OA19704A - Pyrrolo[1,2-f][1,2,4]Triazines useful for treating respiratory syncitial virus infections - Google Patents

Abstract

Provided herein are formulations, methods and substituted tetrahydrofuranyl-pyrrolo[1,2-/][1,2,4]triazine-4-amine compounds of formula (I) for treating Pneumovirinae virus infections, including respiratory syncytial virus infections, as well as methods and intermediates for synthesis of tetrahydrofuranyl-pyrrolo[1,2-f][1,2,4]triazine-4amine compounds.

Description

PYRROLO[1,2-f][1,2,4]TRIAZINES USEFUL FOR TREATING RESPIRATORY SYNCITIAL VIRUS INFECTIONS

FIELD

Provided herein are substituted tetrahydrofuranyl-pyrrolo[1,2-/j[1,2,4]triazine-4-amine compounds, methods and pharmaceutical formulations for treating Pneumovirinae virus infections, particularly including respiratory syncytial virus infections, as well as methods and intermediates useful for preparing the compounds.

BACKGROUND

Pneumovirinae viruses are negative-sense, single-stranded, RNA viruses that are responsible for many prévalent human and animal diseases. The Pneumovirinae sub-family of viruses is a part of the family Paramyxoviridae and includes human respiratory syncytial virus (HRSV). Almost ail children will hâve had an HRSV infection by their second birthday. H RSV is the major cause of lower respiratory tract infections in infancy and childhood with 0.5% to 2% of those infected requiring hospitalization. The elderly and adults with chronic heart, lung disease or those that are immunosuppressed also hâve a high risk for developing severe HRSV disease (http://www.cdc.gov/rsv/index.html). No vaccine to prevent HRSV infection is currently available. The monoclonal antibody palivizumab is available for immunoprophylaxis, but its use is restricted to infants at high risk, e.g., prématuré infants or those with either congénital heart or lung disease, and the cost for general use is often prohibitive. In addition, nucleoside analog ribavirin has been approved as the only antiviral agent to treat HRSV infections but has limited efficacy. Therefore, there is a need for antiPneumovirinae therapeutics.

Examples of pyrrolo[2,3-d]pyrimidine compounds useful for treating viral infections are described in U.S. 2012/0009147 A1 (Cho et al.), U.S. 2012/0020921 A1 (Cho et al.), WO 2008/089105 A2 (Babu et al.), WO 2008/141079 A1 (Babu et al.), WO 2009/132135 A1 (Butler et al.), WO 2010/002877 A2 (Francom), WO 2011/035231 A1 (Cho et al.), WO 2011/035250 A1 (Butler et al.), WO 2011/150288 A1 (Cho et al.), WO 2012/012465 (Cho et al.), WO 2012/012776 A1 (Mackman et al.), WO 2012/037038 (Clarke et al.), WO 2012/087596 A1 (Delaney et al.), and WO 2012/142075 A1 (Girijavallabhan et al.).

There remains a need for new antiviral agents useful in treating Paramyxoviridae viral infections, including Pneumovirinae viral infections, such as H RSV infections, that are effective and hâve acceptable toxicity profiles.

SUMMARY

Provided are compounds, methods, and pharmaceutical formulations for the treatment of infections caused by the Pneumovirinae virus family, including treatment of infections caused by human respiratory syncytial virus.

Provided is a compound of the Formula (I), or a pharmaceutically acceptable sait thereof:

wherein:

R¹ is H or F;

R² is H or F;

R³ is OH or F;

R⁴ is CN, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C4 cycloalkyl, azido, halogen, or C1-C2 haloalkyl;

R⁶ is OH;

R⁵ is selected from the group of H and:

wherein:

n’ is selected from 1, 2, 3, and 4;

R⁸ is selected from Ci-Cs alkyl, -O-Ci-Cs alkyl, benzyl, -O-benzyl, -CH2-C3-C6 cycloalkyl, -O-CH2-C3-C6 cycloalkyl, and CF3;

R⁹ is selected from phenyl, 1-naphthyl, 2-naphthyl,

R¹⁰ is selected from H and CH3;

R¹¹ is selected from H or Ch-Ce alkyl;

R¹² is selected from H, Ch-Cs alkyl, benzyl, C3-C6 cycloalkyl, and -CH2-C3-C6 cycloalkyl.

DETAILED DESCRIPTION

An embodiment herein comprises a compound of Formula (I), or a pharmaceutically acceptable sait thereof, wherein R¹ is H, and ail other variables, including R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², and ri are as defined above for Formula (I).

Another embodiment herein comprises a compound of Formula (I), or a pharmaceutically acceptable sait thereof, wherein R² is H, and ail other variables, including R¹, R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², and ri are as defined above for Formula (I).

A further embodiment herein comprises a compound of Formula (I), or a pharmaceutically acceptable sait thereof, wherein both R¹ and R² are H, and ail other variables, including R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², and ri are as defined above for Formula (I).

Still another embodiment herein comprises a compound of Formula (I), or a pharmaceutically acceptable sait thereof, wherein both R¹, R², and R⁵ are H, and ail other variables, including R³, R⁴, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², and ri are as defined above for Formula (I).

Another separate embodiment herein comprises a compound of Formula (I), or a pharmaceutically acceptable sait thereof, wherein both R¹ and R² are H, R³ is OH, and ail other variables, including R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², and ri are as defined above for Formula (I).

Another separate embodiment herein comprises a compound of Formula (I), or a pharmaceutically acceptable sait thereof, wherein both R¹ and R² are H, R³ is F, and ail other variables, including R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², and ri are as defined above for Formula (I).

