Pyrrolo [1, 2-f] [1,2,4] triazines useful for treating respiratory syncytial virus infections

Abstract

To provide a new antiviral agent useful for treating Paramyxoviridae virus infections, including Pneumovirinae virus infections such as HRSV infections, having an effective and acceptable toxicity profile.SOLUTION: Provided herein are formulations, methods, and substituted tetrahydrofuranyl-pyrrolo [1, 2-f] [1,2,4] triazin-4-amine compounds of formula (I), and methods and intermediates for synthesizing tetrahydrofuranyl-pyrrolo [1, 2-f] [1,2,4] triazin-4-amine compounds, for treating Pneumovirinae virus infections, including respiratory syncytial virus infections.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Field Provided herein are substituted tetrahydrofuranyl-pyrrolo[1,2-f][1,2,4]triazin-4-amine compounds, methods and pharmaceutical formulations for treating Pneumovirinae virus infections, including particularly respiratory syncytial virus infections, as well as methods and intermediates useful for preparing the compounds. [Background technology]

[0002] background Pneumovirinae viruses are single-stranded, negative-sense RNA viruses that cause many endemic human and animal diseases. The Pneumovirinae subfamily of viruses is part of the Paramyxoviridae family and includes human respiratory syncytial virus (HRSV). Almost all children will acquire an HRSV infection by their second birthday. HRSV is a leading cause of lower respiratory tract infections in infants and children, with 0.5%–2% of infected individuals requiring hospitalization. Elderly and adults with chronic heart or lung disease or immunosuppression are also at high risk for developing severe HRSV disease (http: / / www.cdc.gov / rsv / index.html). No vaccine is currently available to prevent HRSV infection. The monoclonal antibody palivizumab is available for immunoprophylaxis, but its use is limited to high-risk infants, such as premature infants or those with congenital heart or lung disease, and its cost is often prohibitive for general use. Furthermore, the nucleoside analog ribavirin is the only antiviral agent approved for treating HRSV infection, but its efficacy is limited. Therefore, there is a need for anti-Pneumovirinae therapeutics.

[0003] Examples of pyrrolo[2,3-d]pyrimidine compounds useful for treating viral infections are disclosed in US2012 / 0009147A1 (Cho et al.), US2012 / 0020921A1 (Cho et al.), WO2008 / 089105A2 (Babu et al.), WO2008 / 141079A1 (Babu et al.), WO2009 / 132135A1 (Butler et al.), WO2010 / 002877A2 (Francom), WO2011 / 035231A1 (Cho et al.), WO2011 / 035250A1 (Butler et al.), WO2011 / 150288A1 (Cho et al.), WO2012 / 012465 (Cho et al.), WO2012 / 012776A1 (Mackman et al.), WO2012 / 037038 (Clarke et al.), WO2012 / 087596A1 (Delaney et al.) and WO2012 / 142075A1 (Girijavallabhan et al.).

[0004] There remains a need for new antiviral agents useful for treating Paramyxoviridae virus infections, including Pneumovirinae virus infections such as HRSV infections, that are effective and have an acceptable toxicity profile. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] US Patent Application Publication No. 2012 / 0009147 [Patent Document 2] US Patent Application Publication No. 2012 / 0020921 [Patent Document 3] International Publication No. 2008 / 089105 [Patent Document 4] International Publication No. 2008 / 141079 [Patent Document 5] International Publication No. 2009 / 132135 [Patent Document 6] International Publication No. 2010 / 002877 [Patent Document 7] International Publication No. 2011 / 035231 [Patent Document 8] International Publication No. 2011 / 035250 [Patent Document 9] International Publication No. 2011 / 150288 [Patent Document 10] International Publication No. 2012 / 012465 [Patent Document 11] International Publication No. 2012 / 012776 [Patent Document 12] International Publication No. 2012 / 037038 [Patent Document 13] International Publication No. 2012 / 087596 [Patent Document 14] International Publication No. 2012 / 142075 Summary of the Invention [Means for solving the problem]

[0006] overview Compounds, methods and pharmaceutical formulations are provided for treating infections caused by the Pneumovirinae virus family, including the treatment of infections caused by human respiratory syncytial virus.

[0007] A compound of formula (I) or a pharmaceutically acceptable salt thereof [ka] is provided, wherein R 1 is H or F, R 2 is H or F, R 3 is OH or F, R 4 is CN, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C4 cycloalkyl, azido, halogen or C1-C2 haloalkyl; R 6 is OH, R5 is H and [ka] wherein: n' is selected from 1, 2, 3 and 4; R 8 is selected from C1-C8 alkyl, —O—C1-C8 alkyl, benzyl, —O-benzyl, —CH2—C3-C6 cycloalkyl, —O—CH2—C3-C6 cycloalkyl, and CF3; R 9 is phenyl, 1-naphthyl, 2-naphthyl, [ka] is selected from R 10 is selected from H and CH3; R 11 is selected from H or C1-C6 alkyl; R 12 is selected from H, C1-C8 alkyl, benzyl, C3-C6 cycloalkyl and —CH2—C3-C6 cycloalkyl. DETAILED DESCRIPTION OF THE INVENTION

[0008] Detailed Description One embodiment herein is R 1 is H and R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 and n', or a pharmaceutically acceptable salt thereof, wherein all other variables, including n, are as defined above for formula (I).

[0009] Another embodiment herein is R 2 is H and R1 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 and n', or a pharmaceutically acceptable salt thereof, in which all other variables, including n, are as defined above for formula (I).

[0010] Further embodiments herein include R 1 and R 2 Both are H and R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 and n', or a pharmaceutically acceptable salt thereof, in which all other variables, including n, are as defined above for formula (I).

[0011] Yet another embodiment herein is R 1 , R 2 and R 5 are H, and R 3 , R 4 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 and n', or a pharmaceutically acceptable salt thereof, in which all other variables, including n, are as defined above for formula (I).

[0012] Another separate embodiment herein is R 1 and R 2 Both are H and R 3 is OH and R 4 , R5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 and n', or a pharmaceutically acceptable salt thereof, in which all other variables, including n, are as defined above for formula (I).

[0013] Another separate embodiment herein is R 1 and R 2 Both are H and R 3 is F and R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 and n', all other variables, including n, are as defined above for Formula (I), including compounds of Formula (I) or pharmaceutically acceptable salts thereof.

[0014] Another embodiment provided herein is a compound of formula (II) or a pharmaceutically acceptable salt thereof: [ka] wherein R 3 is OH or F, R 4 is CN, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C4 cycloalkyl, azido, halogen or C1-C2 haloalkyl; R 5 is H and [ka] wherein: n' is selected from 1, 2, 3 and 4; R 8is selected from C1-C8 alkyl, —O—C1-C8 alkyl, benzyl, —O-benzyl, —CH2—C3-C6 cycloalkyl, —O—CH2—C3-C6 cycloalkyl, and CF3; R 9 is phenyl, R 10 is selected from H and CH3; R 11 is selected from H or C1-C6 alkyl; R 12 is selected from H, C1-C8 alkyl, benzyl, C3-C6 cycloalkyl and —CH2—C3-C6 cycloalkyl.

[0015] A further embodiment is R 3 is OH or F, R 4 is CN, methyl, ethyl, ethenyl, ethynyl, azido, F, Cl, -CH2Cl, -CH2F, -CHF2 or -CF3, R 5 and all other groups are as defined for formula (II). The compound includes a compound of formula (II) or a pharmaceutically acceptable salt thereof:

[0016] Similarly, R 3

[0023] Embodiments are provided which include compounds of formula (II), as described above, or pharmaceutically acceptable salts thereof, wherein is F.

[0017] Similarly, R 3 Embodiments are provided which include compounds of formula (II), as described above, or pharmaceutically acceptable salts thereof, wherein is OH.

[0018] Similarly, R 3 is F and R 4

[0023] Embodiments are provided which include compounds of formula (II), as described above, or pharmaceutically acceptable salts thereof, wherein is CN.

[0019] Similarly, R 3 is OH and R 4

[0023] Embodiments are provided which include compounds of formula (II), as described above, or pharmaceutically acceptable salts thereof, wherein is CN.

[0020] Similarly, R 1 and R 2 Both are H and R 3 is F and R 4 Embodiments are provided which include compounds of formula (II), as described above, or pharmaceutically acceptable salts thereof, wherein is methyl, ethyl, vinyl, or ethynyl.

[0021] Similarly, R 3 is OH and R 4 Embodiments are provided which include compounds of formula (II), as described above, or pharmaceutically acceptable salts thereof, wherein is methyl, ethyl, vinyl, or ethynyl.

[0022] Similarly, R 3 is F and R 4 Embodiments are provided which include compounds of formula (II), as described above, or pharmaceutically acceptable salts thereof, wherein is halomethyl.

[0023] Similarly, R 3 is OH and R 4 Embodiments are provided which include compounds of formula (II), as described above, or pharmaceutically acceptable salts thereof, wherein is halomethyl.

[0024] Similarly, R 5

[0023] Embodiments are provided which include compounds of formula (II), as described above, wherein is H, or a pharmaceutically acceptable salt thereof.

[0025] Similarly, each R 5 is H and R 3 is OH and R 4 Embodiments are provided which include compounds of formula (II), as described above, or pharmaceutically acceptable salts thereof, wherein is methyl, ethyl, vinyl, or ethynyl.

[0026] Similarly, R 5 is H and R 3 is F and R 4Embodiments are provided which include compounds of formula (II), as described above, or pharmaceutically acceptable salts thereof, wherein is halomethyl.

[0027] Similarly, R 5 is H and R 3 is OH and R 4 Embodiments are provided which include compounds of formula (II), as described above, or pharmaceutically acceptable salts thereof, wherein is halomethyl.

[0028] R 5 Within each of the above embodiments, which include compounds of Formula (II) or a pharmaceutically acceptable salt thereof, where R may be other than H, all other variables are as described for that embodiment, and R 5 but, [ka] [ka] is selected from the group of R 8 is selected from C1-C8 alkyl, —O—C1-C8 alkyl, benzyl, and —CH2—C3-C6 cycloalkyl; R 12 There are further embodiments where is selected from C1-C8 alkyl, benzyl, C3-C6 cycloalkyl, and -CH2-C3-C6 cycloalkyl.

[0029] Within each of the immediately preceding embodiments, R 8 and R 9 are C1 to C 8 alkyl. Within each of the embodiments described in the immediately preceding sentence, there are further embodiments which include compounds of formula (II) or a pharmaceutically acceptable salt thereof, wherein all of the other variables are as immediately above described, except that R is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 5 8 and R 9Additional embodiments exist which include compounds of formula (II) or a pharmaceutically acceptable salt thereof, wherein all of the other variables are as immediately above described, except that each R is selected from C1-C6 alkyl. Within each of the embodiments described in the immediately above, 8 and R 9 Additional embodiments exist which include compounds of formula (II) or a pharmaceutically acceptable salt thereof, wherein all of the other variables are as immediately above described, except that each R is selected from C1-C5 alkyl. Within each of the embodiments described in the immediately above, 8 and R 9 Additional embodiments exist which include compounds of formula (II) or a pharmaceutically acceptable salt thereof, wherein all of the other variables are as immediately above described, except that each is selected from C1-C4 alkyl.

[0030] Within each of the embodiments described herein, including compounds of Formula (I) or Formula (II), all variables are as defined for the particular embodiment, and R 3 If F, then R 4 Further embodiments exist which further include the proviso that is not methyl.

[0031] definition The terms halo and halogen refer to halogen atoms selected from F, Cl, Br and I.

[0032] "Azide" refers to an azide group, i.e., an -N3 group. The term "n," as used herein, refers to an integer such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, i.e., an integer selected from 2 to 20 or 2-20. In some examples, "n" refers to a group of integers such as 1-3, 1-4, 1-6, 1-8, 2-4, 2-6, 2-8, etc.

[0033] The term "haloalkyl," as used herein, refers to an alkyl, as defined herein, in which one or more hydrogen atoms are each replaced by a halo substituent. For example, a (C-C)haloalkyl is a (C-C)alkyl in which one or more hydrogen atoms are replaced by a halo substituent. This range includes from one halo substituent on the alkyl group to complete halogenation of the alkyl group.

[0034] The term "(C 1~n "Haloalkyl," as used herein, is intended to mean an alkyl radical having 1 to n carbon atoms, as defined above, in which one or more hydrogen atoms are each replaced by a halo substituent. (C 1~n ) Examples of haloalkyl include, but are not limited to, chloromethyl, chloroethyl, dichloroethyl, bromomethyl, bromoethyl, dibromoethyl, fluoromethyl, difluoromethyl, trifluoromethyl, fluoroethyl, and difluoroethyl.

[0035] The term "(C 1~n "(C ) alkyl," as used herein, is intended to mean an acyclic, straight-chain or branched-chain alkyl radical containing 1 to n carbon atoms. 1~4 )Alkyl" includes, but is not limited to, methyl, ethyl, propyl (n-propyl), butyl (n-butyl), 1-methylethyl (iso-propyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (iso-butyl), and 1,1-dimethylethyl (tert-butyl or t-butyl). The abbreviation Me refers to a methyl group. Et means an ethyl group, Pr means a propyl group, iPr means a 1-methylethyl group, Bu means a butyl group, and tBu means a 1,1-dimethylethyl group.

[0036] The term "alkyl" refers to a hydrocarbon containing normal, secondary, or tertiary atoms. For example, an alkyl group can have 1 to 4 carbon atoms (i.e., (C1-C4) alkyl), 1 to 3 carbon atoms (i.e., (C1-C3) alkyl), or 1 or 2 carbon atoms (i.e., (C1-C2) alkyl). Examples of suitable alkyl groups include, but are not limited to, methyl (Me, -CH), ethyl (Et, -CHCH), 1-propyl (n-Pr, n-propyl, -CHCHCH), 2-propyl (i-Pr, i-propyl, -CH(CH)), 1-butyl (n-Bu, n-butyl, -CHCHCHCH), 2-methyl-1-propyl (i-Bu, i-butyl, -CHCH(CH)), 2-butyl (s-Bu, s-butyl, -CH(CH)CHCH), and 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH)). "Alkyl" also refers to a saturated branched or straight-chain hydrocarbon radical having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkyl 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.

[0037] "Alkenyl" means an alkenyl group having at least one site of unsaturation, i.e., carbon-carbon sp 2 It is a straight-chain or branched-chain hydrocarbon containing normal, secondary, or tertiary carbon atoms with a double bond. For example, an alkenyl group can have 2 to 4 carbon atoms (i.e., C2-C4 alkenyl) or 2 to 3 carbon atoms (i.e., C2-C3 alkenyl). Examples of suitable alkenyl groups include, but are not limited to, ethylene or vinyl (-CH=CH2) and allyl (-CH2CH=CH2).

[0038] The term "(C 2~n "(C)alkenyl," as used herein, is intended to mean an unsaturated, acyclic, straight- or branched-chain radical containing 2 to n carbon atoms, at least two of which are joined to each other by a double bond. Examples of such radicals include, but are not limited to, ethenyl (vinyl), 1-propenyl, 2-propenyl, and 1-butenyl. Unless otherwise specified, the term "(C)alkenyl" is intended to mean an unsaturated, acyclic, straight- or branched-chain radical containing 2 to n carbon atoms, at least two of which are joined to each other by a double bond. Examples of such radicals include, but are not limited to, ethenyl (vinyl), 1-propenyl, 2-propenyl, and 1-butenyl. 2~n (C)alkenyl" is understood to encompass the possible individual stereoisomers, including but not limited to the (E) and (Z) isomers, and mixtures thereof. 2~n When a (C) alkenyl group is substituted, unless otherwise specified, the substitutions should be such that the resulting compound is chemically stable, such as would be recognized by one of ordinary skill in the art. 2~n ) An alkenyl group is understood to be substituted on any carbon atom thereof that would otherwise bear a hydrogen atom.

[0039] "Alkynyl" refers to a straight or branched chain hydrocarbon containing normal, secondary, or tertiary carbon atoms with at least one site of unsaturation, i.e., a carbon-carbon triple bond. For example, an alkynyl group can have 2 to 4 carbon atoms (i.e., a C2-C4 alkynyl) or 2 to 3 carbon atoms (i.e., a C2-C3 alkyne). Examples of suitable alkynyl groups include, but are not limited to, acetylene (-C≡CH), propargyl (-CH2C≡CH), and the like.

[0040] The term "(C 2~n )alkynyl" as used herein refers to an unsaturated alkyl group containing 2 to n carbon atoms. It is intended to mean an acyclic, straight-chain or branched-chain radical, at least two of whose carbon atoms are joined to each other by a triple bond. Examples of such radicals where n is 4 include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and 1-butynyl. (C 2~n When a (C) alkynyl group is substituted, unless otherwise specified, the substitutions should be such that the resulting compound is chemically stable, such as would be recognized by one of ordinary skill in the art. 2~n ) An alkynyl group is understood to be substituted on any carbon atom thereof that would otherwise bear a hydrogen atom.

[0041] The term cycloalkyl refers to a cyclic aliphatic group. Cycloalkyl groups may be referred to herein by the number of carbon atoms in their ring, such as "C-C cycloalkyl," which refers to a cycloalkyl ring having three or four carbon ring atoms, or "C-C cycloalkyl," which indicates a cycloalkyl ring having three, four, five, or six carbon ring atoms, i.e., a cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl ring.

[0042] The term "carbocycle" or "carbocyclyl" refers to a saturated (i.e., cycloalkyl) or partially unsaturated (e.g., cycloalkenyl, cycloalkadienyl, etc.) ring having a specified number of carbon atoms, such as 3 to 4 carbon atoms or 3 to 6 carbon atoms, as a monocyclic ring system. In one embodiment, a carbocycle is a monocycle containing 3 to 6 ring carbons (i.e., a (C3-C6)carbocycle). 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 cyclohexa-1,3-dienyl rings.

[0043] Each carbocyclyl group is optionally substituted by 0, 1, 2, or 3 substituents independently selected from halogen, —OH, —CN, —NO2, —NH2, —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, C1-C6 alkyl, C1-C6 alkoxy, and —CF3.

[0044] Pharmaceutical preparations Similarly, provided herein is a pharmaceutical formulation comprising a pharmaceutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, solvate, and / or ester thereof, and a pharmaceutically acceptable carrier or excipient. Similarly, provided are separate pharmaceutical formulations, each comprising a pharmaceutically effective amount of a compound of formula (II) or one of the specific compounds of the Examples herein, or a pharmaceutically acceptable salt, solvate, and / or ester thereof, and a pharmaceutically acceptable carrier or excipient.

[0045] The compounds herein are formulated with conventional carriers and excipients, selected in accordance with routine practice. Tablets contain additives, glidants, fillers, binders, and the like. Aqueous formulations are prepared in sterile form and, if intended for delivery by routes other than oral administration, are generally isotonic. All formulations optionally contain additives, such as those described in the "Handbook of Pharmaceutical Excipients" (1986). Additives include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextran, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, and stearic acid. The pH of the formulations ranges from about 3 to about 11, but is usually about 7 to 10.

[0046] While it is possible for the active ingredients to be administered alone, it may be desirable to present them as pharmaceutical formulations. Formulations for both veterinary and human use may be as defined above. The formulation comprises at least one active ingredient as described above, together with one or more acceptable carriers, and optionally other therapeutic ingredients, particularly additional therapeutic ingredients discussed herein. The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and physiologically innocuous to the recipient thereof.

[0047] Formulations include those suitable for the above-mentioned administration routes. Formulations can be conveniently provided in unit dosage form and can be prepared by any method well known in the field of pharmacy. Techniques and formulations are generally found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). Such methods include the step of bringing active ingredient into association with a carrier that constitutes one or more accessory ingredients. Generally, formulations are prepared by bringing active ingredient into uniform and intimate association with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0048] Formulations suitable for oral administration may be presented as discrete units such as capsules, cachets, or tablets, each containing a predetermined amount of the active ingredient as a powder or granules, as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion. The active ingredient may also be administered as a bolus, electuary, or paste.

[0049] Tablets are made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing the active ingredient in a free-flowing form, such as a powder or granules, optionally mixed with a binder, lubricant, inert excipient, preservative, surfactant, or dispersant, in a suitable machine. Molded tablets can be made by molding a mixture of powdered active ingredient moistened with an inert liquid excipient in a suitable machine. Tablets can optionally be coated or scored, and are optionally formulated to delay or control the release of the active ingredient therefrom.

[0050] For infections of the eye or other external tissues, e.g., mouth and skin, formulations are preferably applied as topical ointments or creams containing the active ingredient in an amount of, for example, 0.075 to 20% w / w (including ranges of between 0.1% and 20% active ingredient, in 0.1% w / w increments such as 0.6% w / w, 0.7% w / w, etc.), preferably 0.2 to 15% w / w, and most preferably 0.5 to 10% w / w. When formulated as an ointment, the active ingredient can be used with either a paraffinic or water-miscible ointment base. Alternatively, the active ingredient can be formulated in a cream with an oil-in-water cream base.

[0051] If desired, the aqueous phase of the cream base can contain, for example, at least 30% w / w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups, such as propylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol, and polyethylene glycol (including PEG 400), and mixtures thereof. It may be desirable for topical formulations to include a compound that enhances absorption or penetration of the active ingredient through the skin or other affected area. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogs.

[0052] The oil phase of the emulsion may be composed of known ingredients in a known manner. The phase may comprise a simple emulsifier (otherwise known as an emulgent), but desirably comprises a mixture of at least one emulsifier with a fat or oil, or both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier, which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifiers, with or without stabilizers, constitute a so-called emulsifiable wax, and the wax together with the oil and fat is The ointment base constitutes the so-called emulsifiable ointment base, which forms the oily dispersed phase of the cream formulation.

[0053] Emulgents and emulsion stabilizers suitable for use in the formulation include Tween® 60, Span® 80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.

[0054] The selection of suitable oils or fats for the formulation is based on achieving the desired aesthetic properties. Creams should preferably be non-sticky, non-staining, and washable, with a viscosity suitable for preventing leakage from tubes or other containers. Linear or branched mono- or dibasic alkyl esters such as diisoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, and 2-ethylhexyl palmitate, or a blend of branched esters known as Crodamol CAP, the latter three being preferred esters, may be used. These may be used alone or in combination depending on the desired properties. Alternatively, high-melting lipids such as white soft paraffin and / or liquid paraffin, or other mineral oils, may be used.

[0055] The pharmaceutical preparations herein comprise one or more pharmaceutically acceptable carriers or additives, and optionally other therapeutic agents in combination.The pharmaceutical preparations containing active ingredients can be in any form suitable for the intended method of administration.For oral use, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, liquids, syrups or elixirs can be prepared.Compositions for oral use can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and these compositions may contain one or more agents, including sweeteners, flavoring agents, coloring agents and preservatives, to produce palatable preparations.Tablets containing active ingredients in admixture with non-toxic pharmaceutically acceptable additives suitable for the manufacture of tablets are acceptable. These additives may be, for example, inert fillers such as calcium or sodium carbonate, lactose, calcium or sodium phosphate, granulating and disintegrating agents such as corn starch or alginic acid, binders such as starch, gelatin or acacia, and lubricants such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or may be coated by known techniques, including microencapsulation, to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate, alone or with a wax, may be employed.

[0056] Formulations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid excipient, for example, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.

[0057] Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions, such as suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, dispersing or wetting agents such as natural phospholipids (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long-chain aliphatic alcohols, and the like. Examples of suitable esters include ethylene oxide (e.g., heptadecaethyleneoxycetanol), and condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). Aqueous suspensions may also contain one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0058] Oily suspensions can be prepared by suspending active ingredients in vegetable oils such as peanut oil, olive oil, sesame oil or coconut oil, or in mineral oils such as liquid paraffin.Oral suspensions can contain thickening agents such as beeswax, hard paraffin or cetyl alcohol.Sweeteners and flavoring agents such as those described above can be added to make oral preparations palatable.These compositions can be preserved by adding antioxidants such as ascorbic acid.

[0059] Dispersible powders and granules suitable for preparing aqueous suspension by adding water provide the active ingredient in a 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 disclosed above.Additional additives, such as sweeteners, flavoring agents, and coloring agents, may also be present.

[0060] Pharmaceutical compositions can also be in the form of oil-in-water emulsions.The oil phase can be vegetable oils such as olive oil or peanut oil, mineral oils such as liquid paraffin, or mixtures thereof.Suitable emulsifiers include natural gums such as gum acacia and gum tragacanth, natural phospholipids such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and the condensation products of these partial esters with ethylene oxide, such as polyoxyethylenesorbitan monooleate.Emulsions can also contain sweeteners and flavoring agents.Syrups and elixirs can be formulated with sweeteners such as glycerol, sorbitol, or sucrose.These formulations can also contain demulcents, preservatives, flavorings, or coloring agents.

[0061] The pharmaceutical compositions may be in the form of a sterile injectable or intravenous preparation, such as a sterile injectable aqueous or oleaginous suspension. Such suspensions can be formulated according to known techniques using the above-mentioned dispersing or wetting agents and suitable suspending agents. Sterile injectable or intravenous preparations can also be prepared as sterile injectable solutions or suspensions in non-toxic parenterally acceptable excipients or solvents, such as 1,3-butanediol solution, or as lyophilized powders. Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile, fixed oils can be conventionally used as solvents or suspending media. For this purpose, any non-irritating, fixed oil, including synthetic monoglycerides or diglycerides, can be used. Additionally, fatty acids such as oleic acid can also be used in the preparation of injectable substances.

[0062] The amount of active ingredient that can be combined with carrier materials to produce a single dosage form will vary depending on the host treated and the particular mode of administration. For example, a sustained-release formulation intended for oral administration to humans may contain about 1 to 1,000 mg of active ingredient, combined with an appropriate and convenient amount of carrier material, which may vary from about 5 to about 95% (weight:weight) of the total composition. Pharmaceutical compositions can be prepared to provide easily measurable amounts for administration. For example, an aqueous solution intended for intravenous infusion may contain about 3 to 500 μg of active ingredient per milliliter of solution, with the aim that infusion of a suitable volume can occur at a rate of about 30 mL / hour.

[0063] Formulations suitable for topical administration to the eye also include eye drops, in which the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent, for the active ingredient. The active ingredient is preferably present in such formulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10%, especially about 1.5% w / w.

[0064] Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0065] Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.

[0066] Formulations suitable for pulmonary or nasal administration have particle sizes ranging from 0.1 to 500 microns, e.g., 0.5, 1, 30, or 35 microns, and are administered by rapid inhalation through the nasal passages or by inhalation through the oral cavity to reach the alveolar sacs. Suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol or dry powder administration can be prepared according to conventional methods and may be delivered with other therapeutic agents, such as compounds previously used in the treatment or prevention of Pneumovirinae infections, as described below.

[0067] Another embodiment provides novel, effective, safe, non-irritating, and physiologically compatible inhalable compositions comprising a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof, suitable for treating Pneumovirinae infections and potentially related bronchiolitis. Preferred pharmaceutically acceptable salts are inorganic acid salts, including hydrochloride, hydrobromide, sulfate, or phosphate, which are less likely to cause significant pulmonary irritation. Preferably, the inhalable formulation is delivered to the endobronchial space in an aerosol containing particles having a mass median aerodynamic diameter (MMAD) of about 1 to about 5 μm. Preferably, the compound of Formula (I) or Formula (II) is formulated for aerosol delivery using a nebulizer, a pressurized metered dose inhaler (pMDI), or a dry powder inhaler (DPI).

[0068] Non-limiting examples of nebulizers include spray, jet, ultrasonic, pressurized, vibrating porous plate, or equivalent nebulizers, including those utilizing adaptive aerosol delivery technologies (Denyer, J. Aerosol medicine Pulmonary Drug Delivery 2010, Vol. 23, Suppl. 1, pp. S1-S10). Jet nebulizers use air pressure to break up liquid solutions into aerosol droplets. Ultrasonic nebulizers work by using piezoelectric crystals to shear liquids into small aerosol droplets. Pressurized nebulization systems force solutions under pressure through small pores to generate aerosol droplets. Vibrating porous plate devices use rapid vibrations to shear a liquid stream into the appropriate droplet size.

[0069] In a preferred embodiment, the nebulized formulation is delivered to the endobronchial space in an aerosol containing primarily particles having an MMAD between about 1 μm and about 5 μm using a nebulizer capable of aerosolizing a formulation of a compound of Formula (I) or Formula (II) into particles of the required MMAD. For optimal therapeutic efficacy and to avoid upper airway and systemic side effects, the majority of the aerosolized particles should not have an MMAD greater than about 5 μm. If the aerosol contains a large number of particles with an MMAD greater than 5 μm, the particles will deposit in the upper airways, thereby limiting the amount of drug delivered to sites of inflammation and bronchoconstriction in the lower airways. If the MMAD of the aerosol is smaller than about 1 μm, the particles tend to remain suspended in the inhaled air and are subsequently exhaled during exhalation.

[0070] When formulated and delivered according to the methods herein, the aerosol formulation for nebulization delivers a therapeutically effective dose of a Formula (I) or Formula (II) compound to the site of a Pneumovirinae infection sufficient to treat the Pneumovirinae infection. The amount of drug administered should be adjusted to reflect the efficiency of delivering a therapeutically effective dose of a Formula (I) or Formula (II) compound. In preferred embodiments, combining an aqueous aerosol formulation with a nebulizer, jet, pressurized, vibrating porous plate, or ultrasonic nebulizer allows delivery of at least about 20 to about 90%, typically about 70%, of the administered dose of a Formula (I) or Formula (II) compound to the airways, depending on the nebulizer. In preferred embodiments, at least about 30 to about 50% of the active compound is delivered. More preferably, about 70 to about 90% of the active compound is delivered.

[0071] In another embodiment, the compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof is delivered as a dry, inhalable powder. The compound is administered intrabronchially using a dry powder or metered-dose inhaler as a dry powder formulation, which effectively delivers fine particles of the compound to the endobronchial space. For delivery via a DPI, the compound of Formula (I) or Formula (II) is processed into particles having a predominant MMAD between about 1 μm and about 5 μm by milling, spray drying, critical fluid processing, or precipitation from solution. Media milling, jet milling, and spray drying equipment and procedures capable of producing particle sizes having an MMAD between about 1 μm and about 5 μm are well known in the art. In one embodiment, additives are added to the compound of Formula (I) or Formula (II) before processing into particles of the required size. In another embodiment, additives are blended with particles of the required size to aid in the dispersion of the drug particles, for example, by using lactose as an additive.

[0072] Particle size determination is performed using equipment known in the art, such as a multi-stage Anderson cascade impactor, or other suitable methods, such as those specifically cited in U.S. Pharmacopeia Chapter 601 as characterizing devices for aerosols in metered dose nebulizers and dry powder inhalers.

[0073] In another preferred embodiment, the compound of formula (I) or formula (II) is delivered as a dry powder using a device such as a dry powder inhaler or other dry powder dispersion device. Non-limiting examples of dry powder inhalers and devices include those disclosed in US 5,458,135, US 5,740,794, US 5,775,320, 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. One design is a metered dose device in which a drug reservoir is placed inside the device, and the patient adds a dose of drug to the inhalation chamber. The second design is a factory-metered device, where each individual dose is manufactured in a separate container. Both systems rely on formulations that reduce the drug to small particles of 1 μm to approximately 5 μm MMAD, often including a co-formulation with larger excipient particles, such as, but not limited to, lactose. Drug powder is placed in the inhalation chamber (either by metering on the device or by breaking up a factory-metered dose), and the patient's inspiratory flow encourages the powder to exit the device and enter the oral cavity. The non-laminar flow characteristics of the powder pathway cause breakup of excipient-drug agglomerates, with clumps of larger excipient particles causing impaction in the back of the throat, while smaller drug particles are deposited deep in the lungs. In a preferred embodiment, a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof is administered in any of the dry powder inhalers described herein. These are delivered as dry powders using the type, where the MMAD of the dry powder, excluding any additives, is primarily in the range of 1 μm to about 5 μm.

[0074] In another embodiment, the compound of formula (I) or formula (II) is delivered as a dry powder using a metered dose inhaler. Non-limiting examples of metered dose inhalers and devices include those disclosed in US 5,261,538, US 5,544,647, US 5,622,163, US 4,955,371, US 3,565,070, US 3,361306 and US 6,116,234. In a preferred embodiment, the compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof is delivered as a dry powder using a metered dose inhaler, and in this case, the MMAD of the dry powder, excluding any additives, is mainly in the range of about 1 to 5 μm.

[0075] 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, such carriers as are known in the art to be appropriate.

[0076] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

[0077] The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules, and tablets of the type previously described. Preferred unit dosage formulations are those containing a daily or sub-daily dose, or an appropriate fraction thereof, of the active ingredient, as recited hereinabove.

[0078] It should be understood that in addition to the ingredients particularly mentioned above, the formulations may include other agents conventional in the art having regard to the type of formulation in question; for example, those suitable for oral administration may include flavoring agents.

[0079] There is further provided a veterinary composition comprising at least one active ingredient as defined above, together with a veterinary carrier therefor.

[0080] A veterinary carrier is a substance useful for the purpose of administering the composition and may be a solid, liquid, or gaseous substance that is otherwise inert or acceptable in the veterinary art and compatible with the active ingredient. These veterinary compositions may be administered orally, parenterally, or by any other desired route.

[0081] The compounds herein are used to provide controlled-release pharmaceutical formulations ("controlled-release formulations") that contain as active ingredients one or more compounds in which the release of the active ingredient is controlled and tailored to allow for less frequent dosing or to improve the pharmacokinetic or toxicity profile of a given active ingredient.

[0082] The effective dose of the active ingredient will depend at least in part on the nature of the condition being treated, toxicity, whether the compound is being used prophylactically (low dose) or against an ongoing viral infection, the method of delivery, and the pharmaceutical formulation, and will be determined by the clinician using conventional dose-escalation studies. It is expected to be about 0.0001 to about 100 mg / kg body weight per day, typically about 0.01 to about 10 mg / kg body weight per day, more typically about 0.01 to about 5 mg / kg body weight per day, and most typically about 0.05 to about 0.5 mg / kg body weight per day. For example, the daily candidate dose for an adult of approximately 70 kg body weight will range from 1 mg to 1000 mg, preferably between 5 mg and 500 mg, and can take the form of single or multiple doses.

[0083] Route of administration One or more compounds (referred to herein as active ingredients) are administered by any route suitable for the condition to be treated.Suitable routes include oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) and the like.It is understood that the preferred route may vary depending on, for example, the condition of the recipient.The advantage of the compounds herein is that they are orally bioavailable and can be orally administered.

[0084] Combination therapy The composition may also be used in combination with other active ingredients. In the case of treating Pneumovirinae virus infection, preferably, the other active therapeutic agent is active against Pneumovirinae virus infection, particularly respiratory syncytial virus infection. Non-limiting examples of such other active therapeutic agents include ribavirin, palivizumab, motavizumab, RSV-IGIV (RespiGam®), MEDI-557, A-60444 (also known as RSV604), MDT-637, BMS-433771, ALN-RSV0, ALX-0171, and mixtures thereof.

[0085] Most of the infections of Pneumovirinae virus are respiratory infections.Therefore, additional active therapeutic agents used to treat respiratory symptoms and infection sequelae may be used in combination with the compound of formula (I) or formula (II).The additional agent is preferably administered orally or by direct inhalation.For example, other preferred additional therapeutic agents that can be combined with the compound of formula (I) or formula (II) to treat viral respiratory infections include, but are not limited to, bronchodilators and corticosteroids.

[0086] First introduced as a treatment for asthma in 1950 (Carryer, Journal of Allergy, Vol. 21, pp. 282–287, 1950), glucocorticoids remain the most potent and consistently effective treatment for this disease, although their mechanism of action remains poorly understood (Morris, J. Allergy Clin. Immunol., Vol. 75(1), pp. 1–13, 1985). Unfortunately, oral glucocorticoid therapy is associated with serious undesirable side effects, including truncal obesity, hypertension, glaucoma, glucose intolerance, accelerated cataract formation, bone mineral loss, and psychological effects, all of which limit its use as a long-term treatment (Goodman and Gilman, 10th ed., 2001). A solution to systemic side effects is the direct delivery of steroid medications to the site of inflammation. Inhaled corticosteroids (ICS) were developed to mitigate the severe adverse effects of oral steroids. Non-limiting examples of corticosteroids that may be used in combination with a compound of Formula (I) or Formula (II) are dexamethasone, dexamethasone sodium phosphate, fluorometholone, fluorometholone acetate, loteprednol, loteprednol etabonate, hydrocortisone, prednisolone, fludrocortisone, triamcinolone, triamcinolone acetonide, betamethasone, beclomethasone diproprionate, methylprednisolone, fluocinolone, fluocinolone acetonide, flunisolide, fluocortin-21-butyrate, flumethasone, flumethasone pivalate, budesonide, halobetasol propionate, mometasone furoate, fluticasone propionate, ciclesonide, or a pharmaceutically acceptable salt thereof.

