl) - [7-methoxy-6- (3-morpholin-4-yl-propoxy) - quinazolin-4-yl] -amine (Gefitinib, EGFR inhibitor) and 4- (4- methyl-piperazin-1-ylmethyl) -N- [4-methyl-3- (4-pyridin-3-yl-pyrimidin-2-ylamino) -phenyl] -benzamide (Imatinib, EGFR inhibitor); (iii) a β2-adrenoreceptor agonist bronchodilator selected from the group consisting of formoterol, albuterol or salmeterol, (iv) an anticholinergic selected from the group consisting of 1- {4-hydroxy-1- [3,3,3-tris- (4-fluorophenyl) -propionyl] -pyrrolidine-2-carbonyl} - pyrrolidine- 2-carboxylic (1-methyl-piperidin-4-ylmethyl) -amide; 3- [3- (2-diethylaminoacetoxy) -2-phenyl-propionyloxy] -8-isopropyl-8-methyl-8-azonia-bicyclo [3.2.1] octane (Ipratropium-N, N-diethylglycinate); 1-aza-bicyclo [2.2.2] oct-3-yl ester of 1-cyclohexyl-3,4-dihydro-1H-isoquinoline-2-carboxylic acid (Solifenacin); 1-azabicyclo [2.2.2] 2-hydroxymethyl-4-methanesulfinyl-2-phenylbutyric acid oct-3-yl ester (Revatropate); 2- {1- [2- (2,3-dihydro-benzofuran-5-yl) -ethyl] -pyrrolidin-3-yl} -2,2-diphenyl-acetamide (Darifenacin); 4-azepan-1-yl-2,2-diphenyl-butyramide (Buzepide); 7- [3- (2-diethylamino-acetoxy) -2-phenyl-propionyloxy] -9-ethyl-9-methyl-3-oxa-9-azonia-tricycle [3.3.1.02,4] nonane (Oxitrope-N, N-diethylglycinate); 7- [2- (2-diethylamino-acetoxy) -2,2-di-thiophen-2-yl-acetoxy] -9,9-dimethyl-3-oxa-9-azoniatricycle [3.3.1.02,4] nonane ( Tiotropium-N, N-diethylglycinate); 2- (3-diisopropylamino-1-phenyl-propyl) -4-methyl-phenyl ester of dimethylamino-acetic acid (Tolterodine-N, N-dimethylglycinate); 3- [4,4-bis- (4-fluorophenyl) -2-oxo-imidazolidin1-yl] -1-methyl-1- (2-oxo-2-pyridin-2-yl-ethyl) -pyrrolidinium; 1- [1- (3-fluoro-benzyl) -piperidin-4-yl] -4,4-bis- (4-fluorophenyl) -imidazolidin-2-one; 1-cyclooctyl-3- (3-methoxy-1-aza-bicyclo [2.2.2] oct-3-yl) -1-phenyl-prop-2-in-1-ol; 3- [2- (2-diethylaminoacetoxy) -2,2-di-thiophen-2-yl-acetoxy] -1- (3-phenoxy-propyl) -1-azoniabicyclo [2.2.2] octane (Aclidinio-N, N-diethylglycinate) or 1-methyl-1- (2-phenoxy-ethyl) -piperidin-4-yl ester of (2-diethylamino-acetoxy) -di-thiophen-2-yl-acetic acid; (v) ambroxol mucolytic agent; and (vi) hypertonic saline; or mixtures thereof.
[0014]
Pharmaceutical composition according to claim 12 or 13, characterized in that it is for use in the treatment of a Paramyxoviridae virus infection.
[0015]
Pharmaceutical composition according to claim 14, characterized by the fact that the infection by Paramyxoviridae is caused by a Paramyxovirina virus.
[0016]
Pharmaceutical composition according to claim 14, characterized by the fact that the infection by Paramyxoviridae is caused by a Respirovirus virus.
[0017]
Pharmaceutical composition according to claim 14, characterized by the fact that the infection by Paramyxoviridae is caused by a human parainfluenza virus type 1 or 3.
[0018]
Pharmaceutical composition according to claim 14, characterized by the fact that the infection by Paramyxoviridae is caused by a Pneumovirinae virus.
[0019]
Pharmaceutical composition according to claim 14, characterized by the fact that the infection by Paramyxoviridae is caused by a human respiratory syncytial virus.

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RU2756921C1|2020-11-20|2021-10-07|Общество С Ограниченной Ответственностью "Технология Лекарств"|Method for obtaining remdesivir and phosphoramidates|

法律状态:
2020-06-02| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2020-06-09| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-06-23| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-07-14| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2020-11-24| B09A| Decision: intention to grant|

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2021-01-12| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 22/07/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US36660910P| true| 2010-07-22|2010-07-22|

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US61/366,609|2010-07-22|

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PCT/US2011/045102|WO2012012776A1|2010-07-22|2011-07-22|Methods and compounds for treating paramyxoviridae virus infections|

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