[0087] Other anti-inflammatory agents that act through anti-inflammatory cascade mechanisms also have antiviral effects on viral respiratory tract infections. These compounds are useful as additional therapeutic agents in combination with the compounds of formula (I) or formula (II) for the treatment of gastrointestinal infections. The application of "anti-inflammatory signaling modulators" (referred to herein as AISTMs), such as phosphodiesterase inhibitors (e.g., PDE-4, PDE-5, or PDE-7 specific), transcription factor inhibitors (e.g., blocking NFκB by inhibiting IKK), or kinase inhibitors (e.g., blocking P38MAP, JNK, PI3K, EGFR, or Syk), is a logical approach to switch off inflammation, since these small molecules only target a limited number of common intracellular pathways, signaling pathways that are critical for anti-inflammatory therapeutic intervention (see the review by PJ Barnes, 2006). These non-limiting additional therapeutic agents include 5-(2,4-difluoro-phenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (2-dimethylamino-ethyl)-amide (P38 Map kinase inhibitor ARRY-797); 3-cyclopropylmethoxy-N-(3,5-dichloro-pyridin-4-yl)-4-difluoromethoxy-benzamide (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-[(methyl sulfonyl)amino]-1-dibenzofurancarboxamide (oglemilast, a PDE-4 inhibitor); N-(3,5-dichloro-pyridin-4-yl)-2-[1-(4-fluorobenzyl)-5-hydroxy-1H-indol-3-yl]-2-oxo-acetamide (AWD12-281, a PDE-4 inhibitor); 8-methoxy-2-trifluoromethyl-quinoline-5-carboxylic acid (3,5-dichloro-1-oxy-pyridin-4-yl)-amide (Sch351591, a PDE-4 inhibitor); 4-[5-(4-fluorophenyl)-2-(4-methanesulfinyl-phenyl)-1H-imidazol-4-yl]-pyridine (SB-203850, a PDE-4 inhibitor);4-[4-(4-fluoro-phenyl)-1-(3-phenyl-propyl)-5-pyridin-4-yl-1H-imidazol-2-yl]-but-3-yn-1-ol (RWJ-67657, a P38 inhibitor); 4-cyano-4-(3-cyclopentyloxy-4-methoxy-phenyl)-cyclohexanecarboxylic acid 2-diethylamino-ethyl ester (the 2-diethyl-ethyl ester prodrug of cilomilast, PDE-4 inhibitors); (3-chloro-4-fluorophenyl)-[7-methoxy-6-(3-morpholin-4-yl-propoxy)-quinazolin-4-yl]-amine (gefitinib, an EGFR inhibitor); and 4-(4-methyl-piperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-benzamide (imatinib, an EGFR inhibitor).

[0088] Combinations comprising an inhaled β2-adrenoreceptor agonist bronchodilator, such as formoterol, albuterol, or salmeterol, and a compound of Formula (I) or Formula (II) are also suitable, but not limited to, combinations useful for treating respiratory viral infections.

[0089] Combinations of inhaled β2-adrenoreceptor agonist bronchodilators, such as formoterol or salmeterol, with ICS are also used to treat both bronchoconstriction and inflammation (Symbicort® and Advair®, respectively). Combinations including these ICS and β2-adrenoreceptor agonist combinations with compounds of Formula (I) or Formula (II) are also suitable combinations useful, but not limited to, for the treatment of respiratory viral infections.

[0090] Anticholinergics can be used to treat or prevent pulmonary bronchoconstriction and are therefore useful as additional therapeutic agents in combination with the compounds of Formula (I) or Formula (II) to treat viral respiratory infections. These anticholinergics include, but are not limited to, anticholinergics for controlling cholinergic tone in COPD, anticholinergics for treating men ...The antagonists of muscarinic receptors (especially of the M3 subtype) that have shown efficacy (Witek, 1999) include 1-{4-hydroxy-1-[3,3,3-tris-(4-fluoro-phenyl)-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 (ipratropium-N,N-diethylglycinate), 1-cyclohexyl-3,4-dihydro-1H-isoquinoline-2-carboxylic acid 1-aza-bicyclo[2.2.2]oct-3-yl ester (solifenacin), and 2-hydroxymethyl-4-methanesulfinyl-2- Phenyl-butyric acid 1-aza-bicyclo[2.2.2]oct-3-yl ester (Levatropate), 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)-2-benzofuran-5-yl]-ethyl]-pyrrolidin-3-yl}-2,2-diphenyl-acetamide (Darifenacin), no-acetoxy)-2-phenyl-propionyloxy]-9-ethyl-9-methyl-3-oxa-9-azonia-tricyclo[3.3.1.02,4]nonane (oxitropium-N,N-diethylglycinate), 7-[2-(2-diethylamino-acetoxy)-2,2-di-thiophen-2-yl-acetoxy]-9,9-dimethyl-3-oxa-9-azonia-tricyclo[3.3.1.02,4]nonane (tiotropium-N,N-diethylglycinate), dimethylamino-acetic acid 2-(3-diisopropylamino-1-phenyl-propyl)-4-methyl-phenyl ester (tolterodine-N,N-dimethylglycinate), 3-[4,4-bis-(4-fluoro-phenyl)-2-oxo-imidazolidin-1-yl]-1-methyl-1-(2-oxo-2-pyridin-2-yl-ethyl)-pyrrolidinium, 1-[1-(3-fluoro-benzyl)-piperidin-4-yl]-4,4-bis-(4-fluoro-phenyl)-imidazolidin-2-one , 1-cyclooctyl-3-(3-methoxy-1-aza-bicyclo[2.2.2]oct-3-yl)-1-phenyl-prop-2-yn-1-ol, 3-[2-(2-diethylamino-acetoxy)-2,2-di-thiophen-2-yl-acetoxy]-1-(3-phenoxy-propyl)-1-azonia-bicyclo[2.2.2]octane (acridinium-N,N-diethylglycinate), or (2-diethylamino-acetoxy)-di-thiophen-2-yl-acetic acid 1-methyl-1-(2-phenoxy-ethyl)-piperidin-4-yl ester.

[0091] The compound of formula (I) or formula (II) may also be combined with a mucolytic agent to treat both infection and symptoms of respiratory infection. A non-limiting example of a mucolytic agent is ambroxol. Similarly, the compound of formula (I) or formula (II) may also be combined with an expectorant to treat both infection and symptoms of respiratory infection. A non-limiting example of an expectorant is guaifenesin.

[0092] Nebulized hypertonic saline has been used to improve the immediate and long-term clearance of small airways in patients with lung disease (Kuzik, J. Pediatrics 2007, p. 266). The compound of formula (I) or formula (II) can also be combined with nebulized hypertonic saline, especially when Pneumovirinae virus infection is complicated with bronchiolitis. The combination of the compound of formula (I) or formula (II) and hypertonic saline can also include any of the additional agents discussed above. In one embodiment, about 3% nebulized hypertonic saline is used.

[0093] Any compound can be combined with one or more additional active therapeutic agents in a single dosage form for simultaneous or sequential administration to patients.Combined therapy can be administered as a simultaneous or sequential regimen.When administered sequentially, the combination can be administered in two or more doses.

[0094] Co-administration of a compound with one or more other active therapeutic agents herein generally refers to the simultaneous or sequential administration of the compound and one or more other active therapeutic agents such that therapeutically effective amounts of both the compound and the one or more other active therapeutic agents are present in the patient's body.

[0095] Co-administration includes administering a unit dose of the compound before or after administering a unit dose of one or more other active therapeutic agents, e.g., administering the compound within seconds, minutes, or hours after administering the one or more other active therapeutic agents. For example, a unit dose of the compound can be administered first, followed within seconds or minutes by 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 within seconds or minutes by a unit dose of the compound. In some cases, it may be desirable to administer a unit dose of the compound first, followed after a period of time (e.g., 1-12 hours) by a unit dose of one or more other active 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 time (e.g., 1-12 hours) by a unit dose of the compound herein.

[0096] Combination therapy can provide "synergy" and "synergistic." That is, the effect achieved when active ingredients are used together is greater than the sum of the effects produced when the compounds are used separately. Synergistic effects can be achieved when the active ingredients are (1) co-formulated and administered or delivered simultaneously as a combined formulation, (2) delivered alternately or in parallel as separate formulations, or (3) by some other regimen. When delivered in alternation therapy, synergistic effects can be achieved when the compounds are administered or delivered sequentially, for example, in separate tablets, pills, or capsules, or by different injections in separate syringes. Generally, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e., sequentially, while in combination therapy, effective dosages of two or more active ingredients are administered together. A synergistic antiviral effect refers to an antiviral effect that is greater than the expected purely additive effect of the individual compounds of the combination.

[0097] In yet another embodiment, the present application provides a method of treating a Pneumovirinae virus infection in a human, the method comprising administering to the human a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, and / or ester thereof. Similarly, separate methods of treating a Pneumovirinae virus infection in a human are provided, each of which comprises administering to the human a therapeutically effective, pharmaceutically effective amount of a compound of Formula (II) or one of the specific compounds of the Examples herein, or a pharmaceutically acceptable salt, solvate, and / or ester thereof, and a pharmaceutically acceptable carrier or excipient.

[0098] In another embodiment, there is provided a method of treating a Pneumovirinae infection in a human by administering to the human a therapeutically effective amount of a compound of Formula (I), or a racemate, enantiomer, diastereomer, tautomer, polymorph, pseudopolymorph, amorphous form, hydrate, or solvate of a compound of Formula (I), or a pharmaceutically acceptable salt or ester thereof.

[0099] Further provided are separate methods of treating a Pneumovirinae infection in a human in need thereof, each method comprising administering to the human a therapeutically effective amount of a racemate, enantiomer, diastereomer, tautomer, polymorph, pseudopolymorph, amorphous form, hydrate or solvate of a compound of formula (II) or one of the specific compounds of the Examples herein, or a pharmaceutically acceptable salt, solvate and / or ester thereof.

[0100] In yet another embodiment, the present application provides a method of treating a human respiratory syncytial virus infection in a human, comprising administering to the human a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, solvate, and / or ester thereof.

[0101] In yet another embodiment, the present application provides a method of treating a human respiratory syncytial virus infection in a human, the method comprising administering to the human a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, solvate, and / or ester thereof, and at least one additional active therapeutic agent.

[0102] Further provided are separate methods of treating a human respiratory syncytial virus infection in a human in need thereof, each method comprising administering to the human a therapeutically effective amount of a compound of formula (II) or one of the specific compounds of the Examples herein, or a pharmaceutically acceptable salt, solvate and / or ester thereof.

[0103] Similarly, separate methods of treating human respiratory syncytial virus infection in a human in need thereof are provided, each method comprising administering to the human a therapeutically effective amount of a compound of formula (II) or one of the specific compounds of the Examples herein, or a pharmaceutically acceptable salt, solvate and / or ester thereof, and at least one additional active therapeutic agent.

[0104] Similarly, separate methods of treating human respiratory syncytial virus infection in a human in need thereof, wherein the human also has bronchiolitis, are provided, each method comprising administering to the human a therapeutically effective amount of a compound of Formula (I), Formula (II), or one of the specific compounds of the Examples herein, or a pharmaceutically acceptable salt, solvate, and / or ester thereof.

[0105] Similarly, separate methods of treating human respiratory syncytial virus infection in a human in need thereof, wherein the human also has pneumonia, are provided, each method comprising administering to the human a therapeutically effective amount of a compound of Formula (I), Formula (II), or one of the specific compounds of the Examples herein, or a pharmaceutically acceptable salt, solvate, and / or ester thereof.

[0106] Similarly, separate methods of ameliorating respiratory symptoms in a human suffering from a human respiratory syncytial virus infection are provided, each method comprising administering to the human a therapeutically effective amount of a compound of Formula (I), Formula (II), or one of the specific compounds of the Examples herein, or a pharmaceutically acceptable salt, solvate, and / or ester thereof.

[0107] Respiratory symptoms in humans with respiratory syncytial virus infection can include nasal congestion or runny nose, coughing, wheezing, sneezing, rapid or difficult breathing, apnea, bronchiolitis, and pneumonia.

[0108] Similarly, embodiments are provided that include the use of a compound of formula (I), or a pharmaceutically acceptable salt, solvate and / or ester thereof, for the manufacture of a medicament for treating a Pneumovirinae virus infection or a respiratory syncytial virus infection.

[0109] Similarly, the use of a compound of formula (II) or a compound of the invention as described herein for the manufacture of a medicament for treating a Pneumovirinae virus infection or a respiratory syncytial virus infection. Embodiments are provided that include the use of one of the specific compounds of the examples, or a pharmaceutically acceptable salt, solvate and / or ester thereof.

[0110] Similarly, there is provided a pharmaceutical formulation comprising a pharmaceutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, solvate and / or ester thereof, and a pharmaceutically acceptable carrier or excipient. Further provided is a pharmaceutical formulation comprising a pharmaceutically effective amount of a compound of formula (II) or one of the specific compounds of the Examples herein, or a pharmaceutically acceptable salt, solvate and / or ester thereof, and a pharmaceutically acceptable carrier or excipient.

[0111] Similarly, there is provided a pharmaceutical formulation comprising a pharmaceutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt, solvate, and / or ester thereof, and a pharmaceutically acceptable carrier or excipient, and a pharmaceutically effective amount of at least one additional active therapeutic agent. Further provided is a pharmaceutical formulation comprising a pharmaceutically effective amount of a compound of formula (II) or one of the specific compounds of the Examples herein, or a pharmaceutically acceptable salt, solvate, and / or ester thereof, and a pharmaceutically acceptable carrier or excipient, and a pharmaceutically effective amount of at least one additional active therapeutic agent.

[0112] Similarly, separate embodiments are provided that include a compound of Formula (I), Formula (II), or one of the specific compounds of the Examples herein, or a pharmaceutically acceptable salt, solvate, and / or ester thereof, for use in treating Pneumovirinae virus infection or respiratory syncytial virus infection in humans.

[0113] Similarly, there is provided a separate embodiment comprising a compound of Formula (I), Formula (II) or one of the specific compounds of the Examples herein, or a pharmaceutically acceptable salt, solvate and / or ester thereof, for use as a pharmaceutical.

[0114] Similarly, separate embodiments are provided which include methods for manufacturing a medicament intended for the treatment of Pneumovirinae virus infection or respiratory syncytial virus infection in humans, characterized by using a compound of formula (I), formula (II) or one of the specific compounds of the Examples herein, or a pharmaceutically acceptable salt, solvate and / or ester thereof.

[0115] Similarly, there is provided a compound of formula (I), or a pharmaceutically acceptable salt, solvate and / or ester thereof, for treating a Pneumovirinae virus infection or a respiratory syncytial virus infection in a human.

[0116] Similarly, separate embodiments are provided that include a compound of formula (II) or one of the specific compounds of the Examples herein, or a pharmaceutically acceptable salt, solvate and / or ester thereof, for treating Pneumovirinae virus infection or respiratory syncytial virus infection in humans.

[0117] Further provided are compounds described herein.Similarly, provided are pharmaceutical compositions described herein.Similarly, provided are methods for using compounds of formula (I) described herein.Further provided are methods for making compounds of formula (I) described herein.

[0118] Metabolites of the compound Similarly, the in vivo metabolic products of the compounds described herein are within the scope of this specification, so long as the products are novel and not obvious beyond the prior art. The products may result, for example, from the oxidation, reduction, hydrolysis, amidation, esterification, etc., of the administered compound, primarily by enzymatic processes. Thus, included are novel and unobvious compounds produced by a process comprising contacting a compound with a mammal for a period of time sufficient to yield a metabolic product thereof. The products are usually radiolabeled (e.g., 14 C or3 H) Identification is accomplished by preparing the compound, parenterally administering the compound to animals such as rats, mice, guinea pigs, monkeys, or humans at a detectable dose (e.g., greater than about 0.5 mg / kg), allowing sufficient time for metabolism to occur (usually about 30 seconds to 30 hours), and isolating the compound's transformation products from urine, blood, or other biological samples. These products are easily isolated because they are labeled (others are isolated by using antibodies capable of binding to epitopes surviving in the metabolites). The structures of the metabolites are determined by conventional techniques, such as MS or NMR analysis. Metabolite analysis is generally performed in the same manner as conventional drug metabolism studies well known to those skilled in the art. Conversion products, even if they themselves do not possess HSV antiviral activity, are useful in diagnostic assays when the compound is administered therapeutically, unless otherwise found in vivo.

[0119] Strategies and methods for determining the stability of compounds in surrogate gastrointestinal fluids are known. A compound is defined herein as stable in the gastrointestinal tract if less than about 50 mole percent of the protecting groups are deprotected when incubated in surrogate intestinal fluid or gastric fluid at 37°C for 1 hour. Simply because a compound is stable to the gastrointestinal tract does not mean that it cannot be hydrolyzed in vivo. Prodrugs are usually stable in the digestive system, but can be substantially hydrolyzed to the parent drug in the digestive lumen, liver, lungs or other metabolic organs, or generally within cells. As used herein, a prodrug is understood to be a compound that is chemically designed to efficiently release the parent drug after overcoming the biological barrier to oral delivery.

[0120] Abbreviation In describing the experimental details, certain abbreviations and acronyms are used. While most of these will be understood by those skilled in the art, Table 1 contains a listing of many of these abbreviations and acronyms. [Table 1-1] [Table 1-2]

[0121] General Scheme

[0122] [ka] Scheme 1 shows a general synthesis of intermediates starting with an iodination reaction (eg, NIS) to generate nucleobase S1b.

[0123] [ka] Scheme 2 shows a method similar to that described in WO2012037038A1. The general synthesis of intermediates is shown in Method 1, starting with a fluorination reaction (e.g., HBF, NaNO) to give intermediate S2b, which can then be iodinated (e.g., NIS) to give nucleobase S2c.

[0124] [ka] Scheme 3 illustrates a general synthesis of compounds, beginning with lithium-halogen exchange (e.g., n-BuLi, [-CHSiMeCl]) reaction with an appropriate nucleobase S3b, followed by addition to lactone S3a. Reduction of the pendant 1' hydroxyl group under Lewis acidic conditions (e.g., BF·EtO, EtSiH) generates intermediate S3c. Intermediate S3c can be first protected at the nitrogen functionality (e.g., BzCl, Pyr; NHOH), and then the benzyl group can be removed under reducing conditions (e.g., HCOH, Pd / C, BCl, BBr) to yield intermediate S3d. A sequence involving first selective 5' hydroxyl protection (e.g., DMTrCl), then 2' and 3' hydroxyl protection (e.g., TBSCl), followed by selective removal of the 5' hydroxyl protecting group under acidic conditions (e.g., TsOH) provides intermediate S3e. The 5' hydroxyl group can then be converted to the corresponding iodide (e.g., (PhO)3PMeI), which is then exposed to basic conditions (i.e., KOtBu) to induce an elimination reaction to generate intermediate S3f. Oxidation of olefin S3f (e.g., DMDO), followed by treatment with an appropriate nucleophile (e.g., TMSCN) under Lewis acidic conditions (e.g., InBr3), and removal of the hydroxyl protecting group (e.g., CsF) affords intermediate S3g. Removal of the nitrogen protecting group (e.g., NH2Me) affords final compounds of type S3h.

[0125] [ka] Scheme 4 shows a general synthesis of compounds beginning with oxidation of olefin S3f (e.g., DMDO) followed by treatment with a suitable nucleophile (e.g., TMSN3) under Lewis acidic conditions (e.g., InBr3) in a manner similar to that described in J. Med. Chem. 2007, 50, 5463-5470. Subsequent removal of the hydroxyl protecting group (e.g., CsF) affords intermediate S4a. Removal of the nitrogen protecting group (e.g., NH2Me) affords final compounds of type S4b.

[0126] [ka] Scheme 5 shows the reaction of the appropriate alcohol HOR to give intermediate S5a. a The general synthesis of compounds is shown, starting with oxidation of olefin S3f (e.g., DMDO) in the presence of HCl followed by removal of the hydroxyl protecting group (e.g., CsF). Removal of the nitrogen protecting group (e.g., NH2Me) yields final compounds of type S5b.

[0127] [ka] Scheme 6 shows a general synthesis of compounds beginning with oxidation of olefin S3f (e.g., DMDO) followed by treatment with a suitable nucleophile (e.g., (HC≡C)3Al) in a manner similar to that described in Nucleosides, Nucleotides, and Nucleic Acids 2005, 24, 343-347. Subsequent removal of the hydroxyl protecting group (e.g., CsF) affords intermediate S3f. 6a is obtained. Removal of the nitrogen protecting group (e.g., NH2Me) affords final compounds of type S6b. Treatment of this final compound under hydrogenation conditions (e.g., H2, Pd / C, or Lindlar conditions) can selectively afford final compounds of type S6c and S6d, respectively. Treatment of final compound S6d under cyclopropanation conditions (e.g., CH2N2) can afford final compounds of type S6e.

[0128] [ka] Scheme 7 shows a general synthesis of compounds, beginning with a synthetic sequence in which the nitrogen of intermediate S7a, synthesized in a manner similar to that described in WO2012037038A1, is protected (e.g., with TMSCl, Pyr; BzCl, Pyr; NH4OH). Selective protection of the 5' hydroxyl group (e.g., DMTrCl) then generates intermediate S7b. Protection of the 2' hydroxyl group (e.g., with TBSCl) and subsequent removal of the 5' hydroxyl group under acidic conditions (e.g., TsOH) affords intermediate S7e. Conversion of the 5' hydroxyl group to an aldehyde under oxidative conditions (e.g., EDCI·HCl, Pyr, TFA, DMSO), followed by condensation of the corresponding enolate with formaldehyde and reduction (e.g., with NaBH4), affords intermediate S7f. Sequential selective protection of the hydroxyl moiety with orthogonal protecting groups (e.g., DMTrCl and TBSCl), followed by removal of the more labile protecting group under acidic conditions (e.g., TsOH), affords intermediate S7g. Conversion of the hydroxyl group to an aldehyde under oxidative conditions (e.g., EDCI·HCl, Pyr, TFA, DMSO) generates intermediate S7h. Processing of aldehyde S7h to the halo-olefin intermediate S7i can be achieved using Wittig olefination conditions (e.g., [Ph3PCH2Br] + Br - Elimination under basic conditions (e.g., KOtBu) generates the alkyne, and removal of the hydroxyl protecting group (e.g., TBAF) and nitrogen protecting group (e.g., NH4OH) affords final compounds of type S7j.

[0129] [ka] Scheme 8 shows the general synthesis of compounds, starting with oxime formation (e.g., NHOH·HCl) and subsequent conversion of the oxime to a nitrile group (e.g., CDI). Then, removal of the nitrogen protecting group (e.g., NH4OH) and the hydroxyl protecting group (e.g., HF·Pyr) yields final compounds of type S8a.

[0130] [ka] Scheme 9 illustrates the use of Wittig olefination conditions (e.g., [PhPCH] + Br - A general synthesis of compounds is shown, beginning with treatment of aldehyde S7h to an olefin with TBAF (e.g., KOtBu). Removal of the hydroxyl protecting group (e.g., TBAF) and nitrogen protecting group (e.g., NHOH) yields final compounds of type S9a. Reducing conditions (e.g., H, Pd / C) can then generate final compounds of type S9b, and cyclopropanation conditions (e.g., CHN) can generate final compounds of type S9c.

[0131] [ka] Scheme 10 shows the general synthesis of compounds, starting with an Appel reaction (e.g., PhP, CCl) to convert the hydroxyl group to a chloride. Removal of the hydroxyl protecting group (e.g., TBAF) and nitrogen protecting group (e.g., NHOH) yields final compounds of type S10a.

[0132] [ka] Scheme 11 shows a general synthesis of compounds, beginning with protecting the free hydroxyl group of intermediate S7g with a labile protecting group (e.g., PMBCl, K2CO3). Selective removal of the 2' and 5' silyloxy protecting groups (e.g., TBAF) and subsequent reprotection with a robust protecting group (e.g., BnBr, NaH) and removal of the labile hydroxyl protecting group under acidic conditions (e.g., AcOH) affords intermediate S11a. Conversion of the hydroxyl group to a fluorine (e.g., DAST) and subsequent removal of the hydroxyl protecting group (e.g., BBr3) and nitrogen protecting group (e.g., NH4OH) affords final compounds of type S11b.

[0133] [ka] Scheme 12 shows a general synthesis of compounds, beginning with the conversion of the 5' hydroxyl group to the corresponding iodide (e.g., (PhO)3PMeI) and then treating this iodide under basic conditions (e.g., KOtBu) to effect an elimination reaction to generate intermediate S12a. In a manner similar to that described in J. Med. Chem. 2007, 50, 5463-5470, oxidation of olefin S12a (e.g., DMDO) and subsequent treatment with an appropriate nucleophile (e.g., TMSN3) under Lewis acidic conditions (e.g., SnCl4), and removal of the hydroxyl protecting group (e.g., CsF) and nitrogen protecting group (e.g., NH4OH), affords final compounds of type S12b.

[0134] [ka] Scheme 13 shows the reaction of the appropriate alcohol HOR a

[0049] Figure 1 shows a general synthesis of compounds starting with oxidation of olefin S12a (e.g., DMDO) in the presence of HCl and removal of the hydroxyl protecting group (e.g., CsF). Removal of the nitrogen protecting group (e.g., NHMe) yields final compounds of type S13a.

[0135] [ka] Scheme 14 shows a general synthesis of compounds starting with xanthate formation (e.g., PhOC(S)Cl, DMAP) followed by a Barton-McCombie deoxygenation reaction (e.g., (TMS)SiH, AIBN) to generate intermediate S14a. Removal of the hydroxyl protecting group (e.g., TBAF) and nitrogen protecting group (e.g., NHOH) yields final compounds of type S14b.

[0136] [ka] Scheme 15 shows a general synthesis of compounds starting from intermediate S15a, which is prepared in a manner similar to that described in Biosci. Biotech. Biochem. 1993, 57, 1433-1438. Removal of the acetate protecting group using hydrolysis conditions (e.g., K2CO3, MeOH) followed by chemoselective oxidation conditions (e.g., NIS, Bu4NI) and protection of the 2' hydroxyl group (e.g., BnBr, Ag2O) generates intermediate S15b. Lithium-halogen exchange (e.g., n-BuLi, [-CH2SiMe2Cl]2) with an appropriate nucleobase S3b and addition to lactone S15b, followed by reduction of the 1' hydroxyl group under Lewis acidic conditions (e.g., BF3·Et2O, Et3SiH) and deprotection (e.g., H2, Pd black) affords final compounds of type S15c.

[0137] [ka] Scheme 16 illustrates the use of oxidative conditions (e.g., EDCI) to generate intermediate S16a. A general synthesis of compounds is shown, beginning with conversion of the 5' hydroxyl group to an aldehyde under conditions (e.g., HCl, Pyrrolidin, TFA, DMSO) followed by condensation of the corresponding enolate with formaldehyde and reduction (e.g., NaBH). Successive selective protection of the hydroxyl moiety with orthogonal protecting groups (e.g., DMTrCl and TBSCl) followed by removal of the more labile protecting group under acidic conditions (e.g., TsOH) affords intermediate S16b. The hydroxyl group may then be converted to a chloride via an Appel reaction (e.g., PhP, CCl), and removal of the hydroxyl protecting group (e.g., TBAF) and nitrogen protecting group (e.g., NHOH) affords final compounds of type S16c.

[0138] [ka] Scheme 17 shows a general synthesis of compounds, beginning with protecting the free hydroxyl group of intermediate S16b with a labile protecting group (e.g., PMBCl, K2CO3). Selective removal of the 2', 3', and 5' silyloxy protecting groups (e.g., TBAF) and subsequent reprotection with a robust protecting group (e.g., BnBr, NaH) and removal of the labile hydroxyl protecting group under acidic conditions (e.g., AcOH) affords intermediate S17a. Conversion of the hydroxyl group to a fluorine (e.g., DAST) and subsequent removal of the hydroxyl protecting group (e.g., BBr3) and nitrogen protecting group (e.g., NH4OH) affords final compounds of type S17b.

[0139] [ka] Scheme 18 shows a general synthesis of compounds in which an appropriate electrophilic halogenation reaction of intermediate S18a gives final compounds of type S18b (e.g., NCS), S18c (e.g., NIS), and S18d (e.g., Selectfluor).

[0140] [ka] Scheme 19 shows a general synthesis of compounds, starting with a cross-coupling reaction (e.g., Zn(CN), Pd(PtBu)) to give final compounds of type S19a, which are then treated with a nitrile hydrolysis reaction (e.g., HO, NHOH, HO) to give compounds of type S19b.

[0141] [ka] Scheme 20 shows the general synthesis of compounds, starting with a Sonogashira reaction (eg, CuI, PdCl2(PPh3)2) to give final compounds of type S20a.

[0142] [ka] Scheme 21 shows the general synthesis of compounds, starting with a cross-coupling reaction (eg, Pd(dppf)Cl 2 , Cs 2 CO 3 ) to give final compounds of type S21a.

[0143] [ka] Scheme 22 shows the general synthesis of compounds, including the synthesis of phosphorylated analogs of type S22b.

[0144] [ka] Scheme 23 shows the general synthesis of compounds, including the synthesis of phosphorylated analogs of type S23b.

[0145] [ka] Scheme 24 shows the general synthesis of compounds, including the synthesis of phosphorylated analogs of type S24b. [Example]

[0146] experiment [ka] Intermediate 1b To a solution of Intermediate 1a (50 mg, 373 mmol) in DMF (1 mL) was added solid N-iodosuccinimide (84 mg, 373 mmol) at room temperature. After 1.5 h, the reaction mixture was diluted with 1 M NaOH solution (10 mL), and the resulting slurry was stirred at room temperature. After 1 h, the solid was collected by vacuum filtration and dried under reduced pressure to give Intermediate 1b.

[0147] 1 H NMR (400 MHz, DMSO-d6)δ7.89 (s, 1H), 7.78 (br-s, 1H), 6.98 (d, J = 4.4 Hz, 1H), 6.82 (d, J = 4.4 Hz, 1H), 3.30 (br-s, 1H).

[0148] LC / MS:t R = 1.21 min, MS m / z = 261.02 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50×4.6mm Solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 2-100% ACN from 0 min to 2.0 min, 100% ACN from 2.0 min to 3.05 min, 100%-2% ACN from 3.05 min to 3.2 min, 2% ACN from 3.2 min to 3.5 min, 2 μl / min.

[0149] HPLC:t r= 1.536 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 2–98% ACN from 0 min to 5.0 min, 98% ACN from 5.0 min to 6.0 min, 2 mL / min.

[0150] [ka] Intermediate 1d - (2S,3R,4R,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)tetrahydrofuran-2-ol

[0151] To a suspension of 7-iodopyrrolo[1,2-f][1,2,4]triazine-4-amine 1b (6.84 g, 26.3 mmol) and 1,2-bis(chlorodimethylsilyl)ethane (5.66 g, 26.3 mmol) in THF (200 mL) under an argon atmosphere was added n-butyllithium (2.5 M in hexane, 34.4 mL, 86.0 mmol) rapidly at −78° C. During the addition, the internal temperature of the reaction mixture rose to −40.5° C., and the reaction mixture became a clear brown solution. After 15 min, a solution of (3R,4R,5R)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)dihydrofuran-2(3H)-one (1c, purchased from Carbosynth, 10 g, 23.9 mmol) in tetrahydrofuran (40 mL) precooled to -78 °C was rapidly added via cannula. After 1 h, the reaction mixture was quenched with acetic acid (15 mL), and the resulting mixture was allowed to warm to room temperature. The resulting mixture was diluted with ethyl acetate (800 mL) and washed with saturated aqueous sodium bicarbonate (500 mL) and brine (500 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by SiO column chromatography (220 g SiO Combiflash HP Gold Column, 0–100% ethyl acetate / hexanes) to afford intermediate 1d.

[0152] LC / MS:t R = 1.50 min, MS m / z = 553.34 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50×4.6mm Solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 2 to 100% ACN from 0 min to 1.5 min, 100% ACN from 1.5 min to 2.2 min, 100% to 2% ACN from 2.2 min to 2.4 min, 2% ACN from 2.4 min to 2.5 min.

[0153] HPLC:t r = 3.442 min; HPLC system: Agilent 1100 series; Column: Gemini 5μ C18 110A, 50 × 4.6 mm; Solvent: acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 2 to 98% ACN from 0 to 5.0 min, 98% ACN from 5.0 to 6.0 min, 2 mL / min. TLC: Eluent: ethyl acetate, R f =0.5(UV)

[0154] [ka] Intermediate 1e - 7-((2S,3S,4R,5R)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)tetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-amine

[0155] To a solution of intermediate 1d (4.74 g, 8.58 mmol) and triethylsilane (3.56 mL, 22.3 mmol) in DCM (43 mL) was slowly added boron trifluoride diethyl etherate (1.59 mL, 12.9 mmol) via syringe at 0 °C under an argon atmosphere. After 2 h, the reaction mixture was slowly diluted with saturated aqueous sodium bicarbonate (100 mL), and the resulting mixture was extracted with ethyl acetate (2 × 150 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude residue was purified by SiO column chromatography (24 g SiO Combiflash HP Gold Column, 0–100% ethyl acetate / hexanes) to give intermediate 1e.

[0156] 1 H NMR (400 MHz, CDCl3)δ7.88 (s, 1H), 7.37 - 7.22 (m, 15H), 6.73 (d, J = 4.6 Hz, 1H), 6.71 (d, J = 4.6 Hz, 1H), 5.66 (d, J = 4.2 Hz, 1H), 4.71 (s, 2H), 4.60 (d, J = 12.0 Hz, 1H), 4.54 (s, 2H), 4.45 (d, J = 11.9 Hz, 1H), 4.39 (dt, J = 7.1, 3.6 Hz, 1H), 4.25 (t, J = 4.6 Hz, 1H), 4.14 - 4.10 (m, 1H), 3.78 (dd, J = 10.7, 3.4 Hz, 1H), 3.65 (dd, J = 10.7, 4.0 Hz, 1H).

[0157] LC / MS:t R = 2.01 min, MS m / z = 537.41 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50 × 4.6 mm; solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 2 to 100% ACN from 0 min to 1.5 min, 100% ACN from 1.5 min to 2.2 min, 100% to 2% ACN from 2.2 min to 2.4 min, 2% ACN from 2.4 min to 2.5 min, 2 μl / min.

[0158] HPLC:t R = 3.596 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 2 to 98% ACN from 0 to 5.0 min, 98% ACN from 5.0 to 6.0 min, 2 mL / min. TLC: eluent: ethyl acetate, R f =0.3(UV)

[0159] [ka] Intermediate 1f - N-(7-((2S,3S,4R,5R)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)tetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0160] To a solution of Intermediate 1e (3.94 g, 7.34 mmol) in pyridine (36.7 mL) was added benzoyl chloride (1.69 mL, 14.68 mmol) slowly at room temperature under an argon atmosphere. After 1 hour, additional benzoyl chloride (1.69 mL, 14.68 mmol) was added slowly. After 19 hours, water (20 mL) was added slowly, causing the reaction mixture to become slightly cloudy. Ammonium hydroxide (approximately 10 mL) was then added slowly until the reaction mixture was basic (pH = 10). After 1 hour, water (150 mL) was added dropwise via addition funnel; during the addition, a white solid slowly began to precipitate from the reaction mixture. The resulting mixture was stirred for 24 hours, and the white solid was collected by vacuum filtration and azeotropically dried from toluene to give Intermediate 1f.

[0161] 1 H NMR (400 MHz, CDCl3)δ8.23 (br s, 1H), 7.62 (t, J = 7.8 Hz, 1H), 7.53 (t, J = 7.6 Hz, 2H), 7.38 - 7.21 (m, 18H), 7.17 (d, J = 7.6 Hz, 1H), 5.69 (d, J = 4.1 Hz, 1H), 4.71 (s, 2H), 4.63 - 4.44 (m, 4H), 4.43 - 4.39 (m, 1H), 4.22 (t, J = 4.5 Hz, 1H), 4.15 - 4.10 (m, 1H), 3.79 (dd, J = 10.8, 3.2 Hz, 1H), 3.65 (dd, J = 10.7, 3.7 Hz, 1H).

[0162] LC / MS:t R = 1.91 min, MS m / z = 641.18 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50 × 4.6 mm; solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 2 to 100% ACN from 0 min to 1.5 min, 100% ACN from 1.5 min to 2.2 min, 100% to 2% ACN from 2.2 min to 2.4 min, 2% ACN from 2.4 min to 2.5 min, 2 μl / min. TLC: Eluent: 50% ethyl acetate in hexane, R f =0.6(UV)

[0163] [ka] Intermediate 1g - N-(7-((2S,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0164] To a mixture of intermediate 1f (4.39 g, 6.85 mmol) and palladium on carbon (10 wt%, 2.2 g) under an argon atmosphere, ethanol (68.5 mL) and formic acid (51.7 mL, 1.37 mol) were added sequentially at room temperature. After 3 days, the reaction mixture was filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure. The crude residue was azeotroped with toluene (3 × 20 mL) to give intermediate 1g, which was used directly in the next step without further purification.

[0165] 1H NMR (400 MHz, CD3OD)δ8.15 (s, 1H), 7.67 - 7.40 (m, 5H), 7.23 (d, J = 4.7 Hz, 1H), 7.00 (d, J = 4.7 Hz, 1H), 5.40 (d, J = 6.0 Hz, 1H), 4.44 (t, J = 5.7 Hz, 1H), 4.17 (t, J = 5.1 Hz, 1H), 4.03 (q, J = 4.3 Hz, 1H), 3.81 (dd, J = 12.1, 3.5 Hz, 1H), 3.71 (dd, J = 12.0, 4.5 Hz, 1H).

[0166] LC / MS:t R = 1.04 min, MS m / z = 371.15 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50×4.6mm Solvent: acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid; Gradient: 2-100% ACN from 0 to 1.5 min, 100% ACN from 1.5 to 2.2 min, 100%-2% ACN from 2.2 to 2.4 min, 2% ACN from 2.4 to 2.5 min, 2 μl / min. HPLC:t R = 2.055 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 2 to 98% ACN from 0 to 5.0 min, 98% ACN from 5.0 to 6.0 min, 2 mL / min.

[0167] [ka] Intermediate 1h - N-(7-((2S,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0168] To a solution of intermediate 1g (2.44 g, 6.59 mmol) in pyridine (32.5 mL) at room temperature was added solid 4,4'-dimethoxytrityl chloride (2.23 g, 6.59 mmol) in one portion. After 5.5 h, the reaction mixture was diluted with ethyl acetate (300 mL), and the resulting mixture was washed with brine (3 × 200 mL). The organic layer was concentrated under reduced pressure, and the crude residue was purified by SiO column chromatography (80 g SiO Combiflash HP Gold Column, 0–100% ethyl acetate / hexanes) to give intermediate 1h.

[0169] LC / MS:t R = 1.68 min, MS m / z = 673.22 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50×4.6m Solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 2 to 100% ACN from 0 min to 1.5 min, 100% ACN from 1.5 min to 2.2 min, 100% to 2% ACN from 2.2 min to 2.4 min, 2% ACN from 2.4 min to 2.5 min, 2 μl / min. HPLC:t R = 4.270 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 2 to 98% ACN from 0 to 5.0 min, then 98% ACN from 5.0 to 6.0 min, 2 mL / min. TLC: Eluent: 50% ethyl acetate in hexane, R f =0.15(UV)

[0170] [ka] Intermediate 1i - N-(7-((2S,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-bis(tert-butyldimethylsilyloxy)tetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0171] To a solution of intermediate 1h (1.84 g, 2.74 mmol) and imidazole (2.23 g, 32.8 mmol) in N,N-dimethylformamide (28.2 mL) was added tert-butyldimethylsilyl chloride (2.47 g, 16.4 mmol) at room temperature. After 17 h, saturated aqueous sodium bicarbonate (500 mL) was slowly added to the reaction mixture. The resulting mixture was extracted with ethyl acetate (500 mL), and the organic layer was washed with brine (2 × 400 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude residue was purified by SiO column chromatography (80 g SiO Combiflash HP Gold Column, 0–100% ethyl acetate / hexanes) to give intermediate 1i.

[0172] LC / MS:t R = 3.43 min, MS m / z = 901.37 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50×4.6mm Solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 2 to 100% ACN from 0 min to 2.0 min, 100% ACN from 2.0 min to 3.55 min, 100% to 2% ACN from 3.55 min to 4.2 min, 2 μl / min. HPLC:t R= 5.724 min; HPLC system: Agilent 1100 series; Column: Gemini 5μ C18 110A, 50 × 4.6 mm; Solvent: acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 2 to 98% ACN from 0 to 5.0 min, 98% ACN from 5.0 to 6.0 min, 2 mL / min. TLC: Eluent: 50% ethyl acetate in hexane, R f =0.75(UV)

[0173] [ka] Intermediate 1j - N-(7-((2S,3S,4R,5R)-3,4-bis(tert-butyldimethylsilyloxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0174] To a solution of intermediate 1i (2.41 g, 2.67 mmol) in dichloromethane (22.3 mL) was slowly added a solution of p-toluenesulfonic acid monohydrate (509 mg, 2.67 mmol) in methanol (3.7 mL) at 0 °C. After 1.5 h, the reaction mixture was diluted with saturated aqueous bicarbonate solution (100 mL), and the resulting mixture was extracted with dichloromethane (2 × 100 mL). The combined organic extracts were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by SiO column chromatography (120 g of SiO Purification by Combiflash HP Gold Column, 0-100% ethyl acetate / hexanes) gave intermediate 1j.

[0175] 1 H NMR (400 MHz, CDCl3)δ8.72 (br-s, 1H), 8.16 (br-t, J = 7.1 Hz, 2H), 8.07 (br-t, J = 7.7 Hz, 3H), 7.49 - 7.43 (m, 1H), 5.75 (d, J = 8.2 Hz, 1H), 5.28 (dd, J = 8.1, 4.7 Hz, 1H), 4.81 (d, J = 5.0 Hz, 1H), 4.70 - 4.63 (m, 1H), 4.44 (d, J = 12.3 Hz, 1H), 4.24 (d, J = 12.4 Hz, 1H), 1.48 (s, 9H), 1.30 (s, 9H), 0.65 (s, 3H), 0.64 (s, 3H), 0.41 (s, 3H), 0.00 (s, 3H).

[0176] LC / MS:t R = 2.66 min, MS m / z = 599.19 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50×4.6mm Solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 2-100% ACN from 0 min to 2.0 min, 100% ACN from 2.0 min to 3.05 min, 100%-2% ACN from 3.05 min to 3.2 min, 2% ACN from 3.2 min to 3.5 min, 2 μl / min. HPLC:t R = 5.622 min; HPLC system: Agilent 1100 ser ies.; Column: Gemini 5μ C18 110A, 50 × 4.6 mm; Solvent: Acetonitrile with 0.1% TFA, water with 0.1% TFA; Gradient: 2–98% ACN from 0 min to 5.0 min, 98% ACN from 5.0 min to 6.0 min, 2 mL / min. TLC: Eluent: 50% ethyl acetate in hexane, R f =0.55(UV)

[0177] [ka] Intermediate 1k - N-(7-((2S,3S,4R,5S)-3,4-bis(tert-butyldimethylsilyloxy)-5-(iodomethyl)tetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0178] To a solution of methyltriphenoxyphosphonium iodide (0.99 g, 2.19 mmol) in DMF (9.9 mL) was added intermediate 1j (1.19 g, 1.99 mmol) at room temperature. After 3 h, an additional portion of methyltriphenoxyphosphonium iodide (0.99 g, 2.19 mmol) was added. After 1 h, the reaction mixture was diluted with ethyl acetate (200 mL) and washed with brine (3 × 100 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by SiO column chromatography (80 g SiO Combiflash HP Gold Column, 0–100% ethyl acetate / hexanes) to give intermediate 1k.

[0179] 1 H NMR (400 MHz, CDCl3)δ8.21 (br s, 1H), 7.61 (br t, J = 7.2 Hz, 1H), 7.53 (br t, J = 7.5 Hz, 3H), 7.05 (br s, 1H), 5.44 (d, J = 4.5 Hz, 1H), 4.52 (t, J = 4.3 Hz, 1H), 4.08 - 3.99 (m, 2H), 3.55 (dd, J = 10.7, 5.2 Hz, 1H), 3.38 (dd, J = 10.7, 5.0 Hz, 1H), 0.93 (s, 9H), 0.85 (s, 9H), 0.16 (s, 3H), 0.11 (s, 3H), -0.01 (s, 3H), -0.11 (s, 1H).

[0180] LC / MS:t R= 3.06 min, MS m / z = 709.16 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50×4.6mm Solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 2-100% ACN from 0 min to 2.0 min, 100% ACN from 2.0 min to 3.05 min, 100%-2% ACN from 3.05 min to 3.2 min, 2% ACN from 3.2 min to 3.5 min, 2 μl / min. HPLC:t R = 5.837 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 2 to 98% ACN from 0 to 5.0 min, then 98% ACN from 5.0 to 6.0 min, 2 mL / min. TLC: Eluent: 20% ethyl acetate in hexane, R f =0.45(UV)

[0181] [ka] Intermediate 1l - N-(7-((2S,3S,4S)-3,4-bis(tert-butyldimethylsilyloxy)-5-methylenetetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0182] To a solution of intermediate 1k (1.77 g, 2.5 mmol) in pyridine (25 mL) was added potassium t-butoxide (700 mg, 6.24 mmol) at room temperature. After 2 h, the reaction mixture was diluted with saturated aqueous sodium bicarbonate (25 mL) and brine (200 mL). The resulting mixture was extracted with ethyl acetate (300 mL). The organic layer was then washed with brine (200 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude residue was purified by SiO2 column chromatography (40 g SiO2 Combiflash HP Gold Column, 0–100% ethyl acetate / hexanes) to give intermediate 1l.

[0183] LC / MS:t R = 2.87 min, MS m / z = 581.37 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50×4.6mm Solvent: acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid Gradient: 2–100% ACN from 0 min to 2.0 min, 100% ACN from 2.0 min to 3.05 min, 100%–2% ACN from 3.05 min to 3.2 min, 2% ACN from 3.2 min to 3.5 min, 2 μl / min. HPLC:t R = 5.750 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 2–98% ACN from 0 min to 5.0 min, 98% ACN from 5.0 min to 6.0 min, 2 mL / min. TLC: Eluent: 50% ethyl acetate in hexane, R f =0.20(UV)

[0184] [ka] Intermediate 1m - N-(7-((2S,3S,4S)-3,4-bis(tert-butyldimethylsilyloxy)-5-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0185] To a solution of intermediate 1l (560 mg, 0.964 mmol) in acetone (4.82 mL) was added DMDO (0.07 M solution in acetone, 13.8 mL, 0.964 mmol) at 0 °C. After 10 min, the reaction mixture was concentrated under reduced pressure and dried azeotropically with toluene (2×1 mL) to give 1m, which was used immediately in the next step without further purification.

[0186] LC / MS:t R = 2.57 min, MS m / z = 615.14 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50×4.6mm Solvent: acetonitrile containing 0.1% acetic acid, water containing 0.1% acetic acid Gradient: 2–100% ACN from 0 min to 2.0 min, 100% ACN from 2.0 min to 3.05 min, 100%–2% ACN from 3.05 min to 3.2 min, 2% ACN from 3.2 min to 3.5 min, 2 μl / min.

[0187] [ka] Intermediate 1n - N-(7-((2S,3S,4S)-3,4-bis(tert-butyldimethylsilyloxy)-5-cyano-5-((trimethylsilyloxy)methyl)tetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0188] To a solution of crude intermediate 1m (approximately 592 mg, approximately 0.964 mmol) and trimethylsilyl cyanide (640 μL, 4.80 mmol) in dichloromethane (19.2 mL) was added indium(III) bromide (681 mg, 1.92 mmol) under an argon atmosphere at 0° C. After 4.5 h, the reaction mixture was quenched with saturated aqueous sodium bicarbonate (6 mL) and warmed to room temperature. The resulting mixture was partitioned between dichloromethane (20 mL) and saturated aqueous sodium bicarbonate (20 mL). The phases were separated, and the aqueous layer was extracted with dichloromethane (20 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to give intermediate 1n (1:1 diastereomeric mixture) (710 mg), which was used directly in the next step without further purification.

[0189] LC / MS: First eluting isomer r = 2.91 min, MS m / z = 696.28 [M+1], second eluting isomer t R = 3.02 min, MS m / z = 696.19 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50×4.6mm Solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 2-100% ACN from 0 min to 2.0 min, 100% ACN from 2.0 min to 3.05 min, 100%-2% ACN from 3.05 min to 3.2 min, 2% ACN from 3.2 min to 3.5 min, 2 μl / min.

[0190] [ka] Intermediate 1o - N-(7-((2S,3R,4S)-5-cyano-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0191] To a solution of crude intermediate 1n (668.23 mg, 0.96 mmol) in DMF (9.6 mL) was added cesium fluoride (729 mg, 4.8 mmol) at room temperature. After 5 h, the reaction mixture was diluted with brine (100 mL), and the resulting mixture was extracted with dichloromethane (3 × 100 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to give intermediate 1o (1:1 diastereomeric mixture), which was used directly in the next step without further purification.

[0192] LC / MS: First eluting isomer R = 1.31 min, MS m / z = 396.19 [M+1], second eluting isomer t R = 1.32 min, MS m / z = 396.19 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50×4.6mm Solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 2-100% ACN from 0 min to 2.0 min, 100% ACN from 2.0 min to 3.05 min, 100%-2% ACN from 3.05 min to 3.2 min, 2% ACN from 3.2 min to 3.5 min, 2 μl / min.

[0193] [ka] Example 1—(2R,3S,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-2-(hydroxymethyl)tetrahydrofuran-2-carbonitrile

[0194] To a solution of crude intermediate 1o in methanol (1 mL) at room temperature was added methylamine (40% in water, 0.3 mL). After 2.5 h, the reaction mixture was concentrated under reduced pressure and directly purified by preparative HPLC (Phenominex Luna 5u C18 100Å 100×30 mm column, 5-15% acetonitrile / water gradient, 25 min). Fractions containing the desired product and the 4' anomer were combined and concentrated under reduced pressure. The 4' anomer was then separated by preparative HPLC (Phenominex Luna 5u C18 100Å 100×30 mm column, 5-15% acetonitrile / water gradient, 25 min). Fractions containing the desired product were combined and lyophilized to give Example 1.

[0195] 1 H NMR (400 MHz, CD3OD)δ7.79 (s, 1H), 6.85 (d, J = 4.5 Hz, 1H), 6.76 (d, J = 4.5 Hz, 1H), 5.45 (d, J = 5.9 Hz, 1H), 4.59 (t, J = 5.7 Hz, 1H), 4.40 (d, J = 5.6 Hz, 1H), 3.88 (d, J = 12.0 Hz, 1H), 3.80 (d, J = 12.0 Hz, 1H).

[0196] LC / MS:t R = 0.29 min, MS m / z = 292.16 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50×4.6mm Solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 2 to 100% ACN from 0 min to 1.5 min, 100% ACN from 1.5 min to 2.2 min, 100% to 2% ACN from 2.2 min to 2.4 min, 2% ACN from 2.4 min to 2.5 min, 2 μl / min. HPLC:t R= 0.377 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 2 to 98% ACN from 0 to 5.0 min, 98% ACN from 5.0 to 6.0 min, 2 mL / min. HPLC:t R = 6.643 min; HPLC system: Agilent 1100 series; column: Luna 5μ C18(2) 110A, 250 × 4.6 mm; solvent: acetonitrile, water; gradient: ACN 5–15% over 10 min, 2 mL / min.

[0197] [ka] Intermediate 2b - N-(7-((2S,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0198] To a flask purged with N2, intermediate 2a, (2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol (prepared according to WO2012037038A1, 1.20 g, 4.01 mmol) (dried by coevaporation with pyridine three times), was added, which was then dissolved in pyridine (18 mL). Chlorotrimethylsilane (1.54 mL, 13.13 mmol) was added in one portion at 0 °C, and the resulting mixture was stirred under a N2 atmosphere for 1 h. Benzoyl chloride (675 μL, 5.82 mmol) was added dropwise, and the reaction mixture was stirred for 1 h. An additional portion of benzoyl chloride (100 μL) was added to consume the remaining starting material. A mixture of mono- and bis-Bz protected products was observed. The reaction was quenched with H2O (5 mL) and the resulting mixture was stirred for 5 min. Then, concentrated NH4OH was added. (aq)(8 mL) was added in one portion and stirred for 15 min, at which point the bis-Bz product was converted to the desired product. The solvent was removed under reduced pressure, and the residue was then co-evaporated with CHOH. Intermediate 2b was isolated after purification by silica gel chromatography using an eluent gradient of 50% to 100% EtOAc in hexanes.

[0199] 1 H NMR (400 MHz, DMSO-d6)δ8.26 - 7.91 (m, 2H), 7.88 - 7.79 (m, 1H), 7.61 (t, J = 7.5 Hz, 1H), 7.56 - 7.36 (m, 2H), 7.37 - 7.24 (m, 1H), 7.10 (d, J = 4.6 Hz, 1H), 7.01 (s, 1H), 5.53 (d, J = 23.4 Hz, 1H), 5.45 (d, J = 6.5 Hz, 1H), 5.16 - 4.91 (m, 1H), 4.86 (t, J = 5.6 Hz, 1H), 4.21 - 4.04 (m, 1H), 3.82 (dd, J = 8.0, 3.9 Hz, 1H), 3.71 (ddd, J = 12.3, 5.6, 2.6 Hz, 1H), 3.52 (ddd, J = 12.2, 5.7, 4.5 Hz, 1H). 19 F NMR (376 MHz, DMSO-d6)δ-196.33 (dt, J = 55.1, 22.5 Hz).

[0200] LC / MS:t R = 0.77 min, MS m / z = 373.14 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ C18 100A, 50×3.00mm Solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid Gradient: 2 to 100% ACN from 0 to 1.4 min, 100% ACN from 1.4 to 1.80 min, 100% to 2% ACN from 1.8 to 1.85 min, and 2% ACN from 1.85 to 2 min.

[0201] [ka] Intermediate 2c - N-(7-((2S,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0202] Intermediate 2b (1.3 g, 3.49 mmol) was dried by coevaporation with pyridine. The dried material was then dissolved in pyridine (15 mL) under a N atmosphere. 4,4'-Dimethoxytrityl chloride (1.71 g, 5.0 mmol) was added in one portion at room temperature and stirred for 2 hours. Ethanol (2 mL) was added, and the resulting solution was stirred for 5 minutes. The solvent was removed under reduced pressure. The residue was purified by silica gel chromatography using an eluent gradient of 0% to 100% EtOAc in hexanes to give intermediate 2c.

[0203] 1 H NMR (400 MHz, DMSO-d6)δ8.28 - 8.02 (m, 2H), 7.61 (t, J = 7.5 Hz, 1H), 7.51 (t, J = 7.6 Hz, 2H), 7.36 (ddt, J = 6.0, 4.7, 2.0 Hz, 2H), 7.31 - 7.04 (m, 7H), 6.90 - 6.73 (m, 4H), 5.62 (d, J = 24.4 Hz, 1H), 5.49 (d, J = 7.0 Hz, 1H), 5.27 - 5.01 (m, 1H), 4.32 - 4.13 (m, 1H), 4.06 - 3.95 (m, 1H), 3.69 (s, 4H), 3.28 (s, 3H), 3.12 (dd, J = 10.4, 5.2 Hz, 1H). 19 F NMR (376 MHz, DMSO-d6)δ-195.58 (dt, J = 52.1, 24.7 Hz).

[0204] LC / MS:t R = 1.37 min, MS m / z = 675.29 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ C18 100A, 50×3.00mm Solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid Gradient: 2 to 100% ACN from 0 to 1.4 min, 100% ACN from 1.4 to 1.80 min, 100% to 2% ACN from 1.8 to 1.85 min, and 2% ACN from 1.85 to 2 min.

[0205] [ka] Intermediate 2d - N-(7-((2S,3S,4R,5R)-4-(tert-butyldimethylsilyloxy)-3-fluoro-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0206] To a solution of intermediate 2c (1.47 g, 2.18 mmol) in DMF (8 mL) prepared under a N atmosphere, imidazole (251 mg, 3.70 mmol) was added, followed by tert-butylchlorodimethylsilane (492 mg, 3.27 mmol). The solution was stirred at room temperature for 16 hours. The solution was diluted with HO (5 mL), and then the solvent was removed under reduced pressure. The residue was partitioned between EtOAc and HO. The layers were separated, and then the organic phase was washed with brine. The organics were dried over NaSO, which was removed by filtration, and the filtrate was concentrated under reduced pressure. This crude material was used directly in the next step.

[0207] The crude material was dissolved in CHCl (15 mL) and cooled to 0° C. To the mixture was added p-toluenesulfonic acid hydrate (414 mg, 2.18 mmol) dissolved in CHOH (6 mL) dropwise and stirred for 15 min. The reaction was diluted with saturated NaHCO 3(aq) The mixture was quenched with HCl. The organics were washed with brine, dried over NaSO, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography using an eluent gradient of 0% to 50% EtOAc in hexanes to give intermediate 2d.

[0208] 1 H NMR (400 MHz, DMSO-d6)δ8.21 - 7.98 (m, 2H), 7.51 (dt, J = 39.9, 7.5 Hz, 3H), 7.12 - 6.86 (m, 2H), 5.48 (d, J = 21.9 Hz, 1H), 5.07 (dt, J = 54.6, 3.8 Hz, 1H), 4.86 (t, J = 5.5 Hz, 1H), 4.28 (ddd, J = 17.8, 7.1, 4.4 Hz, 1H), 3.83 - 3.72 (m, 1H), 3.63 (ddd, J = 12.1, 5.2, 3.0 Hz, 1H), 3.43 (ddd, J = 12.1, 6.0, 4.2 Hz, 1H), 0.80 (s, 9H), 0.01 (s, 3H), 0.00 (s, 3H). 19 F NMR (376 MHz, DMSO-d6)δ-198.05 (dt, J = 54.2, 19.8 Hz).

[0209] LC / MS:t R = 1.35 min, MS m / z = 487.24 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ C18 100A, 50×3.00mm Solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid Gradient: 2 to 100% ACN from 0 to 1.4 min, 100% ACN from 1.4 to 1.80 min, 100% to 2% ACN from 1.8 to 1.85 min, and 2% ACN from 1.85 to 2 min.

[0210] [ka] Intermediate 2e - N-(7-((2S,3S,4R)-4-(tert-butyldimethylsilyloxy)-3-fluoro-5,5-bis(hydroxymethyl)tetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0211] To a solution of intermediate 2d (856 mg, 1.75 mmol) in toluene (4 mL) and DMSO (6 mL) prepared under a N atmosphere was added 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDCI) (504 mg, 2.63 mmol). To this mixture was added pyridine (150 μL) and TFA (70 μL), and the mixture was stirred at room temperature for 15 min. Additional EDCI (100 mg) and pyridine (100 μL) were added, and the mixture was stirred for an additional 45 min. The reaction was quenched with HO (10 mL) and CHCl (10 mL). The organics were washed with brine, dried over NaSO, filtered, and the solvent was removed under reduced pressure to give the crude aldehyde, which was used directly in the next step.

[0212] The crude aldehyde was dissolved in dioxane (5 mL) and diluted with 37% formaldehyde. (aq) (925 μL) was added, followed by 2N NaOH (aq) (925 μL) was added. After stirring at room temperature for 3 h, the reaction was quenched with AcOH, diluted with EtOAc, and washed with HO. The organics were dried over NaSO, filtered, and the solvent was removed under reduced pressure to give the crude aldol product, which was carried on directly to the next step.

[0213] The crude aldol product was dissolved in EtOH (9 mL) under a N atmosphere and cooled to 0 °C. NaBH (80 mg, 2.1 mmol) was added in one portion and the reaction was stirred for 10 min. The reaction was quenched with AcOH, diluted with CHCl, and diluted with water and saturated NaHCO 3(aq) The organic phase was dried over NaSO, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography using an eluent gradient of 0% to 100% EtOAc in hexanes to give intermediate 2e.

[0214] 1 H NMR (400 MHz, DMSO-d6)δ8.20 - 7.99 (m, 2H), 7.51 (dt, J = 39.5, 7.5 Hz, 3H), 7.15 - 6.93 (m, 2H), 5.50 (d, J = 14.1 Hz, 1H), 5.24 (dt, J = 54.2, 5.4 Hz, 1H), 4.78 (t, J = 5.6 Hz, 1H), 4.48 (dd, J = 10.7, 4.8 Hz, 1H), 4.34 (dd, J = 6.7, 4.9 Hz, 1H), 3.62 (dd, J = 11.9, 4.9 Hz, 1H), 3.55 - 3.35 (m, 3H), 0.82 (s, 9H), 0.07 (s, 3H), -0.09 (s, 3H). 19F NMR (376 MHz, DMSO-d6)δ-200.37 (d, J = 51.1 Hz).

[0215] LC / MS:t R = 1.25 min, MS m / z = 517.21 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ C18 100A, 50×3.00mm Solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid Gradient: 2 to 100% ACN from 0 to 1.4 min, 100% ACN from 1.4 to 1.80 min, 100% to 2% ACN from 1.8 to 1.85 min, and 2% ACN from 1.85 to 2 min.

[0216] [ka] Intermediate 2f - N-(7-((2S,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy)methyl)-3-fluorotetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0217] Intermediate 2e (370 mg, 0.717 mmol) was dissolved in CHCl (10 mL) and TEA (200 μL) under a N atmosphere and then cooled to 0 °C. 4,4'-Dimethoxytrityl chloride (0.364 g, 1.07 mmol) was added in one portion and the reaction mixture was stirred for 30 min. CHOH (2 mL) was added and the solution was diluted with CHCl and saturated NaHCO 3(aq)The mixture was diluted with HCl. The organic layer was washed with brine, dried over NaSO, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography using an eluent gradient of 5% to 100% EtOAc in hexanes to give the crude product as a mixture of bis-DMTr and the 4'β product. This mixture was used further without further purification.

[0218] To the crude product (574 mg, mixture) in DMF (3 mL) under N2 atmosphere was added imidazole (143 mg, 2.10 mmol), followed by tert-butylchlorodimethylsilane (158 mg, 1.05 mmol). The solution was stirred at room temperature for 2 h. The solution was diluted with CH3OH (1 mL) and EtOAc. The organics were washed with HO and then brine. The organic layer was dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography using an eluent gradient of 0% to 50% EtOAc in hexanes to give intermediate 2f, which contained some bis-DMTr material.

[0219] LC / MS:t R = 2.25 min, MS m / z = 933.52 [M+1]; LC system Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ C18 100A, 50×3.00mm Solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid Gradient: 2 to 100% ACN from 0 to 1.5 min, 100% ACN from 1.5 to 2.8 min, 100% to 2% ACN from 2.8 to 2.85 min, 2% ACN from 2.85 to 3 min.

[0220] [ka] Intermediate 2g - N-(7-((2S,3S,4R,5R)-4-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy)methyl)-3-fluoro-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0221] Intermediate 2f was dissolved in CHCl (5 mL) and cooled to 0 °C. To the mixture was added p-toluenesulfonic acid hydrate (90 mg, 0.474 mmol) dissolved in CHOH (4 mL) dropwise, and the reaction mixture was stirred for 5 min. The reaction mixture was diluted with saturated NaHCO 3(aq) The mixture was quenched with HCl. The organics were washed with brine, dried over NaSO, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography using an eluent gradient of 0% to 40% EtOAc in hexanes to give intermediate 2g.

[0222] 1 H NMR (400 MHz, CDCl3)δ8.09 - 7.88 (m, 2H), 7.48 (dt, J = 35.4, 7.4 Hz, 3H), 7.30 (d, J = 4.6 Hz, 1H), 6.88 (s, 1H), 5.69 (dd, J = 18.8, 4.2 Hz, 1H), 5.05 (dt, J = 54.6, 4.7 Hz, 1H), 4.62 (dd, J = 14.9, 5.1 Hz, 1H), 3.91 - 3.64 (m, 3H), 0.92 - 0.70 (m, 18H), 0.13 - 0.04 (m, 6H), 0.01 (m, 6H). 19 F NMR (376 MHz, CDCl3)δ-196 (m)

[0223] LC / MS:t R = 2.61 min, MS m / z = 631.43 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ C18 100A, 50×3.00mm Solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid Gradient: 2 to 100% ACN from 0 to 1.5 min, 100% ACN from 1.5 to 2.8 min, 100% to 2% ACN from 2.8 to 2.85 min, 2% ACN from 2.85 to 3 min.

[0224] [ka] Intermediate 2h - N-(7-((2S,3S,4R,5R)-5-((E)-2-bromovinyl)-4-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy)methyl)-3-fluorotetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0225] To a solution of intermediate 2g (228 mg, 0.361 mmol) in toluene (0.75 mL) and DMSO (0.15 mL) under a N atmosphere was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (208 mg, 1.08 mmol). To this mixture was added pyridine (30 μL) and TFA (15 μL), and the mixture was stirred at room temperature for 30 minutes. The reaction was diluted with EtOAc and washed with HO and then brine. The organics were dried over NaSO, filtered, and the solvent was removed under reduced pressure to give the crude aldehyde. This material was used directly in the next step.

[0226] To a suspension of bromomethyltriphenylphosphonium bromide (314 mg, 0.72 mmol) in THF (4 mL) at -40 °C was added KOtBu (1.0 M in THF, 1.08 mL, 1.08 mmol), and the reaction mixture was stirred under a N atmosphere for 2 h. The crude aldehyde was dissolved in THF (4 mL) and added dropwise. The reaction mixture was removed from the cold bath and allowed to warm to 10 °C over 1 h. The reaction mixture was recooled to -40 °C, and the reaction mixture was diluted with saturated NH4Cl (aq) The mixture was quenched with HCl. The layers were separated, the organics were dried over NaSO, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography using an eluent of 0% to 50% EtOAc in hexanes to give intermediate 2h.

[0227] 1 H NMR (400 MHz, CDCl3)δ8.08 - 7.85 (m, 2H), 7.62 - 7.36 (m, 2H), 7.34 - 7.01 (m, 3H), 6.92 (s, 1H), 6.60 - 6.45 (m, 1H), 6.35 (d, J = 8.2 Hz, 1H), 5.61 (d, J = 25.1 Hz, 1H), 4.82 (dd, J = 56.3, 4.9 Hz, 1H), 4.69 - 4.46 (m, 1H), 3.97 (d, J = 11.4 Hz, 1H), 3.53 (d, J = 11.4 Hz, 1H), 0.84 (d, J = 3.9 Hz, 18H), 0.13 - -0.10 (m, 12H). 19 F NMR (376 MHz, CDCl3)δ-190.60 (m).

[0228] LC / MS:t R = 2.10 min, MS m / z = 705.54 / 707.29 [M+1]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ C18 100A, 50 × 3.00 mm Solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid Gradient: 2 to 100% ACN from 0 to 1.5 min, 100% ACN from 1.5 to 2.8 min, 100% to 2% ACN from 2.8 to 2.85 min, 2% ACN from 2.85 to 3 min.

[0229] [ka] Intermediate 2i - N-(7-((2S,3S,4R,5R)-4-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy)methyl)-5-ethynyl-3-fluorotetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0230] Intermediate 2h (204 mg, 0.289 mmol) was dissolved in THF (8 mL) under a N atmosphere and cooled to -40 °C. KOtBu (1.0 M in THF, 1.08 mL, 1.08 mmol) was added slowly. Saturated NH4Cl (aq) The reaction was stirred for 20 minutes while being quenched with HCl. The solution was diluted with EtOAc and washed with brine. The organics were dried over NaSO, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography using an eluent gradient of 0% to 50% EtOAc in hexanes to give intermediate 2i.

[0231] 1H NMR (400 MHz, CDCl3)δ8.12 - 7.88 (m, 2H), 7.51 (dt, J = 36.5, 7.5 Hz, 2H), 7.32 (d, J = 4.6 Hz, 1H), 6.88 (s, 1H), 5.79 (d, J = 22.1 Hz, 1H), 5.02 (ddd, J = 55.3, 5.1, 3.2 Hz, 1H), 4.56 (dd, J = 18.1, 5.1 Hz, 1H), 3.91 (d, J = 11.3 Hz, 1H), 3.83 - 3.62 (m, 1H), 2.55 (m, 1H), 0.90 (dd, J = 25.3, 1.6 Hz, 18H), 0.20 - -0.08 (m, 12H). 19 F NMR (376 MHz, CDCl3)δ-193.10 (wide-s).

[0232] LC / MS:t R = 1.88 min, MS m / z = 625.24 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ C18 100A, 50×3.00mm Solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid Gradient: 2 to 100% ACN from 0 to 1.5 min, 100% ACN from 1.5 to 2.8 min, 100% to 2% ACN from 2.8 to 2.85 min, 2% ACN from 2.85 to 3 min.

[0233] [ka] Intermediate 2j - N-(7-((2S,3R,4R,5R)-5-ethynyl-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrrolo [1,2-f][1,2,4]triazin-4-yl)benzamide

[0234] To a solution of intermediate 2i (152 mg, 0.243 mmol) in THF (3.5 mL) was added TBAF (1.0 M in THF, 700 μL, 0.700 mmol) at room temperature under a N atmosphere, and the mixture was stirred for 30 min. The solvent was removed under reduced pressure. The residue was purified by silica gel chromatography using an eluent gradient of 40% to 100% EtOAc in hexane to give intermediate 2j.

[0235] LC / MS:t R = 0.88 min, MS m / z = 397.16 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ C18 100A, 50×3.00mm Solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid Gradient: 2 to 100% ACN from 0 to 1.5 min, 100% ACN from 1.5 to 2.8 min, 100% to 2% ACN from 2.8 to 2.85 min, 2% ACN from 2.85 to 3 min.

[0236] [ka] Example 2—(2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-2-ethynyl-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol

[0237] To a solution of intermediate 2j (71 mg, 0.179 mmol) in CH3OH (2 mL), concentrated NH4OH (aq) (0.7 mL) was added, and the resulting solution was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure. The residue was purified by silica gel chromatography using an eluent gradient of 0% to 20% CHOH in CHCl, followed by reverse-phase HPLC using an eluent gradient of 0% to 20% ACN in HO to give Example 2.

[0238] 1 H NMR (400 MHz, CD3OD)δ7.77 (s, 1H), 6.90 - 6.70 (m, 2H), 5.62 (dd, J = 25.5, 2.6 Hz, 1H), 5.18 (ddd, J = 56.0, 5.4, 2.7 Hz, 1H), 4.57 (dd, J = 20.5, 5.4 Hz, 1H), 3.93 - 3.59 (m, 2H), 3.02 (d, J = 0.7 Hz, 1H). 19 F NMR (376 MHz, CD3OD)δ-193.76 (ddd, J = 56.0, 25.5, 20.4 Hz).

[0239] LC / MS:t R = 0.45 min, MS m / z = 293.13 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ C18 100A, 50×3.00mm Solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid Gradient: 2% to 100% ACN from 0 min to 1.4 min, 100% ACN from 1.4 min to 1.80 min, 100% to 2% ACN from 1.8 min to 1.85 min, and ACCN from 1.85 min to 2 min. N is 2%. HPLC:t R = 3.112 min; HPLC system: Agilent 1100 series. Column: Phenomenex Kinetex C18 2.6 μm 100A, 4.6 x 100 mm Solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA

[0240] Gradient: 2–98% ACN from 0 min to 8.0 min, 1.5 mL / min.

[0241] [ka] Intermediate 3a - N-(7-((2S,3S,4R,5R)-4-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy)methyl)-3-fluoro-5-formyltetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0242] To a solution of intermediate 2g (1.13 g, 1.79 mmol) in DMSO (1 mL) and toluene (10 mL), prepared under a N2 atmosphere, was added EDCI·HCl (1.02 g, 5.36 mmol) and pyridine (149 μL, 1.92 mmol). TFA (74 μL, 0.97 mmol) was added dropwise. After 1 h, the reaction was checked by LC / MS. A single peak was observed, with a retention time similar to that of the starting material, but the M+1 peak was equal to that expected for the product. An additional 50 μL of pyridine was added, and the reaction was stirred for an additional 15 min. No change was observed by LC / MS. The reaction was diluted with EtOAc and saturated NaHCO 3(aq.) The reaction mixture was quenched with a 1:1 mixture of HCl and H2O. The mixture was partitioned between EtOAc and additional H2O. The organic layer was separated and washed with H2O, brine, and then dried over Na2SO4. The drying agent was removed by vacuum filtration, and the filtrate was concentrated. The residue was dissolved in CH2Cl2 and concentrated, and the resulting material was placed under high vacuum for 1 hour. The product, Intermediate 3a, was used directly in the next reaction.

[0243] LC / MS:t R = 1.90 min, MS m / z = 629.46 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ C18 100A, 50×3.00mm Solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid Gradient: 2 to 100% ACN from 0 to 1.5 min, 100% ACN from 1.5 to 2.8 min, 100% to 2% ACN from 2.8 to 2.85 min, 2% ACN from 2.85 to 3 min.

[0244] [ka] Intermediate 3b - N-(7-((2S,3S,4R,5R)-4-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy)methyl)-3 -fluoro-5-((E)-(hydroxyimino)methyl)tetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0245] To a solution of intermediate 3a (assuming crude material from the previous step, 1.79 mmol) in pyridine (11 mL) prepared under a N atmosphere was added HONH·HCl in one portion at room temperature. The reaction was checked by LC / MS after 5 min. The starting material was consumed. The reaction was checked again after 25 min. No change from the initial time point was observed. The reaction was concentrated, and the residue was partitioned between EtOAc and H O. The organic layer was separated, washed with brine, dried over Na SO , and filtered. The filtrate was concentrated, dissolved in CHCl and concentrated again, and the residue was placed under high vacuum. The product, intermediate 3b, was used directly in the next reaction.

[0246] LC / MS:t R = 1.83 min, MS m / z = 644.55 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ C18 100A, 50×3.00mm Solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid Gradient: 2 to 100% ACN from 0 to 1.5 min, 100% ACN from 1.5 to 2.8 min, 100% to 2% ACN from 2.8 to 2.85 min, 2% ACN from 2.85 to 3 min.

[0247] [ka] Intermediate 3c - N-(7-((2S,3S,4R,5R)-4-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy)methyl)-5-cyano-3-fluorotetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0248] To a solution of intermediate 3b (assuming crude material from the previous step, 1.79 mmol) in ACN (16 mL) was added CDI (436 mg, 2.69 mmol) in one portion. The reaction was carried out under a N2 atmosphere. After 20 min, the reaction was checked by LC / MS. The peaks with the masses of the starting material and product were barely resolved in time. The reaction was checked after 1.5 h. The UV peak corresponding to the starting material had almost disappeared, and the mass peak intensity had decreased. The reaction was diluted with CHCl and saturated NaHCO 3(aq.) The mixture was quenched with a 1:1 mixture of brine and HO. The layers were separated, the aqueous layer was back-extracted with CHCl, and the combined organic layers were extracted with a 1:1 mixture of brine and HO, dried over NaSO, and filtered. The filtrate was concentrated and intermediate 3c was isolated by silica gel column chromatography using the following solvent gradient: 0% EtOAc in hexanes, increasing to 20% EtOAc in hexanes, stopping at 20% EtOAc in hexanes, then increasing to 40% EtOAc in hexanes.

[0249] 1 H NMR (400 MHz, DMSO-d6)δ8.31 (s, 1H), 8.07 (s, 1H), 7.65 (t, J = 8 Hz, 1H), 7.54 (t, J = 8 Hz, 1H), 7.15 (d, 4 Hz, 1H), 7.02 (s, 1H), 5.82 (d, J = 24 Hz, 1H), 5.51 (ddd, J = 52, 4.8, 2.8 Hz, 1H), 4.70 (dd, J = 18.4, 4.4 Hz, 1H), 3.94 (dd, J = 53.2, 11.2 Hz, 2H), 0.93 (s, 9H, ), 0.84 (s, 9H), 0.17 (s, 3H), 0.16 (s, 3H), 0.05 (s, 3H), 0.01 (s, 3H). 19 F NMR (376 MHz, DMSO-d6)δ-194.658 (dt, J = 53, 21.4 Hz).

[0250] LC / MS:t R = 1.84 min, MS m / z = 626.60 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ C18 100A, 50×3.00mm Solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid Gradient: 2 to 100% ACN from 0 to 1.5 min, 100% ACN from 1.5 to 2.8 min, 100% to 2% ACN from 2.8 to 2.85 min, 2% ACN from 2.85 to 3 min.

[0251] [ka] Intermediate 3d - (2R,3R,4S,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3-(tert-butyldimethylsilyloxy)-2-((tert-butyldimethylsilyloxy)methyl)-4-fluorotetrahydrofuran-2-carbonitrile

[0252] To a solution of intermediate 3c (770 mg, 1.23 mmol) in MeOH (11.2 mL) cooled in an ice-water bath was added concentrated NH4OH. (aq)(3.74 mL) was added. The cold bath was removed and the resulting heterogeneous solution was stirred overnight. The next day the reaction was incomplete as determined by LC / MS. Additional concentrated NH4OH (aq) (4 mL) and 2-MeTHF (12 mL) were added. The reaction became homogeneous, but after 20 min, no further reaction progressed. The reaction was concentrated and the residue was dissolved in THF (15 mL). To this mixture was added concentrated NH4OH (aq) (5 mL) and enough MeOH (1.9 mL) were added to make the solution homogeneous and single-phase. The reaction was stirred at room temperature for 22 hours. The reaction was nearly complete (approximately 5% starting material remained). The reaction was diluted with CH2Cl2 and H2O. The layers were separated and the aqueous layer was washed with saturated NaHCO3 3(aq) The aqueous layer was diluted with 2N The mixture was neutralized with HCl and then extracted with CH2Cl2. The combined organic layers were dried over Na2SO4, which was removed by filtration. The filtrate was concentrated, and intermediate 3d was isolated from the residue by silica gel column chromatography using the following solvent gradient: 0% EtOAc in hexanes, increasing to 50% EtOAc in hexanes, stopping at 50% EtOAc in hexanes, and then increasing to 100% EtOAc in hexanes.

[0253] LC / MS:t R = 1.59 min, MS m / z = 522.47 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ C18 100A, 50×3.00mm Solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid Gradient: 2 to 100% ACN from 0 to 1.4 min, 100% ACN from 1.4 to 1.8 min, 100% to 2% ACN from 1.8 to 1.85 min, and 2% ACN from 1.85 to 2 min.

[0254] [ka] Example 3 - (2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-fluoro-3-hydroxy-2-(hydroxymethyl)tetrahydrofuran-2-carbonitrile

[0255] To a solution of intermediate 3d (100 mg, 0.191 mmol) in THF (2 mL) in a polypropylene tube was added 70% HF·pyridine in pyridine (60 μL, 0.478 mmol) under a N atmosphere at 0 °C. After 20 min, the reaction was checked. No reaction had occurred, so additional 70% HF·pyridine in pyridine (150 μL) was added and the cold bath was removed. After 1 h 50 min, additional 70% HF·pyridine in pyridine (150 μL) was added. After an additional 2 h, additional 70% HF·pyridine in pyridine (300 μL) was added. After an additional 2 h 15 min, additional 70% HF·pyridine in pyridine (1 mL) was added. The reaction became homogeneous and no longer cloudy, at which point the final 70% HF·pyridine in pyridine was added. The reaction was stirred overnight. The next day, the reaction was complete. The reaction was cooled in an ice bath and diluted with HO and a small amount of saturated NaHCO 3(aq) The mixture was quenched with HCl. The mixture was concentrated, and the residue was dissolved in DMSO. The remaining insoluble material was removed by filtration, and the filtrate was semi-purified by HPLC. The isolated material was dissolved in DMF and purified by HPLC. Example 3 was isolated as a TFA salt at 0.5%.

[0256] 1 H NMR (400 MHz, DMF-d7)δ7.92 (s, 1H), 6.99 (d, J = 4.4 Hz, 1H), 6.89 (d, J = 4.9 Hz, 1H), 6.64 (d, J = 6 Hz, 1H), 5.92 (t, J = 6.4 Hz, 1H), 5.83 (dd, J = 25.2, 2 Hz, 1H), 5.40 (ddd, J = 54.8, 4.8, 2.4 Hz, 1H), 4.75 (dt, J = 22, 5.2 Hz, 1H), 4.02 (dd, J = 12, 6.4Hz, 1H), 3.87 (dd, J = 12, 6.4 Hz, 1H). 19 F NMR (376 MHz, DMF-d7)δ-74.92 (s), -193.726 (dt, J = 54.5, 23.3 Hz).

[0257] LC / MS:t R = 0.56 min, MS m / z = 294.10 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ C18 100A, 50×3.00mm Solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid Gradient: 2 to 100% ACN from 0 to 1.4 min, 100% ACN from 1.4 to 1.8 min, 100% to 2% ACN from 1.8 to 1.85 min, and 2% ACN from 1.85 to 2 min. HPLC:t R = 3.220 min; HPLC system: Agilent 1100 series; column: Phenomenex Kinetex C18 2.6 μm 100A, 4.6 × 100 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA Gradient: 2–98% ACN from 0 min to 8.0 min, 1.5 mL / min.

[0258] [ka] Intermediate 4b - (3aR,5R,6aS)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyldihydrofuro[3,2-d][1,3]dioxol-6(3aH)-one

[0259] A 10 L four-neck round-bottom flask was charged with a solution of intermediate 4a, (3aR,5S,6S,6aR)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydrofuro[3,2-d][1,3]dioxol-6-ol (500 g, 1.90 mol) in dichloromethane / water (2.7 L / 2.3 L) at room temperature. To this was added sodium carbonate (290 g, 3.42 mol). Potassium carbonate (451 g, 3.24 mol) was then added. This was followed by the addition of 2,2,6,6-tetramethylpiperidinol (TEMPO, 15.2 g, 96.31 mmol). To this mixture was added tetrabutylammonium bromide (31 g, 95.20 mmol). To the above, N-bromosuccinimide (514 g, 2.86 mol) was added portionwise at 35° C. The resulting solution was reacted at room temperature for 2 hours with stirring. The resulting solution was extracted with 2×1 L of dichloromethane, and the organic layers were combined. The resulting mixture was washed with 1×1.5 L of water. The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum to give (crude) intermediate 4b.

[0260] [ka] Intermediate 4c - (3aR,5S,6R,6aR)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydrofuro[3,2-d][1,3]dioxol-6-ol

[0261] A 2 L four-necked round-bottom flask was charged with a solution of intermediate 4b (370 g, 1.29 mol) in methanol (1300 mL). To the above, sodium borohydride (26.4 g, 706.38 mmol) was added portionwise at room temperature. The resulting solution was reacted at room temperature for 2 hours with stirring. The resulting mixture was concentrated under vacuum. The reaction was then quenched by adding 1000 mL of 5% aqueous ammonium chloride solution. The resulting solution was extracted with 3 x 500 mL of dichloromethane, and the organic layers were combined. The resulting solution was washed with 2 x 300 mL of water. The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. The crude product was purified by recrystallization from petroleum ether. This afforded intermediate 4c.

[0262] [ka] intermediate 4d A 5000 mL four-neck round-bottom flask was charged with a solution of intermediate 4c (350 g, 1.34 mol) in dichloromethane (700 mL). To the mixture was added 50% sodium hydroxide / water (700 g). 2-(bromomethyl)naphthalene (340 g, 1.54 mol) was added in portions. The resulting solution was stirred at room temperature for 4 hours. The reaction was then quenched by adding 1800 mL of dichloromethane / water (1:1). The resulting solution was extracted with 2 x 1 L of dichloromethane and the organic layers were combined. The resulting mixture was washed with 1 x 1000 mL of water. The residue was dissolved in 1000 / 1000 mL of petroleum ether / water. The crude product was purified by recrystallization from petroleum ether. This afforded intermediate 4d.

[0263] [ka] Intermediate 4e - (R)-1-((3aR,5R,6R,6aR)-2,2-dimethyl-6-(naphthalen-2-ylmethoxy)tetrahydrofuro[3,2-d][1,3]dioxol-5-yl)ethane-1,2-diol

[0264] Intermediate 4d (500 g, 1.25 mol) was placed in a 5 L four-neck round-bottom flask. Acetic acid (1.8 L) was added to this. Water (600 mL) was added to the mixture. The resulting solution was reacted overnight at room temperature with stirring. The solid was filtered off. The resulting solution was extracted with 3 x 1 L of petroleum ether and the aqueous layers were combined. The resulting solution was diluted with 2 L of ethyl acetate. The resulting mixture was added with sodium chloride. (aq) The resulting solution was washed with 2×2 L of ethyl acetate. The pH of the solution was adjusted to 8 with sodium carbonate (50%). The resulting solution was extracted with 2×1 L of ethyl acetate, and the combined organic layers were concentrated under vacuum to give intermediate 4e.

[0265] [ka] Intermediate 4f - (3aR,5S,6R,6aR)-2,2-dimethyl-6-(naphthalen-2-ylmethoxy)tetrahydrofuro[3,2-d][1,3]dioxole-5-carbaldehyde

[0266] A 10 L four-necked round-bottom flask was charged with a solution of intermediate 4e (300 g, 833.33 mmol) in 1,4-dioxane (2100 mL) at room temperature. Then, a solution of sodium periodate (250 g) in water (4000 mL) was added at room temperature over 0.5 hours. The resulting solution was stirred at room temperature for 0.5 hours. The solid was filtered off. The resulting solution was extracted with 3 x 1000 mL of ethyl acetate, and the organic layers were combined. The resulting mixture was added with sodium chloride. (aq) Washed with 2×1000 mL. The resulting mixture was concentrated in vacuo to give intermediate 4f.

[0267] [ka] Intermediate 4g - ((3aR,6S,6aR)-2,2-dimethyl-6-(naphthalen-2-ylmethoxy)tetrahydrofuro[3,2-d][1,3]dioxole-5,5-diyl)dimethanol

[0268] A 10 L four-necked round-bottom flask was charged with a solution of intermediate 4f (250 g, 761.36 mmol) in water / tetrahydrofuran (1250 / 1250 mL) at room temperature. 2N sodium hydroxide was added to the mixture at 0-15°C with stirring. (aq) (1500 mL) was added dropwise. To the mixture, formaldehyde (620 mL) was added. The resulting solution was reacted overnight at room temperature with stirring. The resulting solution was extracted with 2×2000 mL of ethyl acetate. The resulting mixture was added with sodium chloride. (aq) The organic layers were combined and dried over anhydrous sodium sulfate. The residue was applied to a silica gel column using petroleum ether:ethyl acetate (2 / 1). The crude product was recrystallized from ethyl acetate:ethanol in a ratio of 1 g / (1 mL:1 mL). This gave 4 g of intermediate.

[0269] [ka] Intermediate 4h - ((3aR,5R,6S,6aR)-5-((tert-butyldiphenylsilyloxy)methyl)-2,2-dimethyl-6-(naphthalen-2-ylmethoxy)tetrahydrofuro[3,2-d][1,3]dioxol-5-yl)methanol

[0270] A 5 L four-necked round-bottom flask, purged with nitrogen and maintaining the inert atmosphere, was charged with a solution of 4 g of intermediate (125 g, 346.84 mmol) in 2500 mL of dichloromethane at room temperature. Triethylamine (157.5 mL) was added to the mixture at room temperature. To the above, tert-butyldiphenylsilyl chloride (157.5 mL) was added dropwise with stirring at 0-10°C. The resulting solution was allowed to react overnight at room temperature with stirring. The reaction was then quenched by adding 37.5 mL of methanol. The resulting mixture was added to 5% hydrogen chloride. (aq) 2 x 500 mL and sodium bicarbonate (aq) The resulting mixture was washed with 2 x 500 mL of 1 N sodium hydroxide. (aq) The mixture was washed with 2×500 mL of hexanes. The mixture was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by recrystallization from dichloromethane / hexanes to give intermediate 4h.

[0271] [ka] Intermediate 4i - tert-butyl(((3aR,5R,6S,6aR)-5-(iodomethyl)-2,2-dimethyl-6-(naphthalen-2-ylmethoxy)tetrahydrofuro[3,2-d][1,3]dioxol-5-yl)methoxy)diphenylsilane

[0272] A 1000 mL three-necked round-bottom flask, purged with nitrogen and maintaining the inert atmosphere, was charged with a solution of intermediate 4h (20 g, 31.73 mmol) in toluene (320 mL), triphenylphosphine (35 g, 132.11 mmol), and imidazole (8.96 g, 132.26 mmol). This was followed by the addition of iodine (16.95 g, 66.8 mmol) in several portions at 60 °C. The resulting solution was stirred overnight at 80 °C in an oil bath. The reaction mixture was cooled to room temperature in a water / ice bath. The resulting solution was diluted with 1000 mL of ethyl acetate. The resulting mixture was added with sodium thiosulfate. (aq) The resulting mixture was washed with 2 x 300 mL of sodium chloride. (aq)The mixture was washed with 1×300 mL of ethyl acetate and concentrated under vacuum. The residue was applied to a silica gel column using ethyl acetate / petroleum ether (1:10). This gave intermediate 4i.

[0273] [ka] Intermediate 4j - tert-butyldiphenyl(((3aR,5R,6S,6aR)-2,2,5-trimethyl-6-(naphthalen-2-ylmethoxy)tetrahydrofuro[3,2-d][1,3]dioxol-5-yl)methoxy)silane

[0274] A 2000 mL round-bottom flask, purged with nitrogen and maintaining the inert atmosphere, was charged with a solution of intermediate 4i (66 g, 88.47 mmol) in ethanol / ethyl acetate (600 / 600 mL), triethylamine (20.7 g, 202.52 mmol), and palladium on carbon (10 wt%, 24.8 g, 23.30 mmol). The resulting solution was stirred at 40° C. for 3 hours. The solids were removed by filtration. The resulting mixture was concentrated under vacuum. The resulting solution was diluted with 1500 mL of ethyl acetate. The resulting mixture was diluted with sodium chloride. (aq) The mixture was washed with 1×500 mL of HCl. The mixture was dried over anhydrous sodium sulfate and concentrated in vacuo to give intermediate 4j.

[0275] [ka] Intermediate 4k - (3aR,5R,6S,6aR)-5-((tert-butyldiphenyl) ((silyloxy)methyl)-2,2,5-trimethyltetrahydrofuro[3,2-d][1,3]dioxol-6-ol

[0276] A 500 mL round-bottom flask was charged with a solution of intermediate 4j (1.0 g, 1.63 mmol) in dichloromethane (15 mL), water (1.25 mL), and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 780 mg, 3.40 mmol). The resulting solution was stirred at room temperature for 1 hour. The resulting solution was diluted with 50 mL of dichloromethane. The resulting mixture was added to 1×30 mL of water and sodium bicarbonate. (aq) The resulting mixture was washed with 2 x 30 mL of sodium chloride. (aq) The mixture was washed with 1×30 mL of ethyl acetate. The mixture was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was applied to a silica gel column using ethyl acetate / petroleum ether (1:20 to 1:10). This gave intermediate 4k.

[0277] [ka] Intermediate 4l - (3aR,5R,6S,6aR)-5-(hydroxymethyl)-2,2,5-trimethyltetrahydrofuro[3,2-d][1,3]dioxol-6-ol

[0278] A 50 mL round-bottom flask was charged with a solution of intermediate 4k (520 mg, 1.12 mmol) in tetrahydrofuran (9 mL) and tetrabutylammonium fluoride (369 mg, 1.40 mmol). The resulting solution was stirred at room temperature for 2 hours. The resulting mixture was concentrated in vacuo. The residue was applied to a silica gel column using dichloromethane / methanol (100:1). This afforded intermediate 4l.

[0279] 1 H NMR (300 MHz, DMSO-d6):δ5.64 (d, J = 3.9Hz, 1H), 4.96 (d, J = 6.6Hz, 1H), 4.67 (m, 1H), 4.55 (m, 1H), 4.05 (m, 1H), 3.24 - 3.30 (m, 1H), 3.11 - 3.18 (m, 1H), 1.50 (s, 3H), 1.27 (s, 3H), 1.16 (s, 1H).

[0280] [ka] Intermediate 4m -(3aR,5R,6S,6aR)-6-(benzyloxy)-5-(benzyloxymethyl)-2,2,5-trimethyltetrahydrofuro[3,2-d][1,3]dioxole

[0281] A 50 mL three-necked round-bottom flask, purged with nitrogen and maintaining the inert atmosphere, was charged with a solution of intermediate 4l (180 mg, 0.84 mmol) in tetrahydrofuran (4 mL). This was followed by the addition of sodium hydride (60 wt%, 140 mg, 3.50 mmol) in small portions at 0°C. The resulting solution was stirred at 0°C for 30 minutes. The resulting solution was allowed to react at room temperature with stirring for an additional 30 minutes. To this was added benzyl bromide (452 ​​mg, 2.62 mmol) dropwise with stirring at 0°C. The resulting solution was allowed to react at room temperature with stirring for an additional 30 minutes. The reaction was then allowed to react for 3 hours. (aq) The mixture was quenched by adding 30 mL of HCl. The resulting solution was extracted with 50 mL of dichloromethane, and the combined organic layers were dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied to a silica gel column using ethyl acetate / petroleum ether (1:30 to 1:20). This afforded intermediate 4m.

[0282] [ka] Intermediate 4n - (2R,3R,4S,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-5-methyltetrahydrofuran-2,3-diyl diacetate

[0283] A 1000 mL round-bottom flask was charged with intermediate 4m (also prepared according to Biosci. Biotech. Biochem. 1993, 57, 1433-1438, 45 g, 111.19 mmol), acetic acid (270 mL), acetic anhydride (90 mL), and sulfuric acid (45 mL). The resulting solution was stirred at room temperature for 30 minutes. The reaction was then quenched by adding 1000 mL of water / ice. The resulting solution was diluted with 3000 mL of ethyl acetate. The resulting mixture was diluted with 2×1000 mL of water and sodium bicarbonate. (aq) The resulting mixture was washed with 4 x 1000 mL of sodium chloride. (aq) The mixture was washed with 2×1000 mL of ethyl acetate and concentrated under vacuum. The residue was applied to a silica gel column using ethyl acetate / petroleum ether (1:30 to 1:20). This gave intermediate 4n.

[0284] 1 H NMR (300 MHz, CDCl3):δ7.28 - 7.38 (m, 10H), 6.13 (s, 1H), 5.37 (d, J = 4.8Hz, 1H), 4.44 - 4.68 (m, 4H), 4.33 (d, J = 5.1Hz, 1H), 3.33 - 3.45 (m, 2H), 2.15 (s, 3H), 1.88 (s, 3H), 1.35 (s, 3H). MS m / z=451[M+Na]

[0285] [ka] Intermediate 4o - (3R,4S,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-hydroxy-5-methyldihydrofuran-2(3H)-one

[0286] Intermediate 4n (1.3 g, 3 mmol) was dissolved in anhydrous MeOH (15 mL). Powdered potassium carbonate (456 mg, 3.3 mmol) was added, and the reaction mixture was stirred for 1 hour. The reaction mixture was then concentrated under reduced pressure. Acetonitrile was added, and the mixture was stirred for 5 minutes. The insoluble material was filtered off and washed with acetonitrile. The filtrate was concentrated under reduced pressure. The resulting material was dissolved in anhydrous DCM (20 mL). Tetrabutylammonium iodide (1.66 g, 4.5 mmol) and N-iodo-succinimide (NIS, 1.69 g, 2.5 mmol) were added. The reaction mixture was stirred in the dark for 16 hours. Additional NIS (0.85 g, 1.25 mmol) was added, and the mixture was stirred for 4 hours. Additional NIS (0.85 g, 1.25 mmol) was added, and the mixture was stirred in the dark for 2 days. The reaction mixture was diluted with EtOAc, and aqueous sodium thiosulfate was added. The organic portion was washed twice with hexanes and then with saturated aqueous sodium chloride. The organic portion was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purification by silica gel column (0-30% EtOAc in hexanes) gave intermediate 4o.

[0287] 1 H NMR (400 MHz, CDCl3):δ7.35 - 7.22 (m, 10H), 4.82 (bs, 1H), 4.75 - 4.66 (m, 2H), 4.55 - 4.44 (m, 2H), 4.13 (d, J = 8Hz, 1H), 3.70-3.45 (m, 2H), 1.38 (s, 3H).

[0288] LC / MS:t R = 2.58 min, MS m / z = 342.9 [M+1], 360.0 [M+HO]; LC / MS system: Thermo LCQ Advantage; Phenomenex Gemini, C 18 , 5u, 110A, 30 x 4.6mm; Buffer A: 0.1% acetic acid in water; Buffer B: 0.1% acetic acid in acetonitrile 5 to 100% buffer B in 2.5 min, then 100% for 0.9 min, 2 mL / min. HPLC:t R= 3.78 min; HPLC system: Agilent 1100; Phenomenex Gemini, C 18 , 5u, 110A, 50 x 4.6mm; Buffer A: 0.05% TFA in water; Buffer B: 0.05% TFA in acetonitrile; Buffer B from 2 to 98% in 5 min, 2 mL / min.

[0289] [ka] Intermediate 4p - (3R,4S,5R)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)-5-methyldihydrofuran-2(3H)-one

[0290] Intermediate 4o (955 mg, 2.79 mmol) was dissolved in EtOAc (10 mL). Benzyl bromide (400 μL, 3.35 mmol) and silver(I) oxide (712 mg, 3.07 mmol) were added. The mixture was stirred at 60°C in the dark under N2(g) for 3 h. Additional benzyl bromide (400 μL, 3.35 mmol) was added and the mixture was stirred at 60°C in the dark under N2(g) for 16 h. Additional silver(I) oxide (350 mg, 1.5 mmol) was added and the mixture was stirred at 60°C in the dark under N2(g) for 8 h. The mixture was cooled to room temperature. The solid was filtered off and washed with EtOAc. The filtrate was concentrated under reduced pressure to give an oil. Hexane was added and stirred for 2 h to give a solid. The solid was collected and washed with hexane. The solid was dried under high vacuum to give intermediate 4p.

[0291] 1 H NMR (400 MHz, CDCl3):δ7.35 - 7.16 (m, 15H), 5.03 (d, J = 12Hz, 1H), 4.79 - 4.71 (m, 2H), 4.52 - 4.40 (m, 4H), 4.06 (d, J = 6Hz, 1H), 3.49 - 3.39 (m, 2H), 1.38 (s, 3H).

[0292] LC / MS:t R= 2.91 min, MS m / z = 433.1 [M+1], 450.1 [M+HO]; LC / MS system: Thermo LCQ Advantage; Phenomenex Gemini, C 18 , 5u, 110A, 30 x 4.6mm; Buffer A: 0.1% acetic acid in water; Buffer B: 0.1% acetic acid in acetonitrile; Buffer B from 5 to 100% in 2.5 min, then 100% for 0.9 min, 2 mL / min. HPLC:t R = 4.54 min; HPLC system: Agilent 1100; Phen omenex Gemini, C 18 , 5u, 110A, 50 x 4.6mm; Buffer A: 0.05% TFA in water; Buffer B: 0.05% TFA in acetonitrile 2 to 98% Buffer B in 5 min, 2 mL / min.

[0293] [ka] Intermediate 4q - (3R,4S,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)-5-methyltetrahydrofuran-2-ol

[0294] Intermediate 1b (148 mg, 0.570 mmol) and 1,2-bis(chlorodimethylsilyl)ethane (123 mg, 0.570 mmol) were dissolved in anhydrous THF (20 mL) and stirred at −70° C. under Ar(g). To the reaction mixture was added n-butyllithium (2.5 M solution in hexanes, 684 μL, 1.71 mmol) dropwise, maintaining the internal temperature below −65° C. The reaction was warmed to −40° C. and maintained for 15 min. Next, to the reaction mixture under Ar(g) was added a solution of intermediate 4p (224 mg, 0.518 mmol) in THF (10 mL) pre-cooled to −70° C. The resulting solution was stirred at −40° C. for 2 h. The reaction mixture was then diluted with EtOAc and citric acid. (aq) The organic layer was poured into a stirred mixture of 100 ml of water and 100 ml of saturated NaCl. The mixture was stirred for 5 minutes. (aq)The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. Purification by preparative HPLC gave intermediate 4q.

[0295] LC / MS:t R = 2.60 min, MS m / z = 567.3 [M+1], 565.1 [M-1]; LC / MS system: Thermo LCQ Advantage; Phenomenex Gemini, C 18 , 5u, 110A, 30 x 4.6mm; Buffer A: 0.1% acetic acid in water; Buffer B: 0.1% acetic acid in acetonitrile; Buffer B from 5 to 100% in 2.5 min, then 100% for 0.9 min, 2 mL / min. HPLC:t R = 3.22 min; HPLC system: Agilent 1100; Phenomenex Gemini, C 18 , 5u, 110A, 50 x 4.6mm; Buffer A: 0.05% TFA in water; Buffer B: 0.05% TFA in acetonitrile; Buffer B from 2 to 98% in 5 min, 2 mL / min.

[0296] [ka] Intermediate 4r - 7-((2S,3S,4S,5R)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)-5-methyltetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-amine

[0297] Intermediate 4q (81 mg, 0.143 mmol) was dissolved in anhydrous DCM (15 mL) and stirred in an ice bath under N2(g). Triethylsilane (114 μL, 0.715 mmol) ) was added in one portion. Boron trifluoride diethyl etherate (27 μL, 0.215 mmol) was added dropwise. Stirred for 15 minutes, then the ice bath was removed. Stirred for 60 minutes. Triethylamine (100 μL, 0.715 mmol) was added and concentrated under reduced pressure. Dissolved in EtOAc and saturated NaHCO 3(aq) (2x), then NaCl (aq)The organics were dried over anhydrous NaSO and concentrated under reduced pressure. Purification by silica gel column (0-80% EtOAc in hexanes) gave intermediate 4r.

[0298] 1 H NMR (400 MHz, CDCl3):δ7.71 (s, 1H), 7.35 - 7.10 (m, 16H), 6.82-6.78 (m, 1H), 5.57 (d, J=4.4Hz, 1H), 4.70 - 4.45 (m, 6H), 4.25 - 4.15 (m, 2H), 3.55 - 3.40 (m, 2H), 1.42 (s, 3H). LC / MS:t R = 2.75 min, MS m / z = 551.4 [M+1]; LC / MS system: Thermo LCQ Advantage Phenomenex Gemini, C 18 , 5u, 110A, 30 x 4.6mm; Buffer A: 0.1% acetic acid in water; Buffer B: 0.1% acetic acid in acetonitrile; Buffer B from 5 to 100% in 2.5 min, then 100% for 0.9 min, 2 mL / min. HPLC:t R = 3.57 min; HPLC system: Agilent 1100; Phenomenex Gemini, C 18 , 5u, 110A, 50 x 4.6mm; Buffer A: 0.05% TFA in water; Buffer B: 0.05% TFA in acetonitrile; Buffer B from 2 to 98% in 5 min, 2 mL / min.

[0299] [ka] Example 4—(2R,3S,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-2-(hydroxymethyl)-2-methyltetrahydrofuran-3,4-diol

[0300] Intermediate 4r (23 mg, 0.042 mmol) was dissolved in a formic acid / MeOH solution (1:9, 10 mL). Palladium black was added and the mixture was stirred at 60° C. for 90 minutes. The mixture was cooled to room temperature and filtered through Celite. The filtrate was concentrated under reduced pressure. Purified by preparative HPLC. Concentrated under reduced pressure. NaHCO 3(aq) and purified by HPLC under neutral conditions to give Example 4.

[0301] Preparative HPLC system: Gilson 215 Liquid Handler; Phenomenex Gemini, C 18 4u, 100×30.0mm Buffer A: 0.1% TFA in water; Buffer B: 0.1% TFA in acetonitrile; 5 to 100% of Buffer B in 13 min, 20 mL / min.

[0302] 1 H NMR (400 MHz, CDCl3):δ8.01 (s, 1H), 7.41 (d, J = 4.8 Hz, 1H), 7.02 (d, J = 4.8 Hz, 1H), 5.33 (d, J = 8 Hz, 1H), 4.53-4.49 (m, 1H), 4.15 (d, J = 5.6 Hz, 1H), 3.50 (m, 2H), 1.27 (s, 3H).

[0303] LC / MS:t R = 0.30 min, MS m / z = 281.3 [M+1], 279.0 [M-1]; LC / MS system: Thermo LCQ Advantage; Phenomenex Gemini, C 18 , 5u, 110A, 30 x 4.6mm; Buffer A: 0.1% acetic acid in water; Buffer B: 0.1% acetic acid in acetonitrile; Buffer B from 5 to 100% in 2.5 min, then 100% for 0.9 min, 2 mL / min. HPLC:t R = 0.42 min; HPLC system: Agilent 1100; Phenomenex Gemini, C 18, 5u, 110A, 50 x 4.6mm; Buffer A: 0.05% TFA in water; Buffer B: 0.05% TFA in acetonitrile; Buffer B from 2 to 98% in 5 min, 2 mL / min.

[0304] [ka] Intermediate 5a - N-(7-((2S,3S,4S,5R)-5-azido-3,4-bis(tert-butyldimethylsilyloxy)-5-((trimethylsilyloxy)methyl)tetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0305] To a solution of crude intermediate 1m, N-(7-((2S,3S,4S)-3,4-bis(tert-butyldimethylsilyloxy)-5-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide (approximately 110 mg, approximately 0.18 mmol) and azidotrimethysilane (242 μL, 1.84 mmol) in dichloromethane (1.5 mL) was added indium(III) bromide (130 mg, 0.369 mmol) under an argon atmosphere at room temperature. After 1 h, the reaction mixture was quenched with saturated aqueous sodium bicarbonate (1 mL). The resulting mixture was partitioned between dichloromethane (20 mL) and saturated aqueous sodium bicarbonate (20 mL). The phases were separated, and the aqueous layer was extracted with dichloromethane (20 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to give intermediate 5a, which was used directly in the next step without further purification.

[0306] LC / MS:t R = 3.52 min, MS m / z = 712.16 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50×4.6mm Solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 2 to 100% ACN from 0 min to 2.0 min, 100% ACN from 2.0 min to 3.55 min, 100% to 2% ACN from 3.55 min to 4.2 min, 2 μl / min.

[0307] [ka] Intermediate 5b - N-(7-((2S,3R,4S,5R)-5-azido-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0308] To a solution of crude intermediate 5a (approximately 120 mg, approximately 0.168 mmol) in DMF (5 mL) was added cesium fluoride (256 mg, 1.68 mmol) at room temperature. After 25 hours, the reaction mixture was diluted with brine (100 mL), and the resulting mixture was extracted with ethyl acetate (3 × 100 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to give intermediate 5b, which was used directly in the next step without further purification.

[0309] LC / MS:t R = 1.40 min, MS m / z = 412.17 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50 × 4.6 mm; Solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 2–100% ACN from 0 min to 2.0 min, 100% ACN from 2.0 min to 3.05 min, 100%–2% ACN from 3.05 min to 3.2 min, 2% ACN from 3.2 min to 3.5 min, 2 μl / min. HPLC:t R= 2.46 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 2 to 98% ACN from 0 to 5.0 min, then 98% ACN from 5.0 to 6.0 min, 2 mL / min.

[0310] [ka] Example 5—(2R,3S,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-2-azido-2-(hydroxymethyl)tetrahydrofuran-3,4-diol

[0311] To a solution of crude intermediate 5b in methanol (1 mL) was added concentrated ammonium hydroxide (1 mL) at room temperature. After 2 days, the reaction mixture was concentrated under reduced pressure and purified directly by preparative HPLC (Phenominex Synergi 4u Hydro-RR 80Å 150 × 30 mm column, 5–100% acetonitrile / water gradient). The desired fractions were combined and concentrated under reduced pressure. The residue was repurified by SiO column chromatography (4 g SiO Combiflash HP Gold Column, 0–20% methanol / dichloromethane) to give Example 5.

[0312] 1 H NMR (400 MHz, CD3OD)δ7.79 (s, 1H), 6.86 (d, J = 4.5 Hz, 1H), 6.77 (d, J = 4.5 Hz, 1H), 5.51 (d, J = 6.0 Hz, 1H), 4.63 (t, J = 5.8 Hz, 1H), 4.37 (d, J = 5.7 Hz, 1H), 3.69 (d, J = 12.0 Hz, 1H), 3.59 (d, J = 12.0 Hz, 1H).

[0313] LC / MS:tR = 0.76 min, MS m / z = 308.08 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50 × 4.6 mm; Solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 2 to 100% ACN from 0 min to 1.5 min, 100% ACN from 1.5 min to 2.2 min, 100% to 2% ACN from 2.2 min to 2.4 min, 2% ACN from 2.4 min to 2.5 min, 2 μl / min. HPLC:t R = 1.287 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 2–98% ACN from 0 min to 5.0 min, then 98% ACN from 5.0 min to 6.0 min, 2 mL / min. TLC: Eluent: 20% methanol in dichloromethane, R f =0.4(UV)

[0314] [ka] Example 6 (also referred to as TP-1) - ((2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-2-cyano-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate

[0315] Example 3 (15.0 mg, 0.05 mmol) was dried in a flask under vacuum overnight. Trimethyl phosphate (0.5 mL) and 1,8-bis(dimethylamino)naphthalene (25 mg, 0.12 mmol) were added to the flask, and the solution was cooled in an ice / water bath and stirred under N2. Distilled phosphorus oxychloride (10 μL, 0.11 mmol) was added, and the reaction was stirred with cooling for 4 hours. Tributylamine (0.1 mL, 0.42 mmol) and tributylammonium pyrophosphate (0.8 mL of a 0.5 M solution in DMF, 0.4 mmol) were added, and the reaction was stirred with cooling for an additional 45 minutes. The reaction was quenched with triethylammonium bicarbonate (0.5 M, 5 mL). The solvent was removed by rotary evaporation, and the remaining crude mixture was dissolved in 2 mL of water. The product was purified using a Sephadex DEAE A-25 column with a linear gradient of triethylammonium bicarbonate from 0 to 1 M. Product-containing fractions were pooled and concentrated to give Example 6 (TP1), which was then dissolved in 1 mL of water to give a 10 mM solution. MS m / z=532.0[M-1] Ion exchange HPLC retention time: 12.015 min; column: DNAPac PA-100 4×250mm SN

[0316] Solvent A: milliQ water; Solvent B: 0.5 M tetraethylammonium bromide; Solvent gradient program: equilibration with 100% A for 10 min, then a gradient of 0-80% B over 14 min; Flow rate: 1 mL / min.

[0317] [ka] (Example 7) (also TP2)—((2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-2-ethynyl-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate

[0318] Example 2 (16.0 mg, 0.055 mmol) was dried in a flask under vacuum overnight. Trimethyl phosphate (0.5 mL) and 1,8-bis(dimethylamino)naphthalene (28 mg, 0.13 mmol) were added to the flask, and the solution was cooled in an ice / water bath and stirred under N2. Distilled phosphorus oxychloride (11 μL, 0.12 mmol) was added, and the reaction was stirred with cooling for 4 hours. Tributylamine (0.11 mL, 0.42 mmol) and tributylammonium pyrophosphate (0.9 mL of a 0.5 M solution in DMF, 0.45 mmol) were added, and the reaction was stirred with cooling for an additional 45 minutes. The reaction was quenched with triethylammonium bicarbonate (0.5 M, 5 mL). The solvent was removed by rotary evaporation, and the remaining crude mixture was dissolved in 2 mL of water. The product was purified using a Sephadex DEAE A-25 column with a linear gradient of triethylammonium bicarbonate from 0 to 1 M. Product-containing fractions were pooled and concentrated to give Example 7 (TP2), which was then dissolved in 1.4 mL of water to give a 10 mM solution.

[0319] MS m / z=531.0[M-1] Ion-exchange HPLC retention time: 19.829 min; column: DNAPac PA-100 4×250mm SN Solvent A: milliQ water; Solvent B: 0.5 M tetraethylammonium bromide; Solvent gradient program: equilibration with 100% A for 10 min, then a gradient of 0-80% B over 14 min; Flow rate: 1 mL / min.

[0320] [ka] Example 8 (TP3) - ((2R,3S,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-2-cyano-3,4-dihydro (2-Oxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate

[0321] To a solution of Example 1 (5.0 mg, 0.017 mmol) in PO(OMe) (0.6 mL) was added POCl (45 mg, 0.29 mmol) at 0° C. The reaction mixture was stirred at 0° C. for 10 hours, at which point ion-exchange HPLC showed approximately 50% conversion. A solution of pyrophosphate tributylamine salt (250 mg) in ACN (0.6 mL) was added, followed by tributylamine (110 mg, 0.59 mmol). The reaction mixture was stirred at 0° C. for 0.5 hours, at which point ion-exchange HPLC showed the reaction was complete. The reaction was quenched with triethylammonium bicarbonate buffer (1 M, 5 mL). The reaction mixture was stirred at room temperature for 0.5 hours, then concentrated and coevaporated twice with water. The residue was dissolved in HO (5 mL) and loaded onto an ion exchange column, eluting with HO, followed by 5-35% triethylammonium bicarbonate buffer (1 M)-HO. The product fractions were combined, concentrated, and co-evaporated with HO. The residue was purified again by ion exchange column to give the crude material. 31 P NMR indicated that this material contained an impurity, so it was repurified on a C-18 column, eluting with 0-15% ACN-HO containing 0.05% TEA, and the product-containing fractions were combined and concentrated to give 3.6 mg of material. 1 H NMR analysis showed that it contained only 1.5 equivalents of TEA. This material was dissolved in HO (1 mL) and triethylammonium bicarbonate buffer (1 M, 0.1 mL) was added. The resulting mixture was concentrated under reduced pressure and coevaporated twice with HO under reduced pressure to give Example 8 (TP3) as the tetra-TEA salt.

[0322] 1H NMR (400 MHz, D2O):δ7.78 (s, 1H), 6.85 (d, J = 2.4 Hz, 1H), 6.82 (d, J = 2.4 Hz, 1H), 5.51 (d, J = 3.0 Hz, 1H), 4.65 - 4.55 (m, 2H), 4.20 - 4.08 (m, 2H), 3.15 - 3.00 (m, 24H), 1.18 - 1.08 (m, 36H). 31 P NMR (162 MHz, D2O):δ-6.25 (d, J = 52 Hz), -12.21 (d, J = 52 Hz), -22.32 (t, J = 52 Hz). MS m / z=530.2[M-1], 532.1[M+1]

[0323] [ka] (Example 9) (TP4)—((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-azido-3,4-dihydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate

[0324] To a solution of Example 5 (6.0 mg, 0.019 mmol) in PO(OMe) (0.6 mL) was added POCl (45 mg, 0.29 mmol) at 0° C. The reaction mixture was stirred at 0° C. for 10 hours, at which point ion exchange HPLC indicated approximately 50% conversion. A solution of pyrophosphate tributylamine salt (250 mg) in ACN (0.6 mL) was added, followed by tributylamine (110 mg, 0.59 mmol). The reaction mixture was stirred at 0° C. for 6 hours. The reaction was purified by triethylammonium bicarbonate. The reaction mixture was quenched with 1M buffer (5 mL). The reaction mixture was stirred at room temperature for 0.5 h, then concentrated and coevaporated twice with water. The residue was dissolved in HO (5 mL) and loaded onto an ion-exchange column, eluting with HO, then 5-35% triethylammonium bicarbonate buffer (1M)-HO. The product fractions were combined, concentrated, and coevaporated with HO. The residue was purified again by ion-exchange column to give the crude material. 31 P NMR indicated that this material contained an impurity, so the material was repurified again using an ion exchange column to give crude material. The material was treated with NaHCO (10 mg) and the mixture was concentrated under reduced pressure. The solid residue was dissolved in 0.5 mL of HO and 40 μL of 1N NaOH was added. The resulting mixture was purified using a C-18 column eluted with HO. The product-containing fractions were combined and concentrated under reduced pressure to give Example 9 (TP4) as the tetrasodium salt.

[0325] 1 H NMR (400 MHz, D2O):δ7.76 (s, 1H), 6.88 (d, J = 4.3 Hz, 1H), 6.81 (d, J = 4.6 Hz, 1H), 5.59 (d, J = 5.5 Hz, 1H), 4.60 (t, J = 5.6 Hz, 1H), 4.55 (d, J = 5.8 Hz, 1H), 3.99 (qd, J = 11.2, 5.5 Hz, 3H). 31 P NMR (162 MHz, D2O): δ-8.13 (d, J = 19.8 Hz), -14.04 (d, J = 18.9 Hz), -24.00 (t, J = 19.3 Hz). MS m / z=546.1[M-1], 547.9[M+1]

[0326] [ka] Intermediate PD1a - S-2-Hydroxyethyl 2,2-dimethylpropanethioate

[0327] To a solution of 2-thioethanol (3.50 mL, 50.0 mmol) and triethylamine (7.02 mL, 50.0 mmol) in CHCl cooled to −78° C. was added pivaloyl chloride (6.15 mL, 50.0 mmol) dropwise over 30 min. The reaction was allowed to warm slowly to room temperature, and progress was monitored by TLC. After 30 min, the reaction was determined to be complete and quenched with water. The layers were separated, and the aqueous layer was washed with CHCl. ​​The organics were combined and dried over sodium sulfate. The solids were filtered, and the solvent was removed under reduced pressure. The crude material was purified by silica gel chromatography with 0–50% EtOAc / hexanes to give intermediate PD1a.

[0328] 1 H NMR (400 MHz, DMSO-d6)δ4.89 (t, J = 5.5 Hz, 1H), 3.49 - 3.36 (m, 2H), 2.86 (t, J = 6.7 Hz, 2H), 1.14 (s, 9H).

[0329] [ka] Phosphorus oxychloride (281 μL, 3.08 mmol) was dissolved in CH2Cl2 (5 mL). The solution was cooled to -78 °C. Thioester PD1a (1.00 g, 6.17 mmol) was dissolved in CHCl (5 mL) and slowly added to the POCl solution. TEA (891 μL, 6.16 mmol) was then added dropwise and stirred for 30 min while cooled. The mixture was then warmed to room temperature and stirred for 2 h. p-Nitrophenol (428 mg, 3.08 mmol) was added in one portion, followed by the slow addition of TEA (449 μL, 3.08 mmol). The mixture was stirred at room temperature for 30 min. TLC (70:30 hexane / EtOAC) showed only one spot, but LC / MS showed two peaks (product and bis-p-nitrophenolate). The solution was diluted with ether, and the solid was removed by filtration and discarded. The mother liquor was concentrated and purified by silica gel chromatography to give a mixture of product and bis-p-nitrophenolate. The mixture was then repurified by HPLC to give intermediate PD1b, S,S′-2,2′-((4-nitrophenoxy)phosphoryl)bis(oxy)bis(ethane-2,1-diyl)bis(2,2-dimethylpropanethioate).

[0330] 1 H NMR (400 MHz, CDCl3)δ8.29 - 8.21 (m, 2H), 7.46 - 7.36 (m, 2H), 4.23 (br q, J = 7.7 Hz, 4H), 3.16 (br t, J = 6.7 Hz, 4H), 1.23 (s, 18H). 31 P NMR (162 MHz, CDCl3)δ-7.72 (s)

[0331] [ka] Example 10 (also referred to as PD1) - S,S'-2,2'-((((2R,3S,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-2-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)phosphoryl)bis(oxy)bis(ethane-2,1-diyl)bis(2,2-dimethylpropanethioate)

[0332] Example 1 (6.0 mg, 0.02 mmol) was dissolved in NMP (0.1 mL) and THF (0.2 mL) was added. Next, under an argon atmosphere, tert-butylmagnesium chloride (1.0 M solution in THF, 0.031 mL, 0.031 mmol) was added at room temperature. After 10 minutes, a solution of intermediate PD1b (15.7 mg, 0.031 mmol) in THF (0.1 mL) was added, and the resulting mixture was heated to 50°C. After 5 hours, the resulting residue was purified by preparative HPLC (Phenominex Synergi 4u Hydro-RR 80Å). The product was purified by column chromatography (150 x 30 mm, 40-100% acetonitrile / water gradient). Fractions containing the desired product were combined and lyophilized to give Example 10 (PD1).

[0333] 1 H NMR (400 MHz, CDCl3)δ7.82 (s, 1H), 6.69 (d, J = 4.5 Hz, 1H), 6.64 (d, J = 4.5 Hz, 1H), 5.56 (d, J = 3.4 Hz, 1H), 4.61 (br s, 2H), 4.45 - 4.32 (m, 2H), 4.22 - 4.06 (m, 4H), 3.13 (dt, J = 11.7, 6.7 Hz, 4H), 1.23 (s, 9H), 1.21 (s, 9H). 31 P NMR (162 MHz, CDCl3)δ-2.34 (s).

[0334] LC / MS:t R= 1.70 min, MS m / z = 660.02 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50×4.6mm Solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 2-100% ACN from 0 min to 2.0 min, 100% ACN from 2.0 min to 3.05 min, 100%-2% ACN from 3.05 min to 3.2 min, 2% ACN from 3.2 min to 3.5 min, 2 μl / min. HPLC:t R = 3.204 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA Gradient: 2-98% ACN from 0 min to 5.0 min, 98% ACN from 5.0 min to 6.0 min, 2 mL / min.

[0335] [ka] (Example 11) (PD2)—S,S′-2,2′-((((2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-2-cyano-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)phosphoryl)bis(oxy)bis(ethane-2,1-diyl)bis(2,2-dimethylpropanethioate)

[0336] Example 3 (10.5 mg, 0.036 mmol) was dissolved in NMP (0.1 mL) and THF (0.1 mL) was added. Next, under an argon atmosphere, tert-butylmagnesium chloride (1.0 M solution in THF, 0.054 mL, 0.054 mmol) was added at room temperature. After 10 min, a solution of intermediate PD1b (27.3 mg, 0.054 mmol) in THF (0.1 mL) was added, and the resulting mixture was warmed to 50 °C. After 24 h, the resulting residue was purified by preparative HPLC (Phenominex Synergi 4u Hydro-RR 80 Å 150 × 30 mm column, 40–100% acetonitrile / water gradient). Fractions containing the desired product were combined and lyophilized to give Example 11 (PD2).

[0337] 1 H NMR (400 MHz, CDCl3)δ7.94 (s, 1H), 6.75 (d, J = 4.5 Hz, 1H), 6.67 (d, J = 4.5 Hz, 1H), 5.77 (dd, J = 27.8, 1.4 Hz, 1H), 5.43 (ddd, J = 55.2, 4.9, 1.3 Hz, 1H), 4.93 (dd, J = 21.2, 4.9 Hz, 1H), 4.49 (dd, J = 11.3, 7.8 Hz, 1H), 4.40 (dd, J = 11.3, 7.8 Hz, 1H), 4.10 (ddt, J = 15.9, 8.0, 6.7 Hz, 4H), 3.16 - 3.04 (m, 4H), 1.23 (s, 9H), 1.21 (s, 9H). 31 P NMR (162 MHz, CDCl3)δ-2.10 (s). 19 F NMR (376 MHz, CDCl3)δ-191.64 (ddd, J = 55.0, 27.8, 21.3 Hz).

[0338] LC / MS:t R= 1.85 min, MS m / z = 662.03 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50 × 4.6 mm; solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 2 to 100% ACN from 0 min to 2.0 min, 100% ACN from 2.0 min to 3.05 min, 100% to 2% ACN from 3.05 min to 3.2 min, 2% ACN from 3.2 min to 3.5 min, 2 μl / min. HPLC:t R = 3.385 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 2–98% ACN from 0 min to 5.0 min, 98% ACN from 5.0 min to 6.0 min, 2 mL / min.

[0339] [ka] Intermediate PD3c - (2S)-2-Ethylbutyl 2-((4-nitrophenoxy)(phenoxy)phosphorylamino)propanoate

[0340] Phenyl dichlorophosphate PD3a (1.5 mL, 10 mmol) was dissolved in 30 mL of anhydrous DCM and stirred in an ice bath under N2(g). The aminoester HCl salt PD3b, (S)-2-ethylbutyl 2-aminopropanoate hydrochloride (prepared according to Eur. J. Med. Chem. 2009, Vol. 44, pp. 3765-3770, 2.1 g, 10 mmol), was added in one portion. TEA (3 mL, 22 mmol) was added dropwise. Stirring was continued at 0 °C for 1 h. p-Nitrophenol (1.4 g, 10 mmol) was added in one portion, followed by TEA (1.5 mL, 11 mmol). The reaction mixture was then stirred at room temperature for 16 h. The mixture was diluted with DCM and saturated NaHCO3 3(aq)The organics were dried over anhydrous Na2SO4 and concentrated under reduced pressure. Purification by silica gel column (0-15% EtOAc in hexanes) gave intermediate PD3c.

[0341] 1 H NMR (400 MHz, CDCl3)δ8.23 (d, J = 8.8 Hz, 2H), 7.41 - 7.30 (m, 4H), 7.25 - 7.19 (m, 3H), 4.10 - 4.00 (m, 3H), 3.90-3.83 (m, 1H), 1.55 - 1.45 (m, 1H), 1.42 - 1.31 (m, 7H), 0.87 (t, J = 7.2Hz, 6H). 31 P NMR (162 MHz, CDCl3)δ-3.04 (s), -3.10 (s).

[0342] LC / MS:t R = 2.87 min, MS m / z = 451.1 [M+1], 449.0 [M-1]; LC / MS system: Thermo LCQ Advantage; Phenomenex Gemini, C 18 , 5u, 110A, 30 x 4.6mm; Buffer A: 0.1% acetic acid in water; Buffer B: 0.1% acetic acid in acetonitrile; Buffer B from 5 to 100% in 2.5 min, then 100% for 0.9 min, 2 mL / min. HPLC:t R = 4.40 min; HPLC system: Agilent 1100; Phenomenex Gemini, C 18 , 5u, 110A, 50 x 4.6mm; Buffer A: 0.05% TFA in water; Buffer B: 0.05% TFA in acetonitrile; Buffer B from 2 to 98% in 5 min, 2 mL / min.

[0343] [ka] Example 12 (PD3) - (2S)-2-Ethylbutyl 2-((((2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-2-cyano-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propanoate

[0344] Example 3 (15 mg, 0.051 mmol) was dissolved in anhydrous DMF (1 mL) and stirred under N2 (g). p-Nitrophenylphosphoamidate PD3c (35 mg, 0.077 mmol) was dissolved in anhydrous DMF (0.5 mL) and added to the reaction mixture in one portion. tBuMgCl in THF (1 M in THF, 77 μL, 0.077 mmol) was added dropwise. Stirred for 2 hours. Additional p-nitrophenylphosphoramidate (35 mg in 0.5 mL anhydrous DMF) and additional tBuMgCl (1 M in THF, 50 μL, 0.050 mmol) were added. Stirred for 2 hours. Additional p-nitrophenylphosphoramidate (35 mg in 0.5 mL anhydrous DMF) and additional tBuMgCl solution (1 M in THF, 50 μL, 0.050 mmol) were added. Stirred for 16 hours. Diluted with EtOAc and saturated NaHCO 3(aq) (3x) with saturated NaCl (aq) The organics were dried over anhydrous Na2SO4 and concentrated under reduced pressure. Purification was performed on a silica gel column (0-5% MeOH in DCM). The fractions were combined and concentrated under reduced pressure. Purification by preparative HPLC using TFA as a modifier gave Example 12 (PD3).

[0345] Preparative HPLC system: Gilson 215 Liquid Handler; Phenomenex Gemini, C 18 4u, 100×30.0mm Buffer A: 0.1% TFA in water; Buffer B: 0.1% TFA in acetonitrile; 5 to 100% of Buffer B in 13 min, 20 mL / min.

[0346] 1H NMR (400 MHz, CDCl3)δ7.89 (s, 1H), 7.31 - 7.13 (m, 6H), 6.80-6.75 (m, 1H), 5.80 - 5.70 (m, 1H), 5.35 - 5.20 (m, 1H), 4.80 - 4.62 (m, 1H), 4.60 - 4.45 (m, 2H), 4.35 - 4.10 (m, 1H), 4.06 - 3.96 (m, 3H), 1.49 - 1.28 (m, 8H), 0.90 - 0.82 (m, 6H). 31 P NMR (162 MHz, CDCl3)δ2.36 (s), 2.22 (s).

[0347] HPLC:t R = 3.00 min; HPLC system: Agilent 1100; Phenomenex Gemini, C 18 , 5u, 110A, 50 x 4.6mm; Buffer A: 0.05% TFA in water; Buffer B: 0.05% TFA in acetonitrile; Buffer B from 2 to 98% in 5 min, 2 mL / min. LC / MS:t R = 2.39 min, MS m / z = 605.1 [M+1], 603.0 [M-1]; LC / MS system: Thermo LCQ Advantage; Phenomenex Gemini, C 18 , 5u, 110A, 30×4.6mm; Buffer A: Water Buffer B: 0.1% acetic acid in acetonitrile; Buffer B: 0.1% acetic acid in acetonitrile; 5 to 100% Buffer B in 2.5 min, then 100% for 0.9 min, 2 mL / min.

[0348] [ka] Intermediate 6a - N-(7-((2S,3S,4R,5R)-4-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy)methyl)-3-fluoro-5-vinyltetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide

[0349] Intermediate 2g, N-(7-((2S,3S,4R,5R)-4-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy)methyl)-3-fluoro-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide (220 mg, 0.35 mmol), was dissolved in 5 mL of anhydrous DMSO and stirred under N2(g). EDCI (100 mg, 0.52 mmol) was added, followed by TFA-pyridine (34 mg, 0.18 mmol). Stirred for 1 hour. Additional EDCI (100 mg, 0.52 mmol) was added and stirred for 1 hour. Monitoring by LC / MS indicated that starting alcohol remained. Additional EDCI (100 mg, 0.52 mmol) was added and stirred for 1 hour. Monitoring by LC / MS showed that the reaction had reached complete conversion. Dilution with ethyl acetate and saturated NaHCO 3(aq) (2x), then saturated NaCl (aq) The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. It was purified by silica gel column (0-20% EtOAc in hexanes). The combined fractions were concentrated under reduced pressure to give the aldehyde as a solid. Methyltriphenylphosphonium bromide (500 mg, 1.40 mmol) was suspended in 10 mL of anhydrous THF and eluted with Ar (g) The mixture was stirred at -78°C under reduced pressure. A 2.5 M solution of n-butyllithium in hexane (560 μL, 1.40 mmol) was added dropwise. The reaction mixture was stirred in an ice bath for 1 hour, resulting in a yellow mixture. The aldehyde prepared above was dissolved in 5 mL of anhydrous THF and added dropwise to the reaction. The ice bath was removed, and the reaction was allowed to warm to room temperature. The mixture was stirred at room temperature for 3 hours. A saturated aqueous solution of NH4Cl was added, and the mixture was extracted with ethyl acetate. The organic extract was washed with saturated NaHCO 3(aq) , then saturated NaCl (aq) The organics were dried over anhydrous Na2SO4 and concentrated under reduced pressure. Purification by silica gel column (0-20% EtOAc in hexanes) gave intermediate 6a.

[0350] 1 H NMR (400 MHz, DMSO-d6)δ8.22 (br s, 1H), 8.03 (br s, 2H), 7.58 (dt, J = 40.4, 7.4 Hz, 3H), 7.12 (d, J = 4.7 Hz, 1H), 6.97 (s, 1H), 6.01 (dd, J = 17.5, 10.9 Hz, 1H), 5.58 (d, J = 22.8 Hz, 1H), 5.46 (dd, J = 17.5, 2.1 Hz, 1H), 5.25 (dd, J = 11.0, 2.0 Hz, 1H), 5.14 (ddd, J = 55.4, 4.9, 2.7 Hz, 1H), 4.61 (dd, J = 20.8, 4.8 Hz, 1H), 3.63 - 3.40 (m, 2H), 0.89 (s, 9H), 0.84 (s, 9H), 0.09 (d, J = 8.4 Hz, 6H), 0.00 (d, J = 14.1 Hz, 6H). 19 F NMR (376 MHz, DMSO-d6)δ-191.86 (d, J = 56.8 Hz). MS m / z=627.3[M+1].

[0351] [ka] Example 13 - (2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-fluoro-2-(hydroxymethyl)-2-vinyltetrahydrofuran-3-ol

[0352] Intermediate 6a (146 mg, 0.23 mmol) was dissolved in THF (10 mL), and the resulting solution was stirred in an ice bath. A 1 M solution of TBAF in THF (700 μL, 0.70 mmol) was added and stirred for 2 hours. Diluted with EtOAc and saturated NaCl (aq)The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The reaction was dissolved in 7M ammonia in MeOH (7 mL) and stirred for 18 hours. The reaction was concentrated under reduced pressure. C with TFA as a modifier 18 Purification was carried out by preparative HPLC. The fractions were combined and concentrated under reduced pressure. 3(aq) The resulting mixture was dissolved in HCl and re-purified by preparative HPLC under neutral conditions. The combined fractions were freeze-dried to give Example 13.

[0353] 1 H NMR (400 MHz, D2O)δ7.54 (s, 1H), 6.62 - 6.49 (m, 2H), 5.98 - 5.79 (m, 1H), 5.55 - 5.36 (m, 2H), 5.31 (d, J = 11.1 Hz, 1H), 5.11 (ddd, J = 54.8, 5.2, 2.9 Hz, 1H), 4.42 (dd, J = 20.6, 4.8 Hz, 1H), 3.62 - 3.43 (m, 2H). 19 F NMR (376 MHz, D2O)δ-193.23 (dd, J = 54.7, 44.2 Hz). MS m / z=295.2[M+1]

[0354] [ka] Example 14 - (2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-2-ethyl-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol

[0355] Example 13 (5 mg, 0.017 mmol) was dissolved in methanol (2 mL). 10% Pd / C Degussa catalyst (2 mg) was then added, and the resulting mixture was stirred under an atmosphere of hydrogen gas. After 40 minutes, the resulting mixture was filtered to remove the Pd / C, and the filtrate was concentrated under reduced pressure. The residue was dissolved in water and lyophilized to give Example 14.

[0356] 1 H NMR (400 MHz, D2O)δ7.67 (s, 1H), 6.79 - 6.55 (m, 2H), 5.54 - 5.12 (m, 2H), 4.46 (dd, J = 15.1, 5.5 Hz, 1H), 3.65 - 3.44 (m, 2H), 1.89 - 1.44 (m, 2H), 0.84 (t, J = 7.6 Hz, 3H). 19 F NMR (376 MHz, D2O)δ-197.62 (ddd, J = 54.5, 20.6, 15.0 Hz). MS m / z=297.3[M+1].

[0357] [ka] (Example 15) (PD4) - S,S'-2,2'-((((2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-fluoro-3-hydroxy-2-vinyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(oxy)bis(ethane-2,1-diyl)bis(2,2-dimethylpropanethioate)

[0358] Example 13 (5 mg, 0.017 mmol) was dissolved in anhydrous DMF (0.5 mL). p-nitro-phenonate (13 mg, 0.026 mmol) was added in one portion. 1 M t-butylmagnesium chloride in THF (25 μL, 0.026 mmol) was added dropwise. Stirred for 1 h. Warmed to 50°C and stirred for 2 h. Additional p-nitro-phenonate (13 mg, 0.026 mmol) was added and stirred for 2 h. Additional 1 M t-butylmagnesium chloride in THF (25 μL, 0.026 mmol) was added and stirred at 50°C for 16 h. Cooled to room temperature. The resulting mixture was directly purified by preparative HPLC column eluted with a linear gradient of 0-100% ACN in water to give Example 15 (PD4).

[0359] 1 H NMR (400 MHz, CD3OD)δ7.84 (s, 1H), 6.92 (d, J = 4.5 Hz, 1H), 6.80 (d, J = 4.5 Hz, 1H), 6.10 (dd, J = 17.4, 10.9 Hz, 1H), 5.67 (dd, J = 5.8, 1.9 Hz, 1H), 5.61 (s, 1H), 5.45 - 5.35 (m, 1H), 5.15 (ddd, J = 55.6, 5.0, 2.2 Hz, 1H), 4.65 (dd, J = 22.5, 5.1 Hz, 1H), 4.13 (dd, J = 11.1, 5.2 Hz, 1H), 4.08 - 3.95 (m, 5H), 3.06 (dd, J = 7.0, 6.1 Hz, 4H), 1.21 (s, 9H), 1.18 (s, 9H). 19 F NMR (376 MHz, CD3OD)δ192.99 (td, J = 55.7, 23.6 Hz). MS m / z=663.0[M+1].

[0360] [ka] Example 16 (PD5) - (2S)-2-Ethylbutyl 2-(((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-azido-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate

[0361] Example 5 (5 mg, 0.016 mmol) was dissolved in anhydrous N-methyl-2-pyrrolidone (0.2 mL) and THF (0.1 mL) was added under an argon atmosphere. Next, tert-butylmagnesium chloride (1 M in THF, 24 μL, 0.024 mmol) was added at room temperature, resulting in the precipitation of a white solid. After 5 minutes, a solution of p-nitrophenyl phosphoramidate PD3c (15 mg, 0.032 mmol) in THF (0.1 mL) was added in one portion to the reaction mixture, and the resulting mixture was heated to 50 °C. After 3.5 hours, the reaction mixture was cooled to room temperature and stirred for 18 hours. Next, p-nitrophenyl phosphoramidate PD3c (50 mg, 0.111 mmol) and tert-butylmagnesium chloride (1 M in THF, 24 μL, 0.024 mmol) were added, and the reaction mixture was stirred for an additional 5 days. The resulting residue was then purified directly by preparative HPLC (Phenominex Synergi 4u Hydro-RR 80Å 150 × 30 mm column, 40 to 100% acetonitrile / water gradient). Fractions containing the desired product were combined and lyophilized to give Example 16 (PD5) (2:1 diastereomeric mixture).

[0362] 1 H NMR (400 MHz, CDCl3)δ7.88 (br s, 1H), 7.33 - 7.22 (br m, 2H), 7.22 - 7.10 (br m, 3H), 6.69 (br d, J = 4.4 Hz, 1H), 6.61 (br d, J = 4.5 Hz, 1H), 5.64 - 5.56 (m, 1H), 4.54 (d, J = 6.3 Hz, 1H), 4.50 - 4.20 (m, 3H), 4.11 - 3.94 (m, 3H), 3.90 - 3.76 (m, 1H), 1.49 (s, J = 6.2 Hz, 1H), 1.40 - 1.24 (m, 7H), 0.86 (t, J = 7.4 Hz, 6H). 31 P NMR (162 MHz, CDCl3)δ2.68 (s), 2.56 (s).

[0363] LC / MS:t R = 1.70 min, MS m / z = 619.09 [M+1]; LC system: Thermo Accela 1250 UHPLC. MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50 × 4.6 mm; solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 2 to 100% ACN from 0 min to 2.0 min, 100% ACN from 2.0 min to 3.05 min, 100% to 2% ACN from 3.05 min to 3.2 min, 2% ACN from 3.2 min to 3.5 min, 2 μl / min. HPLC:t R = 3.010 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 2 to 98% ACN from 0 to 5.0 min, 98% ACN from 5.0 to 6.0 min, 2 mL / min.

[0364] [ka] (Example 17) (TP5) - ((2R,3R,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-4-fluoro-3-hydroxy-2-vinyltetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate

[0365] To a solution of Example 13 (6.0 mg, 0.020 mmol) in PO(OMe) (0.6 mL) was added POCl (50 mg, 0.32 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 6 h, at which point ion-exchange HPLC indicated approximately 90% conversion. A solution of pyrophosphate tributylamine salt (250 mg) in ACN (0.6 mL) was added, followed by tributylamine (110 mg, 0.59 mmol). The reaction mixture was stirred at 0 °C for 1 h. The reaction was quenched with triethylammonium bicarbonate buffer (1 M, 5 mL). The reaction mixture was stirred at room temperature for 0.5 h, then concentrated and coevaporated twice with water. The residue was dissolved in HO (5 mL) and loaded onto an ion-exchange column. The column was eluted with HO, followed by 5–35% triethylammonium bicarbonate buffer (1 M) in HO. The product fractions were combined, concentrated, and co-evaporated with HO. The solid residue was dissolved in 3 mL of HO, and 100 μL of 1N NaOH was added. The resulting mixture was purified on a C-18 column eluted with HO, and the product-containing fractions were combined and concentrated under reduced pressure to give Example 17 (TP5) as the tetrasodium salt.

[0366] 1 H NMR (400 MHz, D2O):δ7.74 (s, 1H), 6.89 (d, J = 4.4 Hz, 1H), 6.81 (d, J = 4.6 Hz, 1H), 6.00 (dd, J = 17.4, 11.1 Hz, 1H), 5.72 (d, J = 23.3 Hz, 1H), 5.49 (d, J = 16.9 Hz, 1H), 5.32 (d, J = 11.1 Hz, 1H), 5.14 (dd, J = 54.0, 4.6 Hz, 1H), 4.72 (dd, J = 23.7, 4.5 Hz, 1H), 4.09 (dd, J = 11.3, 5.8 Hz, 1H), 3.79 (dd, J = 11.6, 3.8 Hz, 1H). 31 P NMR (162 MHz, D2O): δ-8.38 (d, J = 20.5 Hz), -13.67 (d, J = 19.3 Hz), -24.20 (t, J = 19.9 Hz). 19 F NMR (376 MHz, CDCl3)δ-194.58 (dt, J = 55.0, 23.8 Hz). MS m / z=533.0[M-1], 535.0[M+1]

[0367] [ka] (Example 18) (TP6) - ((2R,3R,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-ethyl-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate

[0368] To a solution of Example 14 (5.0 mg, 0.017 mmol) in PO(OMe) (0.6 mL) was added POCl (45 mg, 0.30 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 6 h, at which point ion-exchange HPLC indicated approximately 90% conversion. A solution of pyrophosphate tributylamine salt (250 mg) in ACN (0.6 mL) was added, followed by tributylamine (110 mg, 0.59 mmol). The reaction mixture was stirred at 0 °C for 1 h. The reaction was quenched with triethylammonium bicarbonate buffer (1 M, 5 mL). The reaction mixture was stirred at room temperature for 0.5 h, then concentrated and coevaporated twice with water. The residue was dissolved in HO (5 mL) and loaded onto an ion-exchange column. The column was eluted with HO, followed by 5–35% triethylammonium bicarbonate buffer (1 M) in HO. The product fractions were combined, concentrated, and coevaporated with HO. The solid residue was dissolved in 3 mL of HO, and 100 μL of NaOH (1 N) was added. The resulting mixture was purified on a C-18 column eluted with HO. The product-containing fractions were combined and concentrated under reduced pressure to give 18 (TP6) as the tetrasodium salt.

[0369] 1 H NMR (400 MHz, D2O):δ7.73 (s, 1H), 6.86 (d, J = 4.6 Hz, 1H), 6.80 (d, J = 4.6 Hz, 1H), 5.60 (dd, J = 21.9, 3.5 Hz, 1H), 5.23 (dt, J = 55.2, 4.2 Hz, 1H), 4.65 (dd, J = 20.6, 5.3 Hz, 1H), 4.08 - 3.84 (m, 3H), 1.83 (dq, J = 14.4, 7.4, 6.9 Hz, 1H), 1.62 (dq, J = 15.0, 7.5 Hz, 1H), 0.87 (t, J = 7.5 Hz, 3H). 31P NMR (162 MHz, D2O): -5.72 (d, J = 20.2 Hz), -10.81 (d, J = 19.3 Hz), -21.60 (t, J = 19.8 Hz). 19 F NMR (376 MHz, CDCl3)δ-194.77 (dt, J = 55.2, 21.2 Hz). MS m / z=535.1[M-1], 536.9.0[M+1].

[0370] [ka] Intermediate 8a - N-(7-((2S,3R,4R,5S)-3-fluoro-4-hydroxy-5-(iodomethyl)tetrahydrofuran-2-yl)pyrrolo[2,1-f][1,2,4]triazin-4-yl)benzamide

[0371] To an argon-purged flask was added 2b (68 mg, 0.183 mmol) in DMF (2 mL), followed by methyltriphenoxyphosphonium iodide (124 mg, 0.274 mmol). The reaction was stirred at room temperature for 5 minutes, at which point complete conversion to the product was observed by LCMS. The reaction was quenched with methanol and the solvent removed under reduced pressure. The crude material was partitioned between EtOAc and HO. The organics were separated and washed with brine. The resulting material was dried over NaSO, filtered, and the solvent removed under reduced pressure. The crude material was purified by silica gel chromatography (20-100% EtOAc / hexanes) to give intermediate 8a.

[0372] 1H NMR (400 MHz, DMSO-d6)δ8.17 (m, 3H), 7.63 (t, J = 7.4 Hz, 1H), 7.53 (t, J = 7.6 Hz, 2H), 7.17 - 6.96 (m, 2H), 5.74 (s, 1H), 5.60 (d, J = 24.9 Hz, 1H), 5.19 (ddd, J = 54.6, 4.5, 2.1 Hz, 1H), 4.09 - 3.92 (m, 1H), 3.72 (t, J = 6.4 Hz, 1H), 3.63 (dd, J = 11.0, 3.4 Hz, 1H), 3.44 (dd, J = 11.0, 5.9 Hz, 1H). 19 F NMR (376 MHz, DMSO-d6)δ-194.23 (m).

[0373] LC / MS:t R =1.13 min, MS m / z=483.23[M+1] LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ C18 100A, 50×3.00mm Solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid Gradient: 2 to 100% ACN from 0 to 1.4 min, 100% ACN from 1.4 to 1.80 min, 100% to 2% ACN from 1.8 to 1.85 min, and 2% ACN from 1.85 to 2 min.

[0374] [ka] Intermediate 8b - (3R,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-4-fluoro-2-methylenetetrahydrofuran-3-ol

[0375] Intermediate 8a (80 mg, 0.166 mmol) was dissolved in THF. DBU (0.074 mL, 0.498 mmol) was added in one portion. The reaction was then heated to 60 °C in an oil bath for 16 h. The reaction was cooled to room temperature and the solvent was removed under reduced pressure. The crude material was purified by silica gel chromatography (0-70% EtOAc / Hex) to give intermediate 8b.

[0376] 1 H NMR (400 MHz, DMSO-d6)δ8.34-8.05 (m, 3H), 7.63 (t, J = 7.4 Hz, 1H), 7.53 (t, J = 7.6 Hz, 2H), 7.13 (d, J = 4.7 Hz, 1H), 6.85 (d, J = 4.4 Hz, 1H), 5.97 - 5.82 (m, 2H), 5.39 - 5.13 (m, 1H), 4.89 - 4.69 (m, 1H), 4.38 (d, J = 2.1 Hz, 1H), 4.16 (t, J = 1.8 Hz, 1H). 19 F NMR (376 MHz, DMSO-d6)δ-198.14 (ddd, J = 53.9, 24.7, 20.9 Hz).

[0377] LC / MS:t R =1.05 min, MS m / z=355.15[M+1] LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ C18 100A, 50×3.00mm Solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid Gradient: 2 to 100% ACN from 0 to 1.4 min, 100% ACN from 1.4 to 1.80 min, 100% to 2% ACN from 1.8 to 1.85 min, and 2% ACN from 1.85 to 2 min.

[0378] [ka] Intermediate 8c - (2S,3R,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-azido-4-fluoro-2-(iodomethyl)tetrahydrofuran-3-ol

[0379] Benzyltrimethylammonium chloride (55 mg, 0.296 mmol) and sodium azide (19.3 mg, 0.296 mmol) were dissolved in ACN (1 mL). The resulting mixture was stirred overnight at room temperature, then filtered and added via syringe to a solution of Intermediate 8b (50 mg, 0.141 mmol) in THF (1 mL). Next, N-methylmorpholine (0.078 mL, 0.706 mmol) was added, followed by the dropwise addition of a solution of iodine (65 mg, 0.25 mmol) in THF (1 mL). After 15 min, N-acetylcysteine ​​was added until gas evolution was no longer observable. Next, aqueous saturated sodium thiosulfate was added until the solution turned pale yellow. The crude mixture was partitioned between EtOAc and HO. The phases were separated, and the organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude material was purified by silica gel chromatography (0-60% EtOAc / Hex) to afford intermediate 8c.

[0380] 1 H NMR (400 MHz, DMSO-d6)δ8.31-8.05 (m, 3H), 7.63 (t, J = 7.5 Hz, 1H), 7.53 (t, J = 7.6 Hz, 2H), 7.14 (d, J = 4.6 Hz, 1H), 7.04 (s, 1H), 6.34 (d, J = 6.9 Hz, 1H), 5.80 (d, J = 23.7 Hz, 1H), 5.55 - 5.31 (m, 1H), 4.62 (dt, J = 21.9, 5.9 Hz, 1H), 3.78 - 3.56 (m, 2H). 19 F NMR (376 MHz, DMSO-d6)δ-194.44 (dt, J = 54.7, 22.8 Hz).

[0381] LC / MS:t R = 1.19 min, MS m / z = 524.09 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ C18 100A, 50×3.00mm Solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid Gradient: 2 to 100% ACN from 0 to 1.4 min, 100% ACN from 1.4 to 1.80 min, 100% to 2% ACN from 1.8 to 1.85 min, and 2% ACN from 1.85 to 2 min.

[0382] [ka] Intermediate 8d - (2S,3R,4S,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-azido-4-fluoro-2-(iodomethyl)tetrahydrofuran-3-yl acetate

[0383] To a solution of intermediate 8c (40 mg, 0.076 mmol) in THF (1 mL) was added acetic anhydride (0.009 mL, 0.092 mmol) at room temperature, followed by DMAP (10 mg, 0.082 mmol). After 15 min, the reaction mixture was quenched with methanol, and the resulting mixture was concentrated under reduced pressure. The crude material was purified by silica gel chromatography (0-50% EtOAc / Hex) to give intermediate 8d.

[0384] 1 H NMR (400 MHz, CDCl3)δ8.15-8.02 (m, 3H), 7.62 (t, J = 7.3 Hz, 1H), 7.53 (t, J = 7.6 Hz, 2H), 7.44 (d, J = 4.6 Hz, 1H), 7.00 (d, J = 4.6 Hz, 1H), 5.95 - 5.80 (m, 1H), 5.70 - 5.43 (m, 2H), 3.71 (d, J = 11.3 Hz, 1H), 3.60 (d, J = 11.3 Hz, 1H), 2.25 (s, 3H). 19 F NMR (376 MHz, CDCl3)δ-192.78 (ddd, J = 55.7, 24.6, 18.5 Hz).

[0385] LC / MS:t R = 1.35 min, MS m / z = 566.14 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ C18 100A, 50×3.00mm Solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid Gradient: 2 to 100% ACN from 0 to 1.4 min, 100% ACN from 1.4 to 1.80 min, 100% to 2% ACN from 1.8 to 1.85 min, and 2% ACN from 1.85 to 2 min.

[0386] [ka] Intermediate 8e - ((2R,3R,4S,5S)-3-acetoxy-2-azido-5-(4-benzamidopyrrolo[2,1-f][1,2,4]triazin-7-yl)-4-fluorotetrahydrofuran-2-yl)methyl benzoate

[0387] To a solution of intermediate 8d (30 mg, 0.053 mmol) in DMF (2 mL) was added 15-crown-5 (0.105 mL, 0.531 mmol) and sodium benzoate (77 mg, 0.531 mmol) at room temperature. The reaction mixture was then heated to 105° C. After 30 hours, the reaction mixture was brought to room temperature and 5% LiCl was added. (aq)The mixture was partitioned between HCl and EtOAc. The phases were separated and the aqueous phase was washed with EtOAc (2x). The combined organic extracts were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (0-60% EtOAc / Hex) to give intermediate 8e.

[0388] 1 H NMR (400 MHz, CDCl3)δ8.25 - 7.97 (m, 4H), 7.69 - 7.40 (m, 6H), 7.36 (d, J = 4.7 Hz, 1H), 6.95 - 6.80 (m, 1H), 5.90 (d, J = 25.0 Hz, 1H), 5.65 (d, J = 1.9 Hz, 1H), 5.62 - 5.48 (m, 1H), 4.69 (dd, J = 79.3, 12.0 Hz, 2H), 2.20 (s, 3H). 19 F NMR (376 MHz, CDCl3)δ-192.57 (ddd, J = 53.9, 25.1, 22.0 Hz).

[0389] LC / MS:t R = 1.45 min, MS m / z = 560.14 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ C18 100A, 50×3.00mm Solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid Gradient: 2 to 100% ACN from 0 to 1.4 min, 100% ACN from 1.4 to 1.80 min, 100% to 2% ACN from 1.8 to 1.85 min, and 2% ACN from 1.85 to 2 min.

[0390] [ka] Example 19 - (2R,3R,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-azido-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol

[0391] To Intermediate 8e (24 mg, 0.043 mmol) was added 7N NH3 in CH3OH (2 mL) at room temperature. After 16 h, the resulting mixture was concentrated under reduced pressure. The crude residue was subjected to acid modification. Purification by reverse phase HPLC without modifier gave Example 19.

[0392] 1 H NMR (400 MHz, methanol-d4) δ 7.81 (s, 1H), 6.85 (d, J = 4.5 Hz, 1H), 6.80 (d, J = 4.5 Hz, 1H), 5.80 (dd, J = 24.7, 1.9 Hz, 1H), 5.22 (ddd, J = 55.6, 5.1, 1.9 Hz, 1H), 4.63 (dd, J = 22.9, 5.1 Hz, 1H), 3.81 (d, J = 12.1 Hz, 1H), 3.70 (d, J = 12.2 Hz, 1H). 19 F NMR (376 MHz, methanol-d4) δ-195.30 (ddd, J = 55.5, 24.6, 22.9 Hz).

[0393] LC / MS:t R = 0.61 min, MS m / z = 310.02 [M+1]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ C18 100A, 50×3.00mm Solvent: acetonitrile containing 0.1% formic acid, water containing 0.1% formic acid Gradient: 2 to 100% ACN from 0 to 1.4 min, 100% ACN from 1.4 to 1.80 min, 100% to 2% ACN from 1.8 to 1.85 min, and 2% ACN from 1.85 to 2 min.

[0394] [ka] Intermediate 9b - ((3aR,5R,6S,6aR)-6-(benzyloxy)-5-((benzyloxy)methyl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)methanol

[0395] Under an argon atmosphere, ((3aR,6S,6aR)-6-(benzyloxy)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole-5,5-diyl)dimethanol (9a, purchased from Carbosynth, 10.0 g, 32.2 mmol) was added to a solution of sodium hydride (60 wt%, 1.55 g, 38.7 mmol) in THF (100 mL) at 0 °C. After 10 min, benzyl bromide (4.54 mL, 38.6 mmol) was added, and the reaction mixture was warmed to room temperature. After 2 h, the reaction was quenched with saturated aqueous ammonium chloride solution (500 mL). The resulting mixture was extracted with ethyl acetate (500 mL). The organic phase was then washed with brine (400 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a colorless oil. The crude residue was purified by SiO2 column chromatography (220 g SiO2 Combiflash HP Gold Column, 0-100% ethyl acetate / hexanes) to afford intermediate 9a (9.49 g, 73%) as a colorless oil.

[0396] 1 H NMR (400 MHz, DMSO-d6)δ7.38 - 7.19 (m, 10H), 5.68 (app t, J = 3.6 Hz, 1H), 4.73 (q, J = 4.4 Hz, 1H), 4.63 (d, J = 12.1 Hz, 1H), 4.49 - 4.36 (m, 3H), 4.24 (br s, 1H), 4.20 - 4.13 (m, 1H), 3.81 (d, J = 11.9 Hz, 1H), 3.56 (d, J = 11.9 Hz, 1H), 3.46 (q, J = 10.3 Hz, 2H), 1.47 (s, 3H), 1.25 (s, 3H). LC / MS:t R = 1.88 min, MS m / z = 423.31 [M+Na]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 4.6 mm; Solvent: Acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 2–100% ACN from 0 min to 2.0 min, 100% ACN from 2.0 min to 3.05 min, 100%–2% ACN from 3.05 min to 3.2 min, 2% ACN from 3.2 min to 3.5 min, 2 μl / min. HPLC:t R = 3.79 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 2 to 98% ACN from 0 to 5.0 min, then 98% ACN from 5.0 to 6.0 min, 2 mL / min. TLC: Eluent: 40% ethyl acetate in hexane, R f =0.4(UV)

[0397] [ka] Intermediate 9c - (3aR,5R,6S,6aR)-6-(benzyloxy)-5-((benzyloxy)methyl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole-5-carbaldehyde

[0398] To a solution of intermediate 9b (1.95 g, 4.87 mmol) in dichloromethane (24.5 mL) was added Dess-Martin periodinane (3.1 g, 7.3 mmol) at room temperature. After 1.5 h, the reaction mixture was purified by SiO column chromatography (80 g SiO Combiflash HP Gold Column, 0-100% ethyl acetate / hexane) to give intermediate 9c (1.94 g, 100%) as a colorless oil.

[0399] 1 H NMR (400 MHz, CDCl3)δ9.91 (s, 1H), 7.36 - 7.11 (m, 10H), 5.84 (d, J = 3.4 Hz, 1H), 4.71 (d, J = 12.1 Hz, 1H), 4.59 (d, J = 12.2 Hz, 1H), 4.59 - 4.58 (m, 1H), 4.52 (d, J = 12.0 Hz, 1H), 4.46 (d, J = 12.0 Hz, 1H), 4.37 (d, J = 4.4 Hz, 1H), 3.68 (d, J = 11.0 Hz, 1H), 3.61 (d, J = 11.0 Hz, 1H), 1.60 (s, 3H), 1.35 (s, 3H).

[0400] LC / MS:t R = 1.99 min, MS m / z = 421.25 [M+Na]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 4.6 mm; Solvent: Acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 2 to 100% ACN from 0 to 2.0 min, 100% ACN from 2.0 to 3.05 min, 100% to 2% ACN from 3.05 to 3.2 min, ACN from 3.2 to 3.5 min. CN 2%, 2 μl / min. HPLC:t R= 4.09 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 2 to 98% ACN from 0 to 5.0 min, then 98% ACN from 5.0 to 6.0 min, 2 mL / min. TLC: Eluent: 40% ethyl acetate in hexane, R f =0.6(UV)

[0401] [ka] Intermediate 9d - (3aR,5R,6S,6aR)-6-(benzyloxy)-5-((benzyloxy)methyl)-2,2-dimethyl-5-vinyltetrahydrofuro[2,3-d][1,3]dioxole

[0402] To a solution of methyltriphenylphosphonium bromide (5.38 g, 15.1 mmol) in tetrahydrofuran (20 mL) was added 2.5 M n-butyllithium (6.02 mL) at -78 °C. The reaction was warmed to 0 °C, and a solution of Intermediate 9c (2.00 g, 5.02 mmol) in tetrahydrofuran (5 mL) was added slowly via syringe. The reaction mixture was allowed to warm to room temperature and stirred for 4 h. The reaction mixture was then quenched with saturated aqueous ammonium chloride (10 mL) and partitioned between water (200 mL) and ethyl acetate (200 mL). The layers were separated, and the organic layer was washed with brine (200 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude residue was purified by SiO column chromatography (120 g SiO Combiflash HP Gold Column, 0–50% ethyl acetate / hexanes) to afford Intermediate 9d (1.01 g, 51%) as a colorless oil.

[0403] 1H NMR (400 MHz, CDCl3)δ7.42 - 7.17 (m, 10H), 6.19 (dd, J = 17.6, 11.0 Hz, 1H), 5.76 (d, J = 3.9 Hz, 1H), 5.52 (dd, J = 17.5, 1.9 Hz, 1H), 5.25 (dd, J = 11.1, 1.8 Hz, 1H), 4.76 (d, J = 12.3 Hz, 1H), 4.62 - 4.55 (m, 2H), 4.52 (d, J = 12.1 Hz, 1H), 4.41 (d, J = 12.1 Hz, 1H), 4.25 (d, J = 4.9 Hz, 1H), 3.32 (d, J = 1.5 Hz, 2H), 1.52 (s, 3H), 1.29 (s, 3H)

[0404] LC / MS:t R = 2.13 min, MS m / z = 419.24 [M+Na]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 4.6 mm; Solvent: Acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 2–100% ACN from 0 min to 2.0 min, 100% ACN from 2.0 min to 3.05 min, 100%–2% ACN from 3.05 min to 3.2 min, 2% ACN from 3.2 min to 3.5 min, 2 μl / min. HPLC:t R = 4.37 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 0 min to 5 min 2-98% ACN at 0.0 min, 98% ACN at 2 mL / min at 5.0-6.0 min. TLC: Eluent: 50% ethyl acetate in hexane, R f =0.55(UV)

[0405] [ka] Intermediate 9e - (3R,4S,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-2-methoxy-5-vinyltetrahydrofuran-3-ol

[0406] To a solution of intermediate 9d (1.01 g, 2.55 mmol) in methanol (12.5 mL) was added 4 M HCl in dioxane (320 μL) at room temperature. After 1.25 h, the reaction mixture was partitioned between ethyl acetate (100 mL) and saturated aqueous sodium bicarbonate (100 mL). The phases were separated, and the organic phase was washed with brine (100 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude intermediate 9e (1.05 g, approximately 2.5:1 mixture of 1' anomers) as a colorless oil.

[0407] LC / MS: Major anomer R = 2.00 min, MS m / z = 393.22 [M+Na], secondary anomeric t R = 1.98 min, MS m / z = 393.22 [M+Na]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 4.6 mm; Solvent: Acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 2–100% ACN from 0 min to 2.0 min, 100% ACN from 2.0 min to 3.05 min, 100%–2% ACN from 3.05 min to 3.2 min, 2% ACN from 3.2 min to 3.5 min, 2 μl / min. HPLC: Major anomer R = 4.01 min, subanomeric t R = 3.955 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 2 to 98% ACN from 0 to 5.0 min, then 98% ACN from 5.0 to 6.0 min, 2 mL / min. TLC: Eluent: 25% ethyl acetate in hexane, major anomer R f=0.30(UV), minor anomeric R f =0.25(UV)

[0408] [ka] Intermediate 9f - (2R,3S,4R)-3,4-bis(benzyloxy)-2-((benzyloxy)methyl)-5-methoxy-2-vinyltetrahydrofuran

[0409] To a solution of intermediate 9e (1.0 g, 2.7 mmol) in THF (13.5 mL) at room temperature under an argon atmosphere, solid NaH (60 wt%, 130 mg, 3.2 mmol) was added. After 15 minutes, benzyl bromide (0.38 mL, 3.2 mmol) was added, and the reaction mixture was stirred for 4 hours. The reaction mixture was quenched with saturated aqueous ammonium chloride (5 mL) and acetic acid was added. The mixture was partitioned between ethyl acetate (100 mL) and brine (100 mL). The phases were separated, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude intermediate 9f (1.57 g, approximately 2:1 mixture of 1' anomers) as a colorless oil.

[0410] LC / MS: Major anomer R = 1.88 min, MS m / z = 483.36 [M+Na], secondary anomeric t R = 1.83 min, MS m / z = 483.36 [M+Na]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 4.6 mm; Solvent: Acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 2 to 100% ACN from 0 min to 1.5 min, 100% ACN from 1.5 min to 2.2 min, 100% to 2% ACN from 2.2 min to 2.4 min, 2% ACN from 2.4 min to 2.5 min, 2 μl / min. HPLC: Major anomer R = 4.83 min, subanomeric t R= 4.62 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 2 to 98% ACN from 0 to 5.0 min, 98% ACN from 5.0 to 6.0 min, 2 mL / min.

[0411] [ka] Intermediate 9g - (3R,4S,5R)-3,4-Bis(benzyloxy)-5-((benzyloxy)methyl)-5-vinyltetrahydrofuran-2-ol

[0412] To Intermediate 9f (1.5 g, 3.2 mmol) was added a solution of TFA (16 mL) and water (1.6 mL) at 0 °C, and the reaction mixture was warmed to room temperature. After 9 h, water (1 mL) was added, and the reaction mixture was stirred for an additional 10 h. The reaction mixture was then concentrated under reduced pressure. The crude residue was dissolved in ethyl acetate (200 mL) and washed with saturated aqueous sodium bicarbonate (2 × 150 mL) and brine (150 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by SiO2 column chromatography (24 g SiO2 Combiflash HP Gold Column, 0–100% ethyl acetate / hexanes). Fractions containing the desired product were combined to give Intermediate 9g (580 mg) as a colorless oil, which was a mixture with other impurities. The mixture was used directly in the next step.

[0413] LC / MS:t R = 3.13 min, MS m / z = 463.88 [M+OH]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 4.6 mm; Solvent: Acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 2–100% ACN from 0 min to 2.0 min, 100% ACN from 2.0 min to 3.05 min, 100%–2% ACN from 3.05 min to 3.2 min, 2% ACN from 3.2 min to 3.5 min, 2 μl / min. HPLC:t R = 4.34 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 2 to 98% ACN from 0 to 5.0 min, then 98% ACN from 5.0 to 6.0 min, 2 mL / min.

[0414] [ka] Intermediate 9h - (3R,4S,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)-5-vinyldihydrofuran-2(3H)-one

[0415] To a solution of intermediate 9g (580 mg, 1.30 mmol) and 4Å MS (100 mg) in DCM (6.45 mL) was added tetrapropylammonium perruthenate (45.7 mg, 130 μmol) and 4-methylmorpholine N-oxide (457 mg, 3.89 mmol) at room temperature. After 1 h, silica gel (approximately 500 mg) was added to the reaction mixture, and the resulting slurry was filtered through a silica gel plug (approximately 1 g). The filtrate was concentrated under reduced pressure. The crude residue was purified by SiO2 column chromatography (12 g SiO2 Combiflash HP Gold Column, 0–100% ethyl acetate / hexanes) to afford intermediate 9h (254 mg, 18% over two steps) as a colorless oil.

[0416] 1H NMR (400 MHz, CDCl3)δ7.38 - 7.23 (m, 13H), 7.20 - 7.13 (m, 2H), 5.91 (dd, J = 17.5, 11.2 Hz, 1H), 5.49 (dd, J = 17.5, 0.9 Hz, 1H), 5.33 (dd, J = 11.2, 0.9 Hz, 1H), 4.96 (d, J = 12.0 Hz, 1H). 4.74 - 4.68 (m, 2H), 4.55 - 4.47 (m, 3H), 4.39 (d, J = 11.9 Hz, 1H), 4.20 (d, J = 6.0 Hz, 1H), 3.55 (d, J = 10.8 Hz, 1H), 3.46 (d, J = 10.8 Hz, 1H)

[0417] LC / MS:t R = 2.19 min, MS m / z = 444.78 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 4.6 mm; Solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 2 to 100% ACN from 0 min to 2.0 min, 100% ACN from 2.0 min to 3.05 min, 100% to 2% ACN from 3.05 min to 3.2 min, 2% ACN from 3.2 min to 3.5 min, 2 μl / min. HPLC:t R = 4.53 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 2 to 98% ACN from 0 to 5.0 min, 98% ACN from 5.0 to 6.0 min, 2 mL / min. TLC: Eluent: 25% ethyl acetate in hexane, R f =0.45(UV)

[0418] [ka] Intermediate 9i - (3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)-5-vinyltetrahydrofuran-2-ol

[0419] To a suspension of intermediate 1b (0.21 g, 0.81 mmol) and 1,2-bis(chlorodimethylsilyl)ethane (0.17 g, 0.81 mmol) in THF (4 mL) was added n-butyllithium (2.5 M in hexanes, 1.0 mL, 2.5 mmol) rapidly at −78 °C under an argon atmosphere. The resulting mixture was then transferred via cannula to a solution of 9h (0.18 g, 0.41 mmol) in THF (1 mL) at −78 °C under an argon atmosphere. After 20 min, the reaction mixture was warmed to 0 °C and stirred for 15 min. The reaction mixture was quenched with saturated aqueous ammonium chloride solution (1 mL). The resulting mixture was diluted with ethyl acetate (100 mL) and washed with saturated aqueous sodium bicarbonate solution (100 mL) and brine (100 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by SiO2 column chromatography (12 g SiO2 Combiflash HP Gold Column, 0-100% ethyl acetate / hexanes) to afford intermediate 9i (11.1 mg, 5%, isomer mixture) as a colorless oil.

[0420] LC / MS:t R = 1.97 min, MS m / z = 579.27 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 4.6 mm; Solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 2 to 100% ACN from 0 min to 2.0 min, 100% ACN from 2.0 min to 3.05 min, 100% to 2% ACN from 3.05 min to 3.2 min, 2% ACN from 3.2 min to 3.5 min, 2 μl / min. HPLC:t R= 3.37 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 2 to 98% ACN from 0 to 5.0 min, then 98% ACN from 5.0 to 6.0 min, 2 mL / min. TLC: Eluent: ethyl acetate, R f =0.3(UV)

[0421] [ka] Intermediate 9j - 7-((2S,3S,4S,5R)-3,4-bis(benzyloxy) -5-((benzyloxy)methyl)-5-vinyltetrahydrofuran-2-yl)pyrrolo[2,1-f][1,2,4]triazin-4-amine

[0422] To a solution of intermediate 9i (11.0 mg, 19.0 μmol) and triethylsilane (0.5 mL) in DCM (1 mL) was added boron trifluoride diethyl etherate (0.1 mL) slowly at 0 °C under an argon atmosphere. After 1 h, the reaction mixture was slowly diluted with saturated aqueous sodium bicarbonate (10 mL), and the resulting mixture was extracted with ethyl acetate (2 × 10 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude residue was purified by SiO column chromatography (4 g SiO Combiflash HP Gold Column, 0–100% ethyl acetate / hexanes) to afford intermediate 9j (7.7 mg, 72%) as a colorless film.

[0423] 1 H NMR (400 MHz, CDCl3)δ7.87 (s, 1H), 7.37 - 7.17 (m, 15H), 6.73 (d, J = 4.5 Hz, 1H), 6.51 (d, J = 4.5 Hz, 1H), 6.23 (dd, J = 17.5, 10.9 Hz, 1H), 5.70 (d, J = 3.9 Hz, 1H), 5.59 (dd, J = 17.5, 1.8 Hz, 1H), 5.32 (dd, J = 10.9, 1.7 Hz, 1H), 4.72 - 4.56 (m, 4H), 4.49 (d, J = 11.9 Hz, 2H), 4.43 (d, J = 5.6 Hz, 1H), 4.25 (dd, J = 5.6, 4.0 Hz, 1H), 3.56 (s, 2H).

[0424] LC / MS:t R = 2.32 min, MS m / z = 563.33 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 4.6 mm; Solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 2 to 100% ACN from 0 min to 2.0 min, 100% ACN from 2.0 min to 3.05 min, 100% to 2% ACN from 3.05 min to 3.2 min, 2% ACN from 3.2 min to 3.5 min, 2 μl / min. HPLC:t R = 3.51 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 2 to 98% ACN from 0 to 5.0 min, then 98% ACN from 5.0 to 6.0 min, 2 mL / min. TLC: Eluent: ethyl acetate, R f =0.40(UV)

[0425] [ka] Example 20 - (2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-(hydroxymethyl)-2-vinyltetrahydrofuran-3,4-diol

[0426] Under an argon atmosphere, to a solution of intermediate 9j (7.7 mg, 13.7 μmol) in dichloromethane (1 mL) was added boron tribromide (1 M, 0.06 mL, 60 μmol) dropwise at −78°C. After 1 h, the reaction mixture was warmed to 0° C. and stirred for an additional 1.5 h. The reaction was cooled to −78° C. and quenched with a 2:1 methanol / pyridine solution (1.5 mL). The resulting mixture was warmed to room temperature and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenominex Synergi 4u Hydro-RR 80 Å 150 × 30 mm column, 0-100% acetonitrile / water gradient) to afford Example 20 (0.5 mg, 13%) as a white solid.

[0427] 1 H NMR (400 MHz, CD3OD)δ7.78 (s, 1H), 6.88 (d, J = 4.5 Hz, 1H), 6.76 (d, J = 4.5 Hz, 1H), 6.02 (dd, J = 17.4, 11.0 Hz, 1H), 5.47 (dd, J = 17.4, 2.0 Hz, 1H), 5.23 (dd, J = 10.9, 2.1 Hz, 1H), 5.15 (d, J = 8.3 Hz, 1H), 4.72 (dd, J = 8.2, 5.7 Hz, 1H), 4.34 (d, J = 5.7 Hz, 1H), 3.60 (d, J = 11.8 Hz, 1H), 3.49 (d, J = 11.8 Hz, 1H)

[0428] LC / MS:t R= 0.84 min, MS m / z = 293.19 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 4.6 mm; Solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 2 to 100% ACN from 0 min to 1.5 min, 100% ACN from 1.5 min to 2.2 min, 100% to 2% ACN from 2.2 min to 2.4 min, 2% ACN from 2.4 min to 2.5 min, 2 μl / min. HPLC:t R = 2.181 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 2 to 98% ACN from 0 to 5.0 min, then 98% ACN from 5.0 to 6.0 min, 2 mL / min.

[0429] [ka] Example 21 - (2R,3R,4R,5S)-5-(4-amino-5-fluoropyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-azido-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol

[0430] Example 19 (237 mg, 0.766 mmol) and Selectfluor (407 mg, 1.15 mmol) were suspended in acetonitrile (5 mL) and AcOH (0.2 mL) was added. The resulting mixture was stirred at room temperature for 30 minutes, then neutralized with sodium bicarbonate solution and filtered to remove solids. After concentration in vacuo, the residue was purified by preparative HPLC (0-30% acetonitrile in water) to give Example 21 (27 mg, 11%) as an off-white solid.

[0431] 1 H NMR (400 MHz, CD3OD)δ7.73 (s, 1H), 6. 63 (s, 1H), 5.80 (dd, J = 23.9, 1.7 Hz, 1H), 5.16 (ddd, J = 55.3, 5.0, 1.7 Hz, 1H), 4.56 (dd, J = 23.5, 5.0 Hz, 1H), 3.94 - 3.60 (m, 2H) 19 F NMR (376 MHz, CD3OD)δ-161.76 (s), -195.42 (d, J = 55.4 Hz) MS m / z=328[M+H]. MS system: Thermo LCQ Fleet

[0432] [ka] Intermediate 11b - 2-Fluoropyrrolo[2,1-f][1,2,4]triazin-4-amine

[0433] A polytube vessel was charged with Intermediate 11a (2.0 g, 13.4 mmol). The reaction vessel was then placed in an ice bath, and both 70% HF / Pyr (18 mL) and pyridine (9 mL) were added sequentially. Then, immediately following the addition of pyridine, tBuNO (2.07 mL, 17.43 mmol) was added slowly over 20 minutes. The solution turned from tan to black, accompanied by exothermic and gas evolution. The reaction was then stirred for an additional 20 minutes, after which the reaction mixture was diluted with water and concentrated under reduced pressure. The crude residue was partitioned between ethyl acetate and water. The organics were separated, and the aqueous layer was washed three times with ethyl acetate. The organics were combined and washed with brine. The crude was dried over NaSO, filtered, and the solvent removed under reduced pressure. The crude residue was purified by silica gel chromatography (50-100% EtOAc / Hex) to afford intermediate 11b (1.68 g, 82%) as a tan solid.

[0434] 1H NMR (400 MHz, DMSO-d6)δ8.49 - 8.09 (m, 2H), 7.58 (t, J = 2.0 Hz, 1H), 6.95 (d, J = 4.5, 1H), 6.59 (d, J = 4.5, 1H). 19 F NMR (376 MHz, DMSO-d6)δ-73.42 (s).

[0435] LC / MS:t R = 1.03 min, MS m / z = 153.08 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 1.4 min, 100% ACN from 1.4 min to 1.80 min, 100% to 2% ACN from 1.8 min to 1.85 min, 2% ACN from 1.85 min to 2 min, 1.8 mL / min.

[0436] [ka] Intermediate 11c - 7-Bromo-2-fluoropyrrolo[2,1-f][1,2,4]trimethylsilyl Azin-4-amine

[0437] A solution of 11b (3.36 g, 22.1 mmol) in DMF (50 mL) was cooled to 0 °C in an ice bath. A solution of 1,3-dibromo-5,5-dimethylhydantoin (3.16 g, 11.0 mmol) in DMF (50 mL) was added dropwise via addition funnel over 40 min. After 1 h, the reaction was diluted with saturated NaSO 3(aq) The crude product was quenched with 5% LiCl and EtOAc. (aq) The organics were partitioned between 5% LiCl (aq)The organic layer was dried over NaSO, the solid was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude residue was sonicated with CHCl, and the solid was collected by filtration and dried under high vacuum to give intermediate 11c (3.93 g, 77%) as a yellow solid.

[0438] 1 H NMR (400 MHz, DMSO-d6)δ8.49-8.44 (m, 2H), 7.08 (d, J = 4.6 Hz, 1H), 6.76 (d, J = 4.6 Hz, 1H). 19 F NMR (376 MHz, DMSO-d6)δ-71.45 (s).

[0439] LC / MS:t R = 1.22 min, MS m / z = 232.98 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 1.4 min, 100% ACN from 1.4 min to 1.80 min, 100% to 2% ACN from 1.8 min to 1.85 min, 2% ACN from 1.85 min to 2 min, 1.8 mL / min.

[0440] [ka] Intermediate 11e -(3R,4R,5R)-2-(4-amino-2-fluoropyrrolo[2,1-f][1,2,4]triazin-7-yl)-4-(benzyloxy)-5-((benzyloxy)methyl)-3-fluorotetrahydrofuran-2-ol

[0441] To a solution of 11c (2.09 g, 9.08 mmol) in THF (30 mL), 1,2-bis(chlorodimethylsilyl)ethane (STABASE, 1.96 g, 9.08 mmol) was added in one portion, and the resulting mixture was stirred at ambient temperature for 1 h. The reaction was then cooled to −78 °C using a dry ice bath with methanol. nBuLi (2.5 M in hexanes, 10.9 mL, 27.2 mmol) was added to maintain the internal temperature at −65 °C. Next, a solution of intermediate 11d (prepared according to WO2012012776, 2.5 g, 7.5 mmol) in THF (25 mL) was added over 1 min to the reaction mixture. After 5 min, the reaction mixture was quenched with acetic acid and allowed to reach ambient temperature. The solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate. The organics were washed with water and then brine. The layers were separated and the organics were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give crude 11e as a mixture of isomers which was used directly in the next step.

[0442] LC / MS:t R = 1.32 min and 1.40 min, MS m / z = 483.15 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system : Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: Acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 1.4 min, 100% ACN from 1.4 min to 1.80 min, 100% to 2% ACN from 1.8 min to 1.85 min, 2% ACN from 1.85 min to 2 min, 1.8 mL / min.

[0443] [ka] Intermediate 11f - 7-((2S,3S,4R,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-3-fluorotetrahydrofuran-2-yl)-2-fluoropyrrolo[2,1-f][1,2,4]triazin-4-amine

[0444] Intermediate 11e (1.99 g, 3.72 mmol) was dissolved in CHCl (80 mL), and TES (4.75 mL, 29.7 mmol) was added to the mixture. The reaction mixture was cooled to 0 °C, and BF EtO (1.07 mL, 4.09 mmol) was added slowly. After 15 min, the reaction mixture was diluted with saturated NaHCO 3(aq) The mixture was quenched with HCl and the layers were separated. The aqueous layer was washed with CH2Cl2. The organic layers were combined and washed with saturated NaHCO 3(aq) The organics were dried over NaSO, filtered, and concentrated under reduced pressure. The crude material was purified by silica gel chromatography (0-60% EtOAc / Hex) to give intermediate 11f (1.12 g, 64%, 2:1 mixture of 1' anomers).

[0445] LC / MS:t R = 1.55 min, MS m / z = 467.47 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 1.4 min, 100% ACN from 1.4 min to 1.80 min, 100% to 2% ACN from 1.8 min to 1.85 min, 2% ACN from 1.85 min to 2 min, 1.8 mL / min.

[0446] [ka] Intermediate 11g - (2R,3R,4R,5S)-5-(4-amino-2-fluoropyrrolo[2,1-f][1,2,4]triazin-7-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol

[0447] Intermediate 11f (0.82 g, 1.76 mmol) was dissolved in acetic acid (25 mL). The reaction vessel was purged with argon and 10% Pd / C (468 mg, 0.439 mmol) was added. The vessel was evacuated and H 2(g)The mixture was backfilled (3x). After 1 hour, the reaction vessel was purged with nitrogen. The resulting mixture was filtered through a pad of Celite. The filter cake was washed with CH3OH. The filtrate was concentrated under reduced pressure and then co-evaporated with ethyl acetate followed by hexane to give intermediate 11g (503 mg, 98%, 2:1 mixture of 1' anomers).

[0448] LC / MS:t R = 0.81 min, MS m / z = 286.97 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 1.4 min, 100% ACN from 1.4 min to 1.80 min, 100% to 2% ACN from 1.8 min to 1.85 min, 2% ACN from 1.85 min to 2 min, 1.8 mL / min.

[0449] [ka] Intermediate 11h - (2S,3R,4R,5S)-5-(4-amino-2-fluoropyrrolo[2,1-f][1,2,4]triazin-7-yl)-4-fluoro-2-(iodomethyl)tetrahydrofuran-3-ol

[0450] To an argon-purged flask was added a solution of 11g (283 mg, 0.989 mmol) in 10 mL of DMF, followed by a solution of methyltriphenoxyphosphonium iodide (0.536 g, 1.19 mmol) in 4 mL of DMF. The reaction mixture was stirred at 0 °C for 10 min and then allowed to warm to ambient temperature. After 30 min, the reaction was diluted with saturated NaSO. 3(aq) The crude material was quenched with EtOAc and 5% LiCl (aq)The mixture was partitioned between HCl and HCl. The organics were separated and washed with brine. The organics were dried over NaSO, filtered, and concentrated under reduced pressure. The crude residue was dissolved in ACN and purified by HPLC without acid modifier to give Intermediate 11h (201 mg, 52%, 2:1 mixture of 1' anomers) as a white solid.

[0451] LC / MS:t R = 1.08 min, MS m / z = 397.12 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 1.4 min, 100% ACN from 1.4 min to 1.80 min, 100% to 2% ACN from 1.8 min to 1.85 min, 2% ACN from 1.85 min to 2 min, 1.8 mL / min.

[0452] [ka] Intermediate 11i - (3R,4R,5S)-5-(4-amino-2-fluoropyrrolo[2 ,1-f][1,2,4]triazin-7-yl)-4-fluoro-2-methylenetetrahydrofuran-3-ol

[0453] To a solution of 11h (356 mg, 0.899 mmol) in THF (8 mL) was added DBU (0.403 mL, 2.70 mmol), and the resulting mixture was heated to 60 °C. After 3 h, the reaction mixture was concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (40–100% EtOAc / Hex) to afford intermediate 11i (201 mg, 83%, 2:1 mixture of 1' anomers) as a white solid.

[0454] LC / MS:t R= 1.04 min, MS m / z = 269.14 [M+1]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 1.4 min, 100% ACN from 1.4 min to 1.80 min, 100% to 2% ACN from 1.8 min to 1.85 min, 2% ACN from 1.85 min to 2 min, 1.8 mL / min.

[0455] [ka] Intermediate 11j - (2S,3R,4R,5S)-5-(4-amino-2-fluoropyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-azido-4-fluoro-2-(iodomethyl)tetrahydrofuran-3-ol Benzyltrimethylammonium chloride (292 mg, 1.57 mmol) and sodium azide (102 mg, 1.57 mmol) were dissolved in ACN (4 mL), and the resulting mixture was stirred at ambient temperature for 4 hours. The mixture was filtered, and the filtrate was added to a solution of 11i (0.201 g, 0.749 mmol) in THF (4 mL). NMM (0.412 mL, 3.75 mmol) was added, followed by the dropwise addition of a solution of iodine (0.342 g, 1.35 mmol) in THF (4 mL). After 15 minutes, N-acetylcysteine ​​was added in small portions until no gas evolution was observed. Saturated NaSO was added until the solution became pale yellow. 3(aq) was added. The resulting mixture was partitioned between water and ethyl acetate. The layers were separated, and the organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (20–100% EtOAc / Hex) to afford intermediate 11j (183 mg, 56%) as a single isomer.

[0456] 1H NMR (400 MHz, DMSO-d6)δ8.44 (d, J = 28.6 Hz, 2H), 6.98 (d, J = 4.6 Hz, 1H), 6.79 (d, J = 4.6 Hz, 1H), 6.32 (d, J = 6.9 Hz, 1H), 5.60 (dd, J = 23.8, 2.5 Hz, 1H), 5.36 (ddd, J = 54.9, 5.0, 2.6 Hz, 1H), 4.60 (ddd, J = 21.5, 6.9, 5.0 Hz, 1H), 3.63 (ABq, Δδ= 0.09ppm, J = 8Hz, 2H). 19 F NMR (376 MHz, DMSO-d6)δ-71.74 (s), -194.57 (ddd, J = 54.9, 24.0, 21.7 Hz)

[0457] LC / MS:t R = 1.71 min, MS m / z = 437.93 [M+1]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 2.4 min, 100% ACN from 2.4 min to 2.80 min, 100% to 2% ACN from 2.8 min to 2.85 min, 2% ACN from 2.85 min to 3.0 min, 1.8 mL / min.

[0458] [ka] Intermediate 11k - (2S,3R,4S,5S)-5-(4-amino-2-fluoropyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-azido-4-fluoro-2-(iodomethyl)tetrahydrofuran-3-yl isobutyrate

[0459] To a solution of 11j (0.183 g, 0.419 mmol) in THF (10 mL) was added isobutyric anhydride (0.083 mL, 0.502 mmol), TEA (0.118 mL, 0.837 mmol), and DMAP (10 mg, 0.084 mmol). The reaction was stirred at ambient temperature for 15 min, and the reaction was quenched with CHOH. The reaction mixture was concentrated under reduced pressure, and the crude residue was purified by silica gel chromatography (0–50% EtOAc / Hex) to afford intermediate 11k (0.198 g, 93%) as a white solid.

[0460] 1 H NMR (400 MHz, DMSO-d6)δ8.48 (d, J = 30.7 Hz, 2H), 6.99 (d, J = 4.6 Hz, 1H), 6.84 (d, J = 4.6 Hz, 1H), 5.77 - 5.47 (m, 3H), 3.69 (ABq, Δδ= 0.05ppm, J = 12 Hz, 2H), 2.70 (p, J = 7.0 Hz, 1H), 1.24 - 1.05 (d, J = 7.0 Hz, 6H). 19 F NMR (376 MHz, DMSO-d6)δ-71.58 (s), -194.89 (ddd, J = 55.0, 24.3, 16.8 Hz).

[0461] LC / MS:t R = 1.56 min, MS m / z = 508.13 [M+1]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 2.4 min, 100% ACN from 2.4 min to 2.80 min, 100% to 2% ACN from 2.8 min to 2.85 min, 2% ACN from 2.85 min to 3.0 min, 1.8 mL / min.

[0462] [ka] Intermediate 11l - ((2R,3R,4S,5S)-5-(4-amino-2-fluoropyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-azido-4-fluoro-3-(isobutyryloxy)tetrahydrofuran-2-yl)methyl 3-chlorobenzoate

[0463] Intermediate 11k (0.153 g, 0.302 mmol) was dissolved in CHCl (10 mL) and HO (6 mL). Potassium phosphate dibasic (0.138 g, 0.603 mmol), tetrabutylammonium hydrogen sulfate (0.210 g, 0.618 mmol), and 3-chlorobenzoic acid (0.097 g, 0.618 mmol) were added sequentially. The resulting mixture was cooled to 0 °C, and MCPBA (0.203 g, 0.905 mmol) was added. The reaction mixture was warmed to ambient temperature and stirred for 16 h. The reaction mixture was then diluted with saturated NaSO. 3(aq) The crude aqueous residue was diluted with ACN and purified by preparative HPLC without acid modifier to give intermediate 11l (20 mg, 13%) as a white solid.

[0464] 1 H NMR (400 MHz, DMSO-d6)δ8.46 (d, J = 26.4 Hz, 2H), 7.96 - 7.81 (m, 2H), 7.81 - 7.66 (m, 1H), 7.62 - 7.46 (m, 1H), 6.94 (d, J = 4.5 Hz, 1H), 6.80 (d, J = 4.5 Hz, 1H), 5.73 (s, 3H), 4.60 (ABq, Δδ= 0.08ppm, J = 12 Hz, 2H), 2.66 (p, J = 7.0 Hz, 1H), 1.20 - 1.01 (m, 6H). 19 F NMR (376 MHz, DMSO-d6)δ= -71.45 (s), -193.41 (ddd, J = 54.4, 25.4, 21.2 Hz,).

[0465] LC / MS:t R = 2.22 min, MS m / z = 536.17 [M+1]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 2.4 min, 100% ACN from 2.4 min to 2.80 min, 100% to 2% ACN from 2.8 min to 2.85 min, 2% ACN from 2.85 min to 3.0 min, 1.8 mL / min.

[0466] [ka] Example 22 - (2R,3R,4R,5S)-5-(4-amino-2-fluoropyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-azido-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol To a solution of Intermediate 11l (22 mg, 0.041 mmol) in CHOH (1 mL) was added concentrated NHOH (1 mL) at room temperature. After 30 min, the reaction mixture was concentrated under reduced pressure. The crude residue was diluted with a minimal amount of HO and purified by preparative HPLC without the use of an acid modifier to give Example 22 (10 mg, 77%) as a white solid.

[0467] 1 H NMR (400 MHz, DMSO-d6)δ8.40 (d, J = 30.9 Hz, 2H), 6.95 (d, J = 4.5 Hz, 1H), 6.78 (d, J = 4.5 Hz, 1H), 5.89 (d, J = 7.5 Hz, 1H), 5.61 (dd, J = 23.8, 2.1 Hz, 1H), 5.44 (t, J = 6.1 Hz, 1H), 5.18 (ddd, J = 55.3, 5.1, 2.2 Hz, 1H), 4.44 (ddd, J = 23.7, 7.5, 5.0 Hz, 1H), 3.59 (ddd, J = 48.5, 12.0, 6.1 Hz, 2H). 19 F NMR (376 MHz, DMSO-d6)δ-71.18 (s), -193.48 (dt, J = 55.3, 23.8 Hz).

[0468] LC / MS:t R = 1.13 min, MS m / z = 327.86 [M+1]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 2.4 min, 100% ACN from 2.4 min to 2.80 min, 100% to 2% ACN from 2.8 min to 2.85 min, 2% ACN from 2.85 min to 3.0 min, 1.8 mL / min.

[0469] [ka] Intermediate 12a - (2R,3R,4S,5S)-5-(4-amino-5-fluoropyrrolo[2,1-f][1,2,4]triazin-7-yl)-3-((tert-butyldimethylsilyl)oxy)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-fluorotetrahydrofuran-2-carbonitrile

[0470] To a solution of Intermediate 3d (57 mg, 0.109 mmol) in ACN (3 mL) was added Selectfluor II (52 mg, 0.164 mmol) in one portion. After 1.5 h, the reaction was diluted with saturated NaHCO 3(aq) The reaction was quenched by the addition of ethyl acetate (4 mL) and the biphasic mixture was stirred vigorously for 5 minutes. The reaction was diluted with EtOAc and saturated NaHCO 3(aq)The mixture was further diluted with hexanes. The layers were separated, and the organic phase was extracted with water and then brine. The organic layer was dried over Na2SO4. The drying agent was removed by vacuum filtration, and the filtrate was concentrated under reduced pressure. Intermediate 12a (11 mg, 18.7%) was isolated from the concentrated crude material by silica gel column chromatography using the following solvent gradient: 0% EtOAc in hexanes, increasing to 70% EtOAc in hexanes, quickly increasing to 100% EtOAc once the starting material had eluted from the column.

[0471] 1 H NMR (400 MHz, CD3OD)δ7.72 (s, 1H), 7.55 (s, 1H), 5.65 (dd, J = 24.8, 2.4 Hz, 1H), 5.38 (dq, J = 54.4, 2 Hz, 1H), 4.88 (dd, J = 19.2, 4.4 Hz, 1H), 3.96 (ABq, Δδ AB = 0.141ppm, J = 11 Hz, 2H), 0.99 (s, 9H), 0.86 (s, 9H), 0.21 (s, 6H), 0.07 (s, 3H), -0.02 (s, 3H). 19 F NMR (376 MHz, CD3OD)δ-161.795 (s), -194.806 (ddd, J = 54.5, 19.2, 18.8 Hz).

[0472] LC / MS:R T = 2.06 min, MS m / z = 540.64 [M+1]; LC system: Thermo Accela 1250 UHPLC; MS: Thermo LCQ Fleet; Column: Kinetex 2.6μ C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 2.4 min, 100% ACN from 2.4 min to 2.8 min, 100% to 2% ACN from 2.8 min to 2.85 min, 2% ACN from 2.85 min to 3 min.

[0473] [ka] Example 23 - (2R,3R,4R,5S)-5-(4-amino-5-fluoropyrrolo[2,1-f][1,2,4]triazin-7-yl)-4-fluoro-3-hydroxy-2-(hydroxymethyl)tetrahydrofuran-2-carbonitrile

[0474] To a solution of intermediate 12a (29 mg, 0.054 mmol) in THF (2 mL) in a polypropylene tube was added 70% HF in pyridine·pyridine (51 μL, 1.97 mmol) under a N atmosphere at 0 °C. After 1.5 h, the reaction was removed from the ice bath. Additional portions of 70% HF in pyridine·pyridine were added after 3 h (150 μL), 5 h 45 min (200 μL), and 21 h 15 min (0.7 mL). The reaction mixture was then stirred for an additional 24 h, at which point it was cooled in an ice bath and then dissolved in water and saturated NaHCO 3 . 3(aq) The mixture was concentrated under reduced pressure and the residue was dissolved in DMF. The resulting solution / suspension was filtered through a syringe filter (Whatman 0.45 μm PTFE and GMF). The filtrate was injected into an HPLC, and the semi-purified product was further purified by silica gel column chromatography using the following solvent gradient: 0% MeOH in DCM increasing to 20% MeOH in DCM. The product-containing fractions were concentrated, and the residue was lyophilized to give Example 23 (5 mg, 30%) as a white powder.

[0475] 1 H NMR (400 MHz, DMF-d7)δ7.74 (s, 1H), 6.61 (s, 1H), 5.75 (dd, J = 25.2, 1.6 Hz, 1H), 5.23 (ddd, J = 54.8, 4.8, 1.6 Hz, 1H), 4.64 (dd, J = 22, 4.4 Hz, 1H), 3.90 (ABq, Δδ AB = 0.151 ppm, J = 12 Hz, 2H). 19F NMR (376 MHz, DMF-d7)δ-161.727 (s), -193.726 (ddd, J = 54.5, 22.9, 21.8 Hz).

[0476] LC / MS:R T = 0.81 min, MS m / z = 312.13 [M+1]; LC: Thermo Accela 1250 UHPLC; MS: Thermo LCQ Fleet; Column: Kinetex 2.6μ C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 2.4 min, 100% ACN from 2.4 min to 2.8 min, 100% to 2% ACN from 2.8 min to 2.85 min, 2% ACN from 2.85 min to 3 min.

[0477] [ka] Intermediate 13a - N-(7-((2S,3S,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoro-5-(iodomethyl)tetrahydrofuran-2-yl)pyrrolo[2,1-f][1,2,4]triazin-4-yl)benzamide

[0478] To a solution of triphenylphosphine (973 mg, 3.71 mmol) and imidazole (252 mg, 3.71 mmol) in THF (5 mL) was added iodine (253 mg, 1.86 mmol) at room temperature. Once the iodine was completely dissolved, a solution of compound 2g (650 mg, 0.93 mmol) in THF (5 mL) was slowly added dropwise. The resulting mixture was stirred at 80 °C for 3 days and then concentrated in vacuo. The residue was purified by silica gel column chromatography (0 to 50% EtOAc in hexanes) to give intermediate 13a (230 mg, 33%) as an oil.

[0479] MS m / z=742 [M+H]. MS system: Thermo LCQ Fleet.

[0480] [ka] Intermediate 13b - N-(7-((2S,3S,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3-fluoro-5-methyltetrahydrofuran-2-yl)pyrrolo[2,1-f][1,2,4]triazin-4-yl)benzamide

[0481] Intermediate 13a (200 mg, 0.243 mmol) was dissolved in methanol (10 mL) under a nitrogen atmosphere, and 10% Pd / C (100 mg, 0.094 mmol) and TEA (0.035 mL, 0.243 mmol) were added. The resulting mixture was then heated under a nitrogen atmosphere. (balloon) and stirred at room temperature for 40 min. The resulting mixture was filtered and concentrated in vacuo, and the residue was purified by silica gel column chromatography (0-40% EtOAc in hexanes) to give intermediate 13b (145 mg, 72%) as a white solid in 75% purity.

[0482] MS m / z=616[M+H]. MS system: Thermo LCQ Fleet

[0483] [ka] Example 24 - (2R,3R,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-4-fluoro-2-(hydroxymethyl)-2-methyltetrahydrofuran-3-ol

[0484] Intermediate 13b (145 mg, 75% purity, 0.177 mmol) was dissolved in THF (10 mL) and TBAF (1 M in THF, 0.53 mL, 0.531 mmol) was added. The resulting mixture was stirred at room temperature for 2 hours, and then methanolic ammonia (7 N, 10 mL) was added. The resulting mixture was stirred for 24 hours and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (0 to 35% acetonitrile in water in 20 minutes) to give Example 24 (30 mg, 60%) as a white solid.

[0485] 1 H NMR (400 MHz, CD3OD)δ7.78 (s, 1H), 6.84 (d, J = 4.5 Hz, 1H), 6.76 (d, J = 4.5 Hz, 1H), 5.53 (dd, J = 21.5, 4.0 Hz, 1H), 5.25 (ddd, J = 55.5, 5.3, 4.1 Hz, 1H), 4.44 (dd, J = 17.2, 5.2 Hz, 1H), 3.65 - 3.43 (m, 2H), 1.27 (s, 3H) 19 F NMR (376 MHz, CD3OD)δ-197.08 (ddd, J = 55.4, 21.5, 17.1 Hz) MS m / z=282 [M+H]. MS system: Thermo LCQ Fleet.

[0486] [ka] Intermediate 14a - (2S,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol

[0487] Intermediate 1e (2.64 g, 4.91 mmol) was dissolved in acetic acid (50 mL). The flask was purged with argon and 10% Pd / C (1.05 g, 0.982 mmol) was added. The flask was evacuated and H 2(g)The reaction mixture was backfilled with H 2(g) The mixture was stirred under an atmosphere of 0.05%. After 1 h, the flask was purged with nitrogen and the reaction mixture was filtered through a pad of Celite using a CHOH rinse. The filtrate was concentrated under reduced pressure and then co-evaporated with EtOAc followed by hexanes. The residue was placed under high vacuum to give Intermediate 14a (1.31 g, 99%) as a white solid.

[0488] 1 H NMR (400 MHz, DMSO-d6)δ7.80 (s, 1H), 7.66 (s, 2H), 6.82 (d, J = 4.4 Hz, 1H), 6.66 (d, J = 4.4 Hz, 1H), 5.09 (d, J = 6.5 Hz, 1H), 5.06-4.56 (m, 3H), 4.21 (t, J = 5.9 Hz, 1H), 3.93 (t, J = 4.9 Hz, 1H), 3.77 (q, J = 4.5 Hz, 1H), 3.48 (ddd, J = 38.9, 11.8, 4.4 Hz, 2H).

[0489] LC / MS:t R = 0.47 min, MS m / z = 267.13 [M+H]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2–100% ACN from 0 min to 2.4 min, 100% ACN from 2.4 min to 2.80 min, 100%–2% ACN from 2.8 min to 2.85 min, 2% ACN from 2.85 min to 3.0 min, 1.8 mL / min.

[0490] [ka] Intermediate 14b - ((3aR,4R,6S,6aS)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methanol Intermediate 14a (3.13 g, 11.7 mmol) was dissolved in acetone (80 mL) and TsOH (6.00 g, 31.5 mmol) was added. Triethyl orthoformate (6.0 mL, 36.1 mmol) was added slowly over 10 minutes. The resulting mixture was stirred overnight at ambient temperature. Saturated aqueous sodium carbonate was added until the reaction mixture reached pH = 8. The solids were removed by filtration, and the filtrate was concentrated under reduced pressure. The crude residue was partitioned between EtOAc and water. The phases were separated, and the organics were dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude material was purified by silica gel chromatography (60-100% EtOAc / Hex to 20% MeOH / EtOAc) to afford Intermediate 14b (2.55 g, 71%) as a white solid.

[0491] 1 H NMR (400 MHz, DMSO-d6)δ7.83 (s, 1H), 7.71 (s, 2H), 6.83 (d, J = 4.4 Hz, 1H), 6.73 (d, J = 4.5 Hz, 1H), 5.21 (d, J = 4 .9 Hz, 1H), 5.01 (dd, J = 6.6, 4.9 Hz, 1H), 4.84 (t, J = 5.7 Hz, 1H), 4.71 (dd, J = 6.7, 3.7 Hz, 1H), 3.99 - 3.85 (m, 1H), 3.46 (t, J = 5.5 Hz, 2H), 1.48 (s, 3H), 1.29 (s, 3H).

[0492] LC / MS:t R= 0.87 min, MS m / z = 307.21 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 1.4 min, 100% ACN from 1.4 min to 1.80 min, 100% to 2% ACN from 1.8 min to 1.85 min, 2% ACN from 1.85 min to 2 min, 1.8 mL / min.

[0493] [ka] Intermediate 14c - 7-((3aS,4S,6R,6aR)-6-(((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyrrolo[2,1-f][1,2,4]triazin-4-amine Intermediate 14b (2.55 g, 8.32 mmol) was dissolved in DCM (50 mL) and the mixture was cooled to 0 °C. Imidazole (1.70 g, 24.9 mmol) was added, followed by TBSCl (1.88 g, 12.5 mmol). After 16 h, the reaction was quenched with methanol. The resulting mixture was concentrated under reduced pressure, and the crude residue was partitioned between water and EtOAc. The organics were dried over NaSO, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (50–100% EtOAc / Hex) to afford Intermediate 14c (2.60 g, 74%) as a white solid.

[0494] 1H NMR (400 MHz, DMSO-d6)δ7.83 (s, 1H), 7.74 (s, 2H), 6.82 (d, J = 4.4 Hz, 1H), 6.68 (d, J = 4.4 Hz, 1H), 5.26 (d, J = 4.4 Hz, 1H), 5.00 (dd, J = 6.5, 4.5 Hz, 1H), 4.71 (dd, J = 6.5, 3.7 Hz, 1H), 3.97 (td, J = 5.1, 3.6 Hz, 1H), 3.64 (d, J = 5.2 Hz, 2H), 1.48 (s, 3H), 1.28 (s, 3H), 0.83 (s, 9H), -0.02 (s, 6H).

[0495] LC / MS:t R = 1.91 min, MS m / z = 421.60 [M+H]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 to 2.4 min; 100% ACN from 2.4 min to 2.80 min, 100% to 2% ACN from 2.8 min to 2.85 min, and 2% ACN from 2.85 min to 3.0 min, 1.8 mL / min.

[0496] [ka] Intermediate 14d - tert-Butyl (7-((3aS,4S,6R,6aR)-6-(((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyrrolo[2,1-f][1,2,4]triazin-4-yl)carbamate Intermediate 14c (2.59 g, 6.16 mmol) was dissolved in THF (60 mL) and the resulting solution was cooled to 0 °C. Then, BocO (2.69 g, 12.3 mmol) and DMAP (0.3 g, 2.46 mmol) were added. TEA (2.56 mL, 18.3 mmol) was added slowly and the reaction mixture was allowed to warm to room temperature. After 3 h, the reaction mixture was cooled to 0 °C and MeOH (10 mL) was added followed by concentrated NHOH. (aq) (50 mL) was added. The resulting mixture was warmed to room temperature and stirred overnight. The reaction mixture was concentrated under reduced pressure, and the crude residue was partitioned between EtOAc and water. The layers were separated, and the organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (0-100% EtOAc / Hex) to afford Intermediate 14d (2.82 g, 88%) as a white solid.

[0497] 1 H NMR (400 MHz, DMSO-d6)δ10.46 (s, 1H), 8.20 (s, 1H), 7.19 (d, J = 4.6 Hz, 1H), 6.90 (d, J = 4.6 Hz, 1H), 5.33 (d, J = 4.2 Hz, 1H), 5.02 (dd, J = 6.5, 4.3 Hz, 1H), 4.72 (dd, J = 6.5, 3.6 Hz, 1H), 4.01 (q, J = 5.0 Hz, 1H), 3.64 (d, J = 5.1 Hz, 2H), 1.49 (s, 9H), 1.32 (d, J = 22.7 Hz, 6H), 0.82 (s, 9H), -0.03 (s, 6H).

[0498] LC / MS:t R= 1.89 min, MS m / z = 521.27 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 2.4 min, 100% ACN from 2.4 min to 2.80 min, 100% to 2% ACN from 2.8 min to 2.85 min, 2% ACN from 2.85 min to 3.0 min, 1.8 mL / min.

[0499] [ka] Intermediate 14e - tert-Butyl (7-((3aS,4S,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyrrolo[2,1-f][1,2,4]triazin-4-yl)carbamate

[0500] Intermediate 14d (2.8 g, 5.4 mmol) was dissolved in THF (50 mL) and TBAF (1.0 M in THF, 5.92 mL, 5.92 mmol) was added. After 30 min, additional TBAF (1.0 M in THF, 5.92 mL, 5.92 mmol) was added. After an additional 30 min, the reaction mixture was quenched with water, and the resulting mixture was extracted with EtOAc (2×). The combined organic layers were washed with brine, dried over NaSO, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (10–100% EtOAc / Hex) to afford Intermediate 14e (2.19 g, 86%) as a white solid.

[0501] 1 H NMR (400 MHz, DMSO-d6)δ10.46 (s, 1H), 8.22 (s, 1H), 7.20 (s, 1H), 6.95 (s, 1H), 5.29 (d, J = 4.6 Hz, 1H), 5.03 (dd, J = 6.6, 4.7 Hz, 1H), 4.85 (t, J = 5.7 Hz, 1H), 4.72 (dd, J = 6.6, 3.6 Hz, 1H), 4.05 - 3.90 (m, 1H), 3.46 (t, J = 5.6 Hz, 2H), 1.50 (s, 12H), 1.29 (s, 3H).

[0502] LC / MS:t R = 1.52 min, MS m / z = 407.05 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 2.4 min, 100% ACN from 2.4 min to 2.80 min, 100% to 2% ACN from 2.8 min to 2.85 min, 2% ACN from 2.85 min to 3.0 min, 1.8 mL / min.

[0503] [ka] Intermediate 14f - tert-Butyl (7-((3aS,4S,6aS)-6,6-bis(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyrrolo[2,1-f][1,2,4]triazin-4-yl)carbamate Intermediate 14e (1.78 g, 4.38 mmol) was dissolved in DMSO (20 mL) and toluene (15 mL). Pyridine (0.35 mL, 4.38 mmol) and EDCI (1.26 g, 6.56 mmol) were added, followed by TFA (0.178 mL, 2.39 mmol). After 90 min, additional pyridine (0.35 mL, 4.38 mmol) and EDCI (1.26 g, 6.56 mmol) were added, and the reaction mixture was stirred for an additional 30 min. The reaction was quenched with water, and the resulting mixture was extracted with CHCl. ​​The aqueous layer was back-extracted with CHCl. ​​The combined organic layers were washed with brine, dried over NaSO, filtered, and concentrated under reduced pressure. The crude material was placed under high vacuum for 15 min and then used directly in the next step.

[0504] The crude residue was dissolved in dioxane (15 mL) and formaldehyde (37% in water, 5.0 mL, 37.2 mmol) and 2N NaOH (5.34 mL, 10.7 mmol) were added sequentially. After 10 min, the reaction was quenched with AcOH and the resulting mixture was diluted with saturated NaHCO 3(aq) The crude was partitioned between HCl and CH2Cl2. The aqueous layer was extracted back with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude was placed under high vacuum for 15 minutes and then used directly in the next reaction.

[0505] The crude residue was dissolved in EtOH (50 mL) and NaBH4 (0.324 g, 8.76 mmol) was added portionwise. After 20 min, the reaction mixture was quenched with AcOH and concentrated under reduced pressure. The crude residue was dissolved in EtOAc and saturated NaHCO 3(aq) The organic layer was separated, dried over NaSO, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (50-100% EtOAc / Hex) to give intermediate 14f (1.91 g, 68%) as a white solid.

[0506] 1 H NMR (400 MHz, DMSO-d6)δ10.45 (s, 1H), 8.20 (s, 1H), 7.19 (d, J = 4.3 Hz, 1H), 6.95 (d, J = 4.7 Hz, 1H), 5.35 (d, J = 5.2 Hz, 1H), 5.06 (t, J = 5.7 Hz, 1H), 4.79-4.74 (m, 2H), 4.45 (t, J = 5.8 Hz, 1H), 3.73 - 3.46 (m, 3H), 3.40 - 3.30 (m, 1H), 1.50 (s, 12H), 1.27 (s, 3H).

[0507] LC / MS:t R = 1.45 min, MS m / z = 437.09 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 2.4 min, 100% ACN from 2.4 min to 2.80 min, 100% to 2% ACN from 2.8 min to 2.85 min, 2% ACN from 2.85 min to 3.0 min, 1.8 mL / min.

[0508] [ka] Intermediate 14g - tert-butyl (7-((3aS,4S,6S,6aS)-6-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyrrolo[2,1-f][1,2,4]triazin-4-yl)carbamate Intermediate 14f (1.15 g, 2.63 mmol) was dissolved in CHCl (50 mL) and TEA (0.73 mL, 5.27 mmol) was added. The resulting solution was cooled to 0 °C and DMTrCl (1.35 g, 3.95 mmol) was added. After 10 min, the reaction mixture was quenched with CHOH and then diluted with CHCl. ​​The resulting mixture was diluted with saturated NaHCO 3(aq) The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (0-100% EtOAc / Hex) to afford 14g (1.95 g, 79%) as an off-white solid.

[0509] 1 H NMR (400 MHz, DMSO-d6)δ10.46 (s, 1H), 8.23 (s, 1H), 7.56 - 7.07 (m, 10H), 7.07 - 6.70 (m, 5H), 5.24 (d, J = 5.2 Hz, 1H), 5.04 (t, J = 5.9 Hz, 1H), 4.93 - 4.71 (m, 2H), 3.80 - 3.59 (m, 7H), 3.52 (dd, J = 10.9, 4.8 Hz, 1H), 3.25 (d, J = 9.9 Hz, 1H), 3.09 (d, J = 9.9 Hz, 1H), 1.50 (s, 9H), 1.25 (s, 3H), 1.21 (s, 3H).

[0510] LC / MS:t R = 2.54 min, MS m / z = 739.28 [M+H]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2–100% ACN from 0 min to 2.4 min, 100% ACN from 2.4 min to 2.80 min, 100%–2% ACN from 2.8 min to 2.85 min, 2% ACN from 2.85 min to 3.0 min, 1.8 mL / min.

[0511] [ka] Intermediate 14h - tert-butyl (7-((3aS,4S,6R,6aS)-6-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-6-(((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyrrolo[2,1-f][1,2,4]triazin-4-yl)carbamate

[0512] Intermediate 14g (1.53 g, 2.08 mmol) was dissolved in DMF (10 mL) and imidazole (0.42 g, 6.23 mmol) was added, followed by TBSCl (0.47 g, 3.11 mmol). After 1 h, the reaction was quenched with methanol and diluted with EtOAc and 5% LiCl. (aq) The mixture was partitioned between 14h (1.77 g, 78%) and 150 ml of 14H (1.77 g, 78%). The phases were separated, and the organic layer was washed with brine, dried over NaSO, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (0–50% EtOAc / Hex) to afford 14h (1.77 g, 78%) as an off-white solid.

[0513] 1 H NMR (400 MHz, DMSO-d6)δ10.47 (s, 1H), 8.23 (s, 1H), 7.56 - 6.66 (m, 15H), 5.31 (d, J = 4.9 Hz, 1H), 5.14 (dd, J = 6.5, 4.9 Hz, 1H), 4.73 (d, J = 6.5 Hz, 1H), 3.87 (d, J = 9.7 Hz, 1H), 3.72 (s, 6H), 3.53 (d, J = 9.7 Hz, 1H), 3.31 (m, 1H), 3.08 (d, J = 9.8 Hz, 1H), 1.50 (s, 9H), 1.25 (s, 3H), 1.22 (s, 3H), 0.75 (s, 9H), -0.04 (s, 3H), -0.08 (s, 3H).

[0514] LC / MS:t R = 2.34 min, MS m / z = 853.50 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 1.0 min, 100% ACN from 1.0 min to 2.80 min, 100% to 2% ACN from 2.8 min to 2.85 min, 2% ACN from 2.85 min to 3.0 min, 1.8 mL / min.

[0515] [ka] Intermediate 14i - tert-Butyl (7-((3aS,4S,6R,6aS)-6-(((tert-butyldimethylsilyl)oxy)methyl)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyrrolo[2,1-f][1,2,4]triazin-4-yl)carbamate

[0516] Intermediate 14h (1.38 g, 1.62 mmol) was dissolved in chloroform (20 mL) and the resulting solution was cooled to 0 °C. A solution of TsOH (0.34 g, 1.78 mmol) in CHOH (16 mL) was then added slowly. After 30 min, the reaction was diluted with saturated NaHCO (aq)The mixture was quenched with HCl and the resulting mixture was partitioned between EtOAc and brine. The layers were separated, and the organic layer was dried over NaSO, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (0-100% EtOAc / Hex) to afford intermediate 14i (0.84 g, 94%) as a white solid.

[0517] 1 H NMR (400 MHz, DMSO-d6)δ10.42 (s, 1H), 8.20 (s, 1H), 7.19 (s, 1H), 6.89 (s, 1H), 5.37 (d, J = 4.8 Hz, 1H), 5.05 (dd, J = 6.2, 4.8 Hz, 1H), 4.71 (d, J = 6.2 Hz, 1H), 4.51 (t, J = 5.5 Hz, 1H), 3.70 (d, J = 10.2 Hz, 1H), 3.59 (d, J = 5.5 Hz, 2H), 3.49 (d, J = 10.2 Hz, 1H), 1.49 (s, 12H), 1.28 (s, 3H), 0.82 (s, 9H), -0.01 (s, 3H), -0.02 (s, 3H).

[0518] LC / MS:t R = 1.88 min, MS m / z = 551.25 [M+H]; LC system: Thermo Accela 1250 UHPLC MS system: Thermo LCQ Fleet; column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 1.0 min, 100% ACN from 1.0 min to 2.80 min, 100% to 2% ACN from 2.8 min to 2.85 min, 2% ACN from 2.85 min to 3.0 min, 1.8 mL / min.

[0519] [ka] Intermediate 14j - tert-Butyl (7-((3aS,4S,6R,6aS)-6-(((tert-butyldimethylsilyl)oxy)methyl)-6-cyano-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyrrolo[2,1-f][1,2,4]triazin-4-yl)carbamate

[0520] Intermediate 14i (0.838 g, 1.52 mmol) was dissolved in DMSO (5 mL) and toluene (3 mL). Pyridine (0.14 mL, 1.67 mmol) and EDCI (0.438 g, 2.28 mmol) were added, followed by TFA (0.057 mL, 0.761 mmol). After 30 min, additional pyridine (0.14 mL, 1.67 mmol) and EDCI (0.438 g, 2.28 mmol) were added. After 1 h, additional pyridine (0.14 mL, 1.67 mmol) and EDCI (0.438 g, 2.28 mmol) were added. After 2 h, the reaction mixture was diluted with 1 / 2 saturated NaHCO 3(aq) Quench with EtOAc and 1 / 2 saturated NaHCO 3(aq) The layers were separated, and the organic layer was dried over NaSO, filtered, and concentrated under reduced pressure. The residue was dissolved in CHCl and concentrated under high vacuum for 1 h to give a residue that was used directly in the next step.

[0521] The residue was dissolved in pyridine (8 mL) and hydroxylamine hydrochloride (0.159 g, 2.28 mmol) was added in one portion. After 15 min, the reaction mixture was concentrated under reduced pressure and partitioned between EtOAc and water. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude residue was placed under high vacuum for 30 min and used directly in the third step.

[0522] The crude residue was dissolved in ACN (8 mL). CDI (0.37 g, 2.28 mmol) was added in one portion. After 45 min, additional CDI (0.37 g, 2.28 mmol) was added. After 1 h, the reaction was diluted with 1 / 2 saturated NaHCO 3(aq) The crude material was quenched with EtOAc and 1 / 2 saturated NaHCO 3(aq)The mixture was partitioned between 14j (0.72 g, 87%) and 150 ml of HCl. The layers were separated, and the organic layer was dried over NaSO, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (0–50% EtOAc / Hex) to afford intermediate 14j (0.72 g, 87%) as a white solid.

[0523] 1 H NMR (400 MHz, DMSO-d6)δ10.53 (s, 1H), 8.25 (s, 1H), 7.21 (s, 1H), 7.00 (d, J = 4.6 Hz, 1H), 5.62 (d, J = 3.6 Hz, 1H), 5.28 (dd, J = 6.6, 3.7 Hz, 1H), 4.93 (d, J = 6.6 Hz, 1H), 3.83 (s, 2H), 1.62 (s, 3H), 1.50 (s, 9H), 1.33 (s, 3H), 0.83 (s, 9H), 0.00 (s, 6H).

[0524] LC / MS:t R = 2.50 min, MS m / z = 546.15 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 2.4 min, 100% ACN from 2.4 min to 2.80 min, 100% to 2% ACN from 2.8 min to 2.85 min, 2% ACN from 2.85 min to 3.0 min, 1.8 mL / min.

[0525] [ka] Intermediate 14k - (3aS,4R,6S,6aS)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-4-(((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxole-4-carbonitrile

[0526] Intermediate 14j (0.688 g, 1.26 mmol) was dissolved in CHCl (15 mL). Zinc bromide (0.567 g, 2.52 mmol) was added in one portion, and the reaction mixture was stirred at ambient temperature. After 3 h, the reaction mixture was added to a silica-packed cartridge and purified by silica gel chromatography (40–100% EtOAc / Hex) to give Intermediate 14k (0.56 g, 99%) as a white solid.

[0527] 1 H NMR (400 MHz, DMSO-d6)δ7.86 (s, 1H), 7.80 (s, 2H), 6.85 (d, J = 4.5 Hz, 1H), 6.79 (d, J = 4.5 Hz, 1H), 5.55 (d, J = 3.7 Hz, 1H), 5.25 (dd, J = 6.6, 3.8 Hz, 1H), 4.92 (d, J = 6.6 Hz, 1H), 3.82 (s, 2H), 1.61 (s, 3H), 1.33 (s, 3H), 0.83 (s, 9H), -0.13 (s, 6H).

[0528] LC / MS:t R = 2.27 min, MS m / z = 446.68 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 2.4 min, 100% ACN from 2.4 min to 2.80 min, 100% to 2% ACN from 2.8 min to 2.85 min, 2% ACN from 2.85 min to 3.0 min, 1.8 mL / min.

[0529] [ka] Intermediate 14l - N-(7-((3aS,4S,6R,6aS)-6-(((tert-butyldimethylsilyl)oxy)methyl)-6-cyano-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyrrolo[2,1-f][1,2,4]triazin-4-yl)acetamide

[0530] Intermediate 14k (0.20 g, 0.449 mmol) was dissolved in pyridine (2 mL), then acetic anhydride (0.21 mL, 2.24 mmol) was added, and the reaction was stirred at ambient temperature. After 30 min, the reaction mixture was quenched with methanol and concentrated under reduced pressure. The crude residue was directly purified by silica gel chromatography (0–100% EtOAc / Hex) to afford Intermediate 14l (0.185 g, 85%) as a white solid.

[0531] 1 H NMR (400 MHz, DMSO-d6)δ10.87 (s, 1H), 8.31 (s, 1H), 7.24 (d, J = 4.7 Hz, 1H), 7.05 (d, J = 4.7 Hz, 1H), 5.65 (d, J = 3.6 Hz, 1H), 5.29 (dd, J = 6.6, 3.6 Hz, 1H), 4.93 (d, J = 6.6 Hz, 1H), 3.84 (s, 2H), 2.36 (s, 3H), 1.62 (s, 3H), 1.33 (s, 3H), 0.83 (s, 9H), 0.00 (s, 3H), -0.01 (s, 3H).

[0532] LC / MS:t R = 1.14 min, MS m / z = 488.38 [M+1]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 1.4 min, 100% ACN from 1.4 min to 1.80 min, 100% to 2% ACN from 1.8 min to 1.85 min, 2% ACN from 1.85 min to 2 min, 1.8 mL / min.

[0533] [ka] Intermediate 14m - N-(5-bromo-7-((3aS,4S,6R,6aS)-6-(((tert-butyldimethylsilyl)oxy)methyl)-6-cyano-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyrrolo[2,1-f][1,2,4]triazin-4-yl)acetamide

[0534] Intermediate 14l (80 mg, 0.164 mmol) was dissolved in DMF (2 mL) and NBS (29 mg, 0.164 mmol) was added in one portion. After 45 min, the reaction was diluted with methanol. The solvent was removed under reduced pressure. The crude residue was purified by silica gel chromatography (0–50% EtOAc / Hex) to give Intermediate 14m (50 mg, 54%) as an off-white solid. 1 H NMR (400 MHz, DMSO-d6)δ10.13 (s, 1H), 8.42 (s, 1H), 7.29 (s, 1H), 5.65 (d, J = 3.2 Hz, 1H), 5.29 (dd, J = 6.6, 3.2 Hz, 1H), 4.91 (d, J = 6.5 Hz, 1H), 3.84 (d, J = 1.6 Hz, 2H), 2.27 (s, 3H), 1.62 (s, 3H), 1.33 (s, 3H), 0.83 (s, 9H), 0.02 (s, 3H), 0.00 (s, 3H).

[0535] LC / MS:t R= 1.79 min, MS m / z = 566.40 [M+1]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 1.4 min, 100% ACN from 1.4 min to 1.80 min, 100% to 2% ACN from 1.8 min to 1.85 min, 2% ACN from 1.85 min to 2 min, 1.8 mL / min.

[0536] [ka] Intermediate 14n - N-(7-((3aS,4S,6R,6aS)-6-(((tert -butyldimethylsilyl)oxy)methyl)-6-cyano-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-5-fluoropyrrolo[2,1-f][1,2,4]triazin-4-yl)acetamide Intermediate 14m (50 mg, 0.088 mmol) was dissolved in THF (2 mL) and the solution was cooled to −78 °C. nBuLi (2.5 M in hexanes, 0.071 mL, 0.18 mmol) was added. After 5 min, N-fluorobenzenesulfonimide (NSFI, 33.4 mg, 0.106 mmol) was added and the reaction mixture was stirred for 5 min. The reaction was then quenched with AcOH. The solvent was removed under reduced pressure. The crude residue was purified by reverse-phase HPLC to give intermediate 14n (10 mg, 22%) as a white solid.

[0537] 1 H NMR (400 MHz, methanol-d4) δ 8.13 (s, 1H), 6.80 (s, 1H), 5.65 (d, J = 3.5 Hz, 1H), 5.23 (dd, J = 6.7, 3.6 Hz, 1H), 4.97 (d, J = 6.7 Hz, 1H), 3.92 (d, J = 1.7 Hz, 2H), 2.37 (s, 3H), 1.70 (s, 3H), 1.38 (s, 3H), 0.90 (s, 9H), 0.08 (s, 6H). 19 F NMR (376 MHz, methanol-d4) δ-156.43 (s).

[0538] LC / MS:t R = 1.65 min, MS m / z = 506.18 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 2.4 min, 100% ACN from 2.4 min to 2.80 min, 100% to 2% ACN from 2.8 min to 2.85 min, 2% ACN from 2.85 min to 3.0 min, 1.8 mL / min.

[0539] [ka] Example 25 - (2R,3S,4R,5S)-5-(4-amino-5-fluoropyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-2-(hydroxymethyl)tetrahydrofuran-2-carbonitrile

[0540] Intermediate 14n (11 mg, 0.022 mmol) was dissolved in 50% aqueous TFA at ambient temperature. After 2 h, the reaction mixture was quenched with solid Na2CO3 and allowed to reach pH=8. The solvent was removed under reduced pressure, and the crude residue was purified by reverse-phase HPLC. Fractions containing Example 25 were combined and set aside, while fractions containing N6-acyl were combined and concentrated under reduced pressure. The residue of the N6-acyl intermediate was purified with concentrated NH4OH. (aq)(1 mL), and the mixture was stirred at ambient temperature. After 30 minutes, the resulting mixture was concentrated under reduced pressure, and the crude residue was purified by HPLC. The fractions containing Example 25 were combined with the previously set aside fractions containing Example 25 to give Example 25 (4 mg, 58%) as a white solid.

[0541] 1 H NMR (400 MHz, methanol-d6) δ 7.71 (s, 1H), 6.56 (s, 1H), 5.44 (d, J = 5.6 Hz, 1H), 4.48 (t, J = 5.6 Hz, 1H), 4.36 (d, J = 5.5 Hz, 1H), 3.83 ((ABq, Δδ= 0.05ppm, J = 12 Hz, 2H). 19 F NMR (376 MHz, methanol-d4) δ-161.81 (s).

[0542] LC / MS:t R = 0.47 min, MS m / z = 310.13 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 3.00 mm; Solvent: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; Gradient: 2 to 100% ACN from 0 min to 1.4 min, 100% ACN from 1.4 min to 1.80 min, 100% to 2% ACN from 1.8 min to 1.85 min, 2% ACN from 1.85 min to 2 min, 1.8 mL / min.

[0543] [ka] Intermediate 15a - tert-Butyl (7-((3aS,4S,6R,6aS)-6-(((tert-butyldimethylsilyl)oxy)methyl)-6-(chloromethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyrrolo[2,1-f][1,2,4]triazin-4-yl)carbamate

[0544] Intermediate 14i (100 mg, 0.18 mmol) was dissolved in anhydrous pyridine (5 mL). Trifluoromethanesulfonyl chloride (23 μL, 0.22 mmol) was added in one portion, and the reaction mixture was stirred at room temperature for 45 minutes. Additional trifluoromethanesulfonyl chloride (100 μL) was then added. After 30 minutes, additional trifluoromethanesulfonyl chloride (100 μL) was added. After another 30 minutes, additional trifluoromethanesulfonyl chloride (100 μL) was added, and the reaction was stirred for 30 minutes, at which point the reaction mixture was concentrated under reduced pressure. The crude residue was dissolved in anhydrous DMF (5 mL), and then lithium chloride (153 mg, 3.6 mmol) was added in one portion. The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated aqueous sodium chloride (3×20 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (0-20% ethyl acetate in hexanes) to give intermediate 15a. MS m / z = 569.0 [M+H]. MS system: Thermo LCQ Advantage

[0545] [ka] Example 26 - (2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-(chloromethyl)-2-(hydroxymethyl)tetrahydrofuran-3,4-diol

[0546] Intermediate 15a was dissolved in a solution of TFA and water (1:1, 5 mL), and the resulting mixture was stirred for 16 hours. The reaction mixture was then concentrated under reduced pressure. The crude residue was dissolved in aqueous sodium bicarbonate and acetonitrile and purified by preparative HPLC to give Example 26 (19 mg, 34%) as a white powder.

[0547] 1 H NMR (400 MHz, D2O)δ7.61 (s, 1H), 6.72 - 6.64 (m, 2H), 5.19 (d, J = 9.1 Hz, 1H), 4.73 - 4.66 (m, 1H), 4.28 (d, J = 5.2 Hz, 1H), 3.78 (s, 2H), 3.72 - 3.57 (m, 2H). MS m / z=315.3 [M+H]. MS system: Thermo LCQ Advantage

[0548] [ka] Example 27 (also TP7)—((2R,3R,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-azido-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate

[0549] Example 27 was prepared as the tetrasodium salt starting from Example 19 in a manner similar to that described for Example TP4.

[0550] 1 H NMR (400 MHz, D2O)δ7.76 (s, 1H), 6.83 (d, J = 4.4 Hz, 1H), 6.80 (d, J = 4.8 Hz, 1H), 5.94 (d, J = 25.2 Hz, 1H), 5.24 (dd, J = 55.2, 5.2 Hz, 1H), 4.78 (dd, J = 26.8, 5.2 Hz, 1H), 4.08 - 4.18 (m, 2H). 19 F NMR (376 MHz, D2O)δ-193.74 - -194.02 (m). 31 P NMR (162 MHz, D2O)δ-4.60 (d, J = 53.2 Hz, 1P), -10.25 (d, J = 48.4 Hz, 1P), -20.28 (t, J = 48.4 Hz, 1P).

[0551] [ka] Example 28 - ((2R,3R,4R,5S)-5-(4-amino-5-fluoropyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-azido-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate

[0552] Example 28 was prepared as the tetrasodium salt starting from Example 21 in a manner similar to that described for Example TP4.

[0553] 1 H NMR (400 MHz, D2O)δ7.64 (s, 1H), 6.60 (s, 1H), 5.90 (d, J = 24.4 Hz, 1H), 5.20 (dd, J = 54.8, 4.8 Hz, 1H), 4.72 (dd, J = 27.2, 4.8 Hz, 1H), 4.05 - 4.18 (m, 2H). 19 F NMR (376 MHz, D2O)δ-161.00 (s), -196.39 - -196.69 (m). 31 P NMR (162 MHz, D2O)δ-8.24 (d, J = 50.4 Hz), -14.20 (d, J = 46.0 Hz), -24.08 (t, J = 48.4 Hz). MS m / z=567.87[M+1]. MS system: Thermo LCQ Advantage

[0554] [ka] Example 29 - ((2R,3R,4R,5S)-5-(4-amino-2-fluoropyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-azido-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate

[0555] Example 29 was prepared as the tetrasodium salt starting from Example 22 in a manner similar to that described for Example TP4.

[0556] 1 H NMR (400 MHz, D2O)δ6.81 (d, J = 4.4 Hz, 1H), 6.75 (d, J = 4.8 Hz, 1H), 5.81 (d, J = 24.4 Hz, 1H), 5.16 (dd, J = 54.4, 4.8 Hz, 1H), 4.70 (dd, J = 26.8, 4.4 Hz, 1H), 4.02-4.12 (m, 2H). 19 F NMR (376 MHz, D2O)δ-75.95 (s), -196.51 - -196.80 (m). 31 P NMR (162 MHz, D2O)δ-8.29 (d, J = 53.2 Hz), -14.22 (d, J = 48.4 Hz), -24.09 (t, J = 48.4 Hz). MS m / z=567.59[M+1]. MS system: Thermo LCQ Advantage

[0557] [ka] Example 30 - ((2R,3R,4R,5S)-5-(4-amino-5-fluoropyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-cyano-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate

[0558] Example 30 was prepared as the tetrasodium salt starting from Example 23 in a manner similar to that described for Example TP4.

[0559] 1 H NMR (400 MHz, D2O)δ7.64 (s, 1H), 6.57 (s, 1H), 5.87 (d, J = 24.8 Hz, 1H), 5.26 (dd, J = 53.6, 4.0 Hz, 1H), 4.82 (dd, J = 25.2, 4.4 Hz, 1H), 4.26-4.35 (m, 2H). 19 F NMR (376 MHz, D2O)δ-161.05 (s), -194.92 - -195.19 (m). 31 P NMR (162 MHz, D2O)δ-8.22 (d, J = 50.8 Hz), -14.48 (d, J = 48.4 Hz), -24.01 (t, J = 48.4 Hz). MS m / z=551.91[M+1]. MS system: Thermo LCQ Advantage

[0560] [ka] Example 31 (also TP11) - ((2R,3R,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-4-fluoro-3-hydroxy-2-methyltetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate Example 31 was prepared as the tetrasodium salt starting from Example 24 in a manner similar to that described for Example TP4.

[0561] 1 H NMR (400 MHz, D2O)δ7.66 (s, 1H), 6.78 (d, J = 4.8 Hz, 1H), 6.72 (d, J = 4.4 Hz, 1H), 5.58 (dd, J = 23.6, 2.4 Hz, 1H), 5.16 (ddd, J = 55.2, 5.2, 2.8 Hz, 1H), 4.51 (dd, J = 23.2, 5.2 Hz, 1H), 3.88 (dd, J = 11.6, 6.0 Hz, 1H), 3.78 (dd, J = 10.8, 4.0 Hz, 1H), 1.2 (s, 3H). 19 F NMR (376 MHz, D2O)δ-195.74 - -196.01 (m). 31 P NMR (162 MHz, D2O)δ-8.24 (d, J = 50.4 Hz), -13.54 (d, J = 45.6 Hz), -24.11 (t, J = 48.0 Hz).

[0562] [ka] Example 32 (also TP12) - ((2R,3S,4R,5S)-5-(4-amino-5-fluoropyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate

[0563] Example 32 is a method similar to that described for Example TP4, similar to that described for Example 25. Prepared as the tetrasodium salt starting from

[0564] 1H NMR (400 MHz, D2O)δ7.59 (s, 1H), 6.57 (s, 1H), 5.44 (d, J = 6.0 Hz, 1H), 4.56 (d, J = 5.2 Hz, 1H), 4.48 (dd, J = 5.6 Hz, 1H), 4.16 (dd, J = 11.6, 6.0 Hz, 1H), 4.08 (dd, J = 11.2, 5.2 Hz, 1H). 19 F NMR (376 MHz, D2O)δ-161.25 (s). 31 P NMR (162 MHz, D2O)δ-8.29 (d, J = 48.4 Hz), -14.49 (d, J = 53.2 Hz), -24.15 (t, J = 48.4 Hz). MS m / z=549.90[M+1]. MS system: Thermo LCQ Advantage

[0565] [ka] Example 33 (also TP13) - ((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-(chloromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl tetrahydrogentriphosphate

[0566] Example 33 was prepared as the tetratriethylamine salt starting from Example 26 in a manner similar to that described for Example TP3.

[0567] 1 H NMR (400 MHz, D2O)δ7.76 (s, 1H), 6.92 (br s, 1H), 6.85 (br s, 1H), 5.32 (d, J = 9.6 Hz, 1H), 4.78 (dd, J =8, 6.4 Hz, 1H), 4.53 (d, J = 5.6 Hz, 1H), 4.08 (dd, J = 10.0, 4.0 Hz, 1H), 3.83 - 3.95 (m, 3H), 3.07 (q, J = 7.6 Hz, 24 H), 1.16 (t, J = 7.6 Hz, 36 H). 31 P NMR (162 MHz, D2O)δ-9.44 (d, J = 45.6 Hz), -11.51 (d, J = 48.8 Hz), -22.95 (t, J = 48.4 Hz). MS m / z=555.06[M+1]. MS system: Thermo LCQ Advantage

[0568] [ka] Example 34 (also PD6) - (2S)-2-Ethylbutyl 2-(((((2R,3R,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-cyano-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate

[0569] Example 1 (3.8 mg, 0.013 mmol) was dissolved in anhydrous N-methyl-2-pyrrolidone (0.2 mL) and THF (0.1 mL) was added under an argon atmosphere. Next, tert-butylmagnesium chloride (1 M in THF, 20 μL, 0.024 mmol) was added at room temperature, resulting in the precipitation of a white solid. After 5 min, a solution of p-nitrophenyl phosphoramidate PD3c (12 mg, 0.026 mmol) in THF (0.1 mL) was added in one portion to the reaction mixture, and the resulting mixture was heated to 50 °C. After 20 h, the reaction mixture was cooled to room temperature and then directly purified by preparative HPLC (Phenominex Synergi 4u Hydro-RR 80 Å 150 × 30 mm column, 40–100% acetonitrile / water gradient). Fractions containing the desired product were combined and lyophilized to give Example 34 (2.9 mg, 37%, 3:2 diastereomeric mixture) as a white solid.

[0570] 1 H NMR (400 MHz, CD3OD)δ7.80 (s, 0.3H), 7.78 (s, 0.6H), 7.38 - 7.10 (m, 5H), 6.85 (br dd, J = 4.7, 2.2 Hz, 1H), 6.75 - 6.71 (m, 1H), 5.54 - 5.46 (m, 1H), 4.65 - 4.58 (m, 1H), 4.53 - 4.31 (m, 3H), 4.07 - 3.84 (m, 3H), 1.54 - 1.39 (m, 1H), 1.38 - 1.19 (m, 7H), 0.92 - 0.81 (m, 6H) 29H 31 P NMR (162 MHz, CD3OD)δ3.25 (br s).

[0571] LC / MS:t R= 1.55 min, MS m / z = 603.19 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 4.6 mm; Solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 2 to 100% ACN from 0 min to 2.0 min, 100% ACN from 2.0 min to 3.05 min, 100% to 2% ACN from 3.05 min to 3.2 min, 2% ACN from 3.2 min to 3.5 min, 2 μl / min. HPLC:t R = 2.98 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 2 to 98% ACN from 0 to 5.0 min, then 98% ACN from 5.0 to 6.0 min, 2 mL / min.

[0572] [ka] Example 35 (also PD7) - (2S)-ethyl 2-(((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate

[0573] Example 1 (19 mg, 65.3 μmol) was dissolved in NMP (0.2 mL). Under an argon atmosphere, THF (0.1 mL) was added at room temperature, followed by tert-butylmagnesium chloride (1.0 M solution in tetrahydrofuran, 0.098 mL). After 5 minutes, a solution of intermediate PD7a (prepared according to US20120009147A1, 51.4 mg, 130 μmol) in THF (0.1 mL) was added, and the resulting mixture was warmed to 50°C. After 1 hour, the reaction mixture was cooled to room temperature and directly purified by preparative HPLC (Phenominex Synergi 4u Hydro-RR 80 Å 150 × 30 mm column, 5-100% acetonitrile / water gradient). The product-containing fractions were combined and concentrated, and the resulting residue was repurified by preparative HPLC (Phenominex Luna 5u C18 100 x 30 mm column, 5-100% acetonitrile / water gradient) to give Example 35 (12 mg, 34%, 3:2 mixture of diastereomers) as a white solid.

[0574] 1 H NMR (400 MHz, CD3OD)δ7.80 (d, J = 2.3 Hz, 0.4H), 7.78 (d, J = 2.3 Hz, 0.6H), 7.36 - 7.12 (m, 5H), 6.88 - 6.81 (m, 1H), 6.76 - 6.70 (m, 1H), 5.53 - 5.46 (m, 1H), 4.66 - 4.60 (m, 1H), 4.55 - 4.30 (m, 3H), 4.15 - 3.98 (m, 2H), 3.93 - 3.79 (m, 1H), 1.30 - 1.12 (m, 6H). 31 P NMR (162 MHz, CD3OD)δ3.27 (br s).

[0575] LC / MS: Major diastereomer R = 1.28 min, MS m / z = 547.14 [M+H], minor diastereomer t R= 1.30 min, MS m / z = 547.04 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 × 4.6 mm; Solvent: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; Gradient: 2 to 100% ACN from 0 to 2.0 min, 100% ACN from 2.0 to 3.05 min, 100% to 2% ACN from 3.05 to 3.2 min, 2% ACN from 3.2 to 3.5 min, 2 μl / min. HPLC: Major diastereomer R = 2.44 min, minor diastereomer t R = 2.46 min; HPLC system: Agilent 1100 series; column: Gemini 5μ C18 110A, 50 × 4.6 mm; solvent: acetonitrile containing 0.1% TFA, water containing 0.1% TFA; gradient: 2 to 98% ACN from 0 to 5.0 min, then 98% ACN from 5.0 to 6.0 min, 2 mL / min.

[0576] Similarly, compounds of formula (A), formula (B), formula (C), formula (D) and formula (E) [ka] wherein, in each instance, X 1 represents an oxygen protecting group, and X 2 represents an amine protecting group.

[0577] Useful oxygen protecting groups include silyl ether protecting groups or benzyl-type protecting groups, including methoxybenzyl groups.

[0578] Useful silyl ether protecting groups include trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl (TDS), t-butyldimethylsilyl (TBS or TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylxilyl, triisopropylsilyl (TIPS), diisopropylsilyl (DIIPS), and diisopropylsilyl (DIIPS). These include phenylmethylsilyl (DPMS), di-t-butylmethylsilyl (DTBMS), triphenylsilyl (TPS), methyldiphenylsilyl (MDPS), t-butylmethoxyphenylsilyl, tris(trimethylsilyl)silyl(silyl), (2-hydroxystyryl)dimethylsilyl (HSDMS), (2-hydroxystyryl)diisopropylsilyl (HSDIS), t-butylmethoxyphenylsilyl (TBMPS), and t-butoxydiphenylsilyl (DPTBOS) protecting groups.

[0579] Useful benzyl-type protecting groups include benzyl, benzyl halides, p-methoxybenzyl, benzyloxymethyl, 2,4-dimethoxybenzyl, 3,4-dimethoxybenzyl, 2,6-dimethoxybenzyl, p-CF3-benzyl, p-methylbenzyl, p-methoxylbenzyl, 3,5-dimethylbenzyl, p-tert-butylbenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl (including p-Br-benzyl), 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2,6-difluorobenzyl, and p-acylaminobenzyl (PAB). , p-azidobenzyl (Azb), 4-azido-3-chlorobenzyl, 2-trifluoromethylbenzyl, p-(methylsulfinyl)benzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, 2-quinolinylmethyl, diphenylmethyl (DPM), p,p'-dinitrobenzhydryl, triphenylmethyl, alpha-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4,4',4"-tris(benzoyloxyphenyl)methyl, and 2-naphthylmethyl protecting groups.

[0580] Useful amine protecting groups include p-methoxybenzylcarbonyl (Moz or MeOZ). , acetyl (Ac), benzoyl (Bz), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tosyl (Ts or Tos), trifluoroacetamide, and trityl protecting groups. Useful amine protecting groups also include carbamate and amide protecting groups. Examples of carbamate protecting groups include methyl and ethyl carbamates such as 9-fluorenylmethyloxycarbonyl (FMOC), 9-(2-sulfo)fluorenylmethyl, 9-(2,7-dibromo)fluorenylmethyl, 17-tetrabenzo[a,c,g,i]fluorenylmethyl (Tbfmoc), 2-chloro-3-indenylmethyl (Climoc), benz[f]inden-3-ylmethyl (Bimoc), 2,7-di-t-butyl[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanyl)]methyl (DBD-Tmoc), [2-(1,3-dithianyl)methyl (Dmoc), and 1,1-dioxobenzo[b]thiophen-2-ylmethyl (Bsmoc) carbamates.

[0581] Examples of useful substituted ethyl carbamates include 1,1-dimethyl-2-cyanoethyl, 2-phosphonioethyl (Peoc), 2-methylthioethyl, 2-(p-toluenesulfonyl)ethyl, 2,2,2-trichloroethyl (Troc), 2-(trimethylsilyl)ethyl (Teoc), 2-phenylethyl (hZ), 1-(1-adamantyl)-1-methylethyl (Adpoc), 1,1-dimethyl-2-bromoethyl (Br ... Ethyl, 1,1-dimethyl-2-chloroethyl, 1,1-dimethyl-2,2-dibromoethyl (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl (t-Bumeoc), 2-(2'pyridyl)ethyl, 2-(4'pyridyl)ethyl, 2,2-bis( 4'-nitrophenyl)ethyl (Bnpeoc), N-(2-pivaloylamino)-1,1,dimethylethyl, 2-[(2-nitrophenyl)dithio]-1-phenylethyl (NpSSPeoc), 2-(N,N-dicyclohexylcarboxamido)ethyl, t-butyl (Boc or BOC), 1-adamantyl (1-Adoc), 2-adamantyl (2-Adoc), vinyl (Voc), allyl (Aloc or alloc), 1-isopropylallyl (Ipaoc), cinnamyl (Coc), 4-nitrocinnamyl (Noc), 3-(3'-pyridyl)prop-2-enyl (Paloc), 8-quinolyl, and N-hydroxypiperidinyl, carbamates, and alkyldithiocarbamates including methyldithio, ethyldithio, isopropyldithio, t-butyldithio, and phenyldithiocarbamates.

[0582] Similarly, benzyl, p-methoxybenzyl, p-nitrobenzyl, p-bromobenzyl, p-chlorobenzyl, 2,4-dichlorobenzyl, 4-methylsulfinylbenzyl (Msz), 9-anthrylmethyl, 4-methylthiophenyl (Mtpc), 1-methyl-1-(triphenylphosphonio)ethyl (2-triphenylphosphonioisopropyl) (Ppoc), 2-dansylethyl (Dnseoc), 2-(4-nitrophenyl)ethyl (Npeoc), 4-phenylazoline, Also useful are aryl-containing and substituted aryl-containing carbamates, such as acetoxybenzyl (PhAcOZ), 4-azidobenzyl (ACBZ), 4-azidomethoxybenzyl, m-chloro-p-acyloxybenzyl, p-(dihydroxyboryl)benzyl, carbobenzyloxy (Cbz), 4-benzisoxazolylmethyl (Bic), 2-(trifluoromethyl)-6-chromonylmethyl (Tcroc), phenyl, and diphenylmethyl carbamates. Additional carbamates include butynyl, cyclopentyl, cyclohexyl, cyclopropylmethyl, 1-methylcyclobutyl, 1-methylcyclohexyl, 1,1-dimethylpropynyl, and 1-methyl-1-cyclopropylmethyl carbamate.

[0583] Useful amide protecting groups for amines include N-formyl, N-acetyl, and N-chloroaromatic groups. These include cetyl, N-trichloroacetyl, N-trifluoroacetyl (TFA), N-phenylacetyl, N-3-phenylpropionyl, N-4-pentenoyl, N-picolinoyl, N-3-pyridylcarboxamido, N-benzoylphenylalanyl, N-benzoyl, and Np-phenylbenzoylamido.

[0584] Antiviral activity Another embodiment relates to a method of inhibiting viral infection, comprising treating a sample or subject suspected of being in need of such inhibition with a composition herein.

[0585] Samples suspected of containing viruses are contemplated herein to include natural or man-made materials, such as living organisms; tissue or cell cultures; biological samples, such as samples of biological material (blood, serum, urine, cerebrospinal fluid, tears, sputum, saliva, tissue samples, etc.); laboratory samples; food, water, or air samples; and bioproduct samples, such as extracts of cells, particularly recombinant cells that synthesize the desired glycoprotein. Typically, the sample is 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. Samples include living organisms, such as humans, and man-made materials, such as cell cultures.

[0586] If desired, the antiviral activity of the compound after applying the composition can be observed by any method, including direct and indirect methods for detecting the activity. Quantitative, qualitative, and semi-quantitative methods for determining the activity are all contemplated. Usually, one of the above screening methods is applied, but any other method, such as observing the physiological characteristics of living organisms, can be applied.

[0587] The antiviral activity of a compound can be determined using standard screening protocols known in the art. For example, the antiviral activity of a compound can be determined using the following general protocol.

[0588] Respiratory syncytial virus (RSV) antiviral activity and cytotoxicity assays Anti-RSV activity Antiviral activity against RSV is determined using an infectivity cytopathic cytoprotection assay in HEp-2 cells. In this assay, compounds that inhibit viral infection and / or replication provide a cytoprotective effect against virus-induced cell death, which can be quantified using cell viability reagents. The technique used here is a novel adaptation of a method described in the published literature (Chapman et al., Antimicrob Agents Chemother. 2007, Vol. 51(9):3346-53).

[0589] HEp-2 cells were obtained from ATCC (Manassas, VI) and maintained in MEM medium supplemented with 10% fetal bovine serum and penicillin / streptomycin. Cells were passaged twice a week and maintained at a subconfluent stage. Prior to compound testing, a commercially available stock of RSV strain A2 (Advanced Biotechnologies, Columbia, MD) was titrated to determine the appropriate dilution of the virus stock that produced the desired cytopathic effect in HEp-2 cells.

[0590] For antiviral testing, HEp-2 cells are grown in large cell culture flasks to near, but not fully, confluence. Compounds to be tested are prediluted in DMSO in 384-well compound dilution plates with either 8 or 40 samples per plate, standardized to a dose-response format. 3-fold serial dilutions of each test compound are prepared in the plate, and the test samples are transferred to a sonicator (a The cells are transferred to a cell culture assay 384-well plate at 100 nl per well using a coustic transfer apparatus (Echo, Labcyte). Dilutions of each compound are transferred in serial or quadruplicate samples to the dry assay plate, which is stored until the assay is ready to be performed. Positive and negative controls are placed on opposite ends of the plate (one column) in a vertical block.

[0591] The infectious mixture was then prepared using appropriate dilutions of the virus stock, previously determined by titration using cells at a density of 50,000 cells / ml, and 20 μL / well was added to the compound-containing test plates using an automated system (uFlow, Biotek). Each plate contained negative and positive controls (16 replicates each) to generate 0% and 100% virus inhibition standards, respectively. After infection with RSV, the test plates were incubated for 4 days in a cell culture incubator at 37°C. After incubation, a cell viability reagent, Cell TiterGlo (Promega, Madison, WI), was added to the assay plate, which was then briefly incubated, and luminescence readings were taken (Envision, Perkin Elmer) for all assay plates. The percentage of inhibition of RSV-induced cytopathic effect was determined from the remaining cell viability levels. These values ​​were calculated relative to the 0% and 100% inhibition controls for each test concentration, and the EC values ​​for each compound were calculated. 50 Values ​​are determined by nonlinear regression as the concentration that inhibits RSV-induced cytopathic effect by 50%. Various potent anti-RSV tool compounds are used as positive controls for antiviral activity.

[0592] Cytotoxicity assay in HEp-2 cells The cytotoxicity of test compounds is determined in parallel with antiviral activity in uninfected HEp-2 cells using a cell viability reagent in a manner similar to that previously described for other cell types (Cihlar et al., Antimicrob Agents Chemother. 2008, 52(2):655-65). The same protocol as for determining antiviral activity is used to measure compound cytotoxicity, except that the cells are not infected with RSV. Instead, the same density of uninfected cell mixture is added at 20 μl / well to plates also containing prediluted compound at 100 μl / sample. The assay plates are then incubated for 4 days, followed by cell viability testing using the same CellTiter Glo reagent addition and luminescence readout. Untreated cells and cells treated with 2 μM puromycin (Sigma, St. Louis, MO) serve as controls for 100% and 0% cell viability, respectively. The percentage of cell viability was calculated for each test compound concentration relative to the 0% and 100% controls and CC 50 Values ​​are determined by non-linear regression as the compound concentration at which cell viability is reduced by 50%.

[0593] Cytotoxicity assay in MT-4 cells The MT-4 cell line was obtained from the NIH AIDS Research and Reference Reagent Program (Germantown, MD) and cultured in RPMI-1640 medium (Irvine Scientific, Santa Ana, CA, catalog number 9160) supplemented with 10% FBS, penicillin 100 units / mL, streptomycin 100 units / mL, and 2 mM L-glutamine. MT-4 cells were passaged twice per week, reaching 0.6 × 10 6 Cell densities were maintained below 2 x 10 cells / mL. Complete RPMI-1640 medium containing 100x concentrations of 3-fold serially diluted compounds ranging from 26 nM to 530 μM was stamped in quadruplicate into black 384-well plates. After compound addition, 2 x 10 cells / mL were added. 3MT-4 cells were added to each well using a MicroFlo liquid dispenser (BioTek, Winooski, VT), and the cells were cultured for 5 days at 37°C in a 5% CO2 incubator. After incubation, the cells were equilibrated to 25°C, and cell viability was determined by adding 25 μL of Cell-Titer Glo viability reagent. The mixture was incubated at 25°C for 10 minutes, and the luminescent signal was measured. The CC values ​​were quantified using a Victor Luminescence plate reader. 50 Values ​​are defined as the concentration of compound that reduces cell viability by 50%, as determined by the Cell-Titer Glo signal. Data were analyzed using Pipeline Pilot Plate Data Analytics Collection software (Version 7.0, Accelrys, San Diego, CA). 50 Values ​​are calculated using a 4-parameter sigmoidal dose-response equation: Y = Bottom + (Top - Bottom) / (1 + 10^[(LogCC50-X) * The cell viability was calculated from nonlinear regression analysis using the formula [HillSlope] (where Top and Bottom were fixed at 100% and 0% cell viability, respectively). 50 Values ​​were calculated as the mean ± standard deviation of three independent experiments. [Table 2]

[0594] Another advantage is that R 4 Compounds lacking substitution (i.e., R 4 R compared to compounds where ═H 4 The present invention relates to the advantages that compounds having the substitutions offer in terms of MT-4 cytotoxicity. For example, the structure [ka] The compound (2S,3R,4S,5R)-2-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (Patil, SA, Otter, RA, Klein, RS Tetrahedron Lett. 1994, 35, 5339-5342) has the formula: 50 =0.007 μM, while Examples 1, 4, 5, 20 and 26 all exhibited CC 50 Furthermore, the structure [ka] The compound (2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol (WO2012037038A1) has the formula: 50 = 30 μM, while Examples 2, 3, 13 and 14 all showed CC 50 >106μM.

[0595] Another advantage is that R 4 Compounds lacking substitution (i.e., R 4 R compared to compounds where ═H 4 R with substitution 3 The compound =F has advantages in terms of HEp-2 anti-RSV activity. For example, the compound having the above structure, (2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol (WO2012037038A1), exhibits EC 50 ≥ 100 μM, while Examples 2, 3, 13, 14, 19, 21, 22, 23 and 24 all have an EC 50 Indicates ≦100uM.

[0596] RSV RNP preparation RSV ribonucleoprotein (RNP) complexes were prepared using a modified method from Mason et al. (1). HEp-2 cells were cultured at 7.1 × 10 in MEM + 10% fetal bovine serum (FBS). 4 cells / cm 2 The cells were plated at a density of 1000 x g and allowed to attach overnight at 37°C (5% CO). After attachment, the cells were incubated in 35 mL MEM + 2% FBS with RSV A2 (MOI 1000 x g). =5). 20 hours post-infection, the medium was replaced with MEM + 2% FBS supplemented with 2 μg / mL actinomycin D and returned to 37°C for 1 hour. Cells were then washed once with PBS and treated with 35 mL of PBS + lyso-lecithin 250 μg / mL for 1 minute, after which all liquid was aspirated. Cells were harvested by scraping into 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 repeated passage (10 times) using an 18-gauge needle. The cell lysate was placed on ice for 10 minutes and then centrifuged at 2400 g at 4°C for 10 minutes. The supernatant (S1) was removed, and the pellet (P1) was triturated with an 18-gauge needle 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 (10 times). The resuspended pellet was placed on ice for 10 minutes and then centrifuged at 2400 g and 4°C for 10 minutes. The supernatant (S2) was removed, and the pellet (P2) was triturated 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 2400 g and 4°C for 10 minutes. The supernatant (S3) fraction, enriched for RSV RNP complexes, was collected and the protein concentration determined by UV absorbance at 280 nm. Aliquots of the RSV RNP S3 fraction were stored at -80°C.

[0597] RSV RNP assay The transcription reaction was carried out in 30 μL of reaction buffer [50 mM TRIS acetate (pH 8.0), 120 mM potassium acetate, 5% glycerol, 4.5 mM MgCl, 3 mM DTT, 2 mM ethylene glycol-bis(2-aminoethyl ether)-tetraacetic acid (EGTA), 50 μg / mL BSA, 2.5 U RNasin (Promega), ATP, GTP, UTP, CTP, and 1.5 uCi [α- 32 The assay contained 25 μg of crude RSV RNP complex in [P]NTP (3000 Ci / mmol). The radiolabeled nucleotides used in the transcription assay were selected to match the nucleotide analogs being evaluated for inhibition of RSV RNP transcription. The chilled competitor NTP was added at its K m The remaining three nucleotides were added to a final concentration of 100 μM.

[0598] To determine whether the nucleotide analogs inhibited RSV RNP transcription, compounds were added using six 5-fold serial dilutions. After 90 min of incubation at 30°C, the RNP reaction was stopped with 350 μL of Qiagen RLT lysis buffer, and RNA was purified using a Qiagen RNeasy 96 kit. Purified RNA was denatured in RNA sample loading buffer (Sigma) at 65°C for 10 min and run on a 1.2% agarose / MOPS gel containing 2 M formaldehyde. The agarose gel was dried, exposed to a Storm phosphorimager screen, and developed using a Storm phosphorimager (GE Healthcare). The compound concentration (IC) that reduced the total radiolabeled transcript by 50% was determined. 50 ) was calculated by two-fold nonlinear regression analysis.

[0599] References Mason, S., Lawetz, C., Gaudette, Y., Do, F., Scouten, E., Lagace, L., Simoneau, B. and Liuzzi, M. (2004) Polyadenylation-dependent screening assay for respiratory syncytial virus RNA transcriptase activity and identification of an inhibitor. Nucleic Acids Research, 32, 4758-4767. [Table 3]

[0600] Further considerations relate to the advantages of the exemplary compounds, which exhibit potent inhibition of RSV RNP transcription compared to compounds with 2'CMe substitutions. For example, the structure [ka] ((2R,3R,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate (WO2008089105A2 and WO2010002877A2) has the formula: 50 = 8.5 μM, while Example TP3 shows IC 50 = 0.025 μM. Furthermore, the structure [ka] The compound ((2R,3R,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate (WO2011035231A1) has the formula: 50 ≥ 100 μM, while Example TP1 exhibits an IC 50= 0.086 μM, and TP2 is IC 50 = 1 μM.

[0601] In one embodiment, for example, the following items are provided: (Item 1) A compound of formula (I) or a pharmaceutically acceptable salt thereof [ka] wherein R 1 is H or F, R 2 is H or F, R 3 is OH or F, R 4 is CN, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C4 cycloalkyl, azido, halogen or C1-C2 haloalkyl; R 6 is OH, R 5 is H and [ka] [ka] wherein: n' is selected from 1, 2, 3 and 4; R 8 is selected from C1-C8 alkyl, —O—C1-C8 alkyl, benzyl, —O-benzyl, —CH2—C3-C6 cycloalkyl, —O—CH2—C3-C6 cycloalkyl, and CF3; R 9 is phenyl, 1-naphthyl, 2-naphthyl, [ka] is selected from R 10 is selected from H and CH3; R 11is selected from H or C1-C6 alkyl; R 12 is selected from H, C1-C8 alkyl, benzyl, C3-C6 cycloalkyl and —CH2—C3-C6 cycloalkyl; The compound or a pharmaceutically acceptable salt thereof. (Item 2) R 1 is H, or a pharmaceutically acceptable salt thereof. (Item 3) R 2 is H, or a pharmaceutically acceptable salt thereof. (Item 4) R 1 and R 2 and n is H. (Item 5) R 1 , R 2 and R 5 5. The compound according to any one of items 1, 2, 3 or 4, or a pharmaceutically acceptable salt thereof, wherein each is H. (Item 6) R 1 and R 2 Both are H and R 3 6. The compound according to any one of items 1, 2, 3, 4 or 5, or a pharmaceutically acceptable salt thereof, wherein is OH. (Item 7) R 1 and R 2 Both are H and R 3 6. The compound according to any one of items 1, 2, 3, 4 or 5, wherein is F, or a pharmaceutically acceptable salt thereof. (Item 8) Formula (II) [ka] 5. A compound according to any one of items 1, 2, 3 or 4, or a pharmaceutically acceptable salt thereof, wherein R 3 is OH or F, R 4is CN, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C4 cycloalkyl, azido, halogen or C1-C2 haloalkyl; R 5 is H and [ka] [ka] wherein: n' is selected from 1, 2, 3 and 4; R 8 is selected from C1-C8 alkyl, —O—C1-C8 alkyl, benzyl, —O-benzyl, —CH2—C3-C6 cycloalkyl, —O—CH2—C3-C6 cycloalkyl, and CF3; R 9 is phenyl, R 10 is selected from H and CH3; R 11 is selected from H or C1-C6 alkyl; R 12 is selected from H, C1-C8 alkyl, benzyl, C3-C6 cycloalkyl and —CH2—C3-C6 cycloalkyl, or a pharmaceutically acceptable salt thereof. (Item 9) R 4 9. The compound according to any one of items 1, 2, 3, 4, 5 or 8, wherein is CN, methyl, ethyl, ethenyl, ethynyl, azido, F, Cl, —CH2Cl, —CH2F, —CHF2 or —CF3, or a pharmaceutically acceptable salt thereof. (Item 10) R 3 10. The compound according to any one of items 1, 2, 3, 4, 5, 7, 8 or 9, or a pharmaceutically acceptable salt thereof, wherein (Item 11) R 3 10. The compound according to any one of items 1, 2, 3, 4, 5, 6, 8 or 9, or a pharmaceutically acceptable salt thereof, wherein is OH. (Item 12) R 3 is F and R 4 11. The compound according to any one of items 1, 2, 3, 4, 5, 7, 8, 9 or 10, or a pharmaceutically acceptable salt thereof, wherein (Item 13) R 3 is OH and R 4 12. The compound according to any one of items 1, 2, 3, 4, 5, 7, 8, 9 or 11, or a pharmaceutically acceptable salt thereof, wherein (Item 14) R 1 and R 2 Both are H and R 3 is F and R 4 11. The compound according to any one of items 1, 2, 3, 4, 5, 7, 8, 9 or 10, or a pharmaceutically acceptable salt thereof, wherein is methyl, ethyl, vinyl or ethynyl. (Item 15) R 3 is OH and R 4 is methyl, ethyl, vinyl or ethynyl, or a pharmaceutically acceptable salt thereof. The salt is acceptable. (Item 16) R 3 is F and R 4 11. The compound according to any one of items 1, 2, 3, 4, 5, 7, 8, 9 or 10, or a pharmaceutically acceptable salt thereof, wherein (Item 17) R 3 is OH and R 4 12. The compound according to any one of items 1, 2, 3, 4, 5, 7, 8, 9 or 11, or a pharmaceutically acceptable salt thereof, wherein (Item 18) R 5 18. The compound according to any one of items 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17, or a pharmaceutically acceptable salt thereof, wherein (Item 19) R 5 but, [ka] is selected from the group of R 8 is selected from C1-C8 alkyl, —O—C1-C8 alkyl, benzyl, and —CH2—C3-C6 cycloalkyl; R 12 is selected from C1-C8 alkyl, benzyl, C3-C6 cycloalkyl and —CH2—C3-C6 cycloalkyl, or a pharmaceutically acceptable salt thereof. (Item 20) R 8 and R 9 20. The compound according to any one of items 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 19, or a pharmaceutically acceptable salt thereof, wherein each is C1-C8 alkyl. (Item 21) R 8 and R 9 or a pharmaceutically acceptable salt thereof. (Item 22) R 8 and R 9 or a pharmaceutically acceptable salt thereof. (Item 23) R 8 and R 9 are each selected from C1-C4 alkyl, items 1, 2, 3, and 4 24. A compound according to any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof. (Item 24) R 3 If F, then R 4 24. The compound of any of items 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23, or a pharmaceutically acceptable salt thereof, further comprising the proviso that (Item 25) R 5 But H and [ka] 25. The compound according to any of items 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24, or a pharmaceutically acceptable salt thereof, selected from the group consisting of: (Item 26) R 5 but [ka] 26. The compound according to any one of items 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24 or 25, or a pharmaceutically acceptable salt thereof, (Item 27) R 5 but [ka] 26. The compound according to any one of items 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24 or 25, or a pharmaceutically acceptable salt thereof, (Item 28) R 5 but [ka] 26. The compound according to any one of items 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24 or 25, or a pharmaceutically acceptable salt thereof, (Item 29) [ka] 29. The compound according to any of items 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28, or a pharmaceutically acceptable salt thereof, selected from the group consisting of (Item 30) [ka] 29. The compound according to any of items 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28, or a pharmaceutically acceptable salt thereof, selected ...

Claims

[Claim 1] Acceptable toxicity profile.

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