Another embodiment provided herein comprises a compound of Formula (II), or a pharmaceutically acceptable sait thereof:

wherein:

R³ is OH or F;

R⁴ is CN, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C4 cycloalkyl, azido, halogen, 5 or C1-C2 haloalkyl;

R⁵ is selected from the group of H and:

wherein:

n’ is selected from 1, 2, 3, and 4;

R⁸ is selected from Ci-Cs alkyl, -O-Ci-Cs alkyl, benzyl, -O-benzyl, -CH2-C3-C6 cycloalkyl, -O-CH2-C3-C6 cycloalkyl, and CF₃;

R⁹ is phenyl;

R¹⁰ is selected from H and CH3;

R¹¹ is selected from H or Ci-Ce alkyl;

R¹² is selected from H, Ci-Cs alkyl, benzyl, C3-C6 cycloalkyl, and -CH2-C3-C6 cycloalkyl.

A further embodiment comprises a compound of Formula (II), or a pharmaceutically acceptable sait thereof, wherein:

R³ is OH or F;

R⁴ is CN, methyl, ethyl, ethenyl, ethynyl, azido, F, Cl, -CH2CI, -CH2F, -CHF2, or-CF₃;

and R⁵ and ail other groups are as defined for Formula (II).

Also provided is an embodiment comprising a compound of Formula (II) or a pharmaceutically acceptable sait thereof, as described above, wherein R³ is F.

Also provided is an embodiment comprising a compound of Formula (II) or a pharmaceutically acceptable sait thereof, as described above, wherein R³ is OH.

Also provided is an embodiment comprising a compound of Formula (II) or a pharmaceutically acceptable sait thereof, as described above, wherein R³ is F and R⁴ is CN.

Also provided is an embodiment comprising a compound of Formula (II) or a pharmaceutically acceptable sait thereof, as described above, wherein R³ is OH and R⁴ is CN.

Also provided is an embodiment comprising a compound of Formula (II) or a pharmaceutically acceptable sait thereof, as described above, wherein both R¹ and R² are H, R³ is F, and R⁴ is methyl, ethyl, vinyl, or ethynyl.

Also provided is an embodiment comprising a compound of Formula (II) or a pharmaceutically acceptable sait thereof, as described above, wherein R³ is OH, and R⁴ is methyl, ethyl, vinyl, or ethynyl.

Also provided is an embodiment comprising a compound of Formula (II) or a pharmaceutically acceptable sait thereof, as described above, wherein R³ is F and R⁴ is halomethyl.

Also provided is an embodiment comprising a compound of Formula (II) or a pharmaceutically acceptable sait thereof, as described above, wherein R³ is OH and R⁴ is halomethyl.

Also provided is an embodiment comprising a compound of Formula (II) or a pharmaceutically acceptable sait thereof, as described above, wherein R⁵ is H.

Also provided is an embodiment comprising a compound of Formula (II) or a pharmaceutically acceptable sait thereof, as described above, wherein each of R⁵ is H, R³ is OH, and R⁴ is methyl, ethyl, vinyl, orethynyl.

Also provided is an embodiment comprising a compound of Formula (II) or a pharmaceutically acceptable sait thereof, as described above, wherein R⁵ is H, R³ is F, and R⁴ is halomethyl.

Also provided is an embodiment comprising a compound of Formula (II) or a pharmaceutically acceptable sait thereof, as described above, wherein R⁵ are H, R³ is OH, and R⁴ is halomethyl.

Within each of the embodiments described above comprising a compound of Formula (II), or a pharmaceutically acceptable sait thereof, wherein R⁵ may be other than H, there is a further embodiment wherein ail other variables are as described for the embodiment and R⁵ is selected from the group of:

O il £

HO—P--$I f

HO ²

wherein:

R⁸ is selected from Ci-C₈ alkyl, -O-Ci-C₈ alkyl, benzyl, and -CH₂-C3-C₆ cycloalkyl; and

R¹² is selected from Ci-C₈ alkyl, benzyl, C₈-C₆ cycloalkyl, and -CH2-C3-C6 cycloalkyl.

Within each ofthe embodiments described immediately above there is a further embodiment comprising a compound of Formula (II), or a pharmaceutically acceptable sait thereof, wherein ail other variables are as described immediately above, except that R⁸ and R⁹ are each selected from Ci-C8 alkyl. Within each of the embodiments described in the last sentence there is a further embodiment comprising a compound of Formula (II), or a pharmaceutically acceptable sait thereof, wherein ail other variables are as described immediately above, except that R⁸ and R⁹ are each selected from Ci-C8 alkyl. Within each of the embodiments described in the last sentence there is a further embodiment comprising a compound of Formula (II), or a pharmaceutically acceptable sait thereof, wherein ail other variables are as described immediately above, except that R⁸ and R⁹ are each selected from C1-C5 alkyl. Within each of the embodiments described in the last sentence there is a further embodiment comprising a compound of Formula (II), or a pharmaceutically acceptable sait thereof, wherein ail other variables are as described immediately above, except that R⁸ and R⁹ are each selected from C1-C4 alkyl.

Within each of the embodiments described herein comprising a compound of Formula (I) or of Formula (II) there is a further embodiment wherein ail variables are as defined for the particular embodiment and further comprising the proviso that when R³ is F, R⁴ is not methyl.

Définitions

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

“Azido” refers to an azide group, i.e. the group -N₃. The term “n” as used herein refers to an integer, such as an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, i.e. 2 to 20 or 2-20. In some instances, “n” refers to groups of integers such as 1 to 3, 1 to 4, 1 to 6, 1 to 8, 2 to 4, 2 to 6, 2 to 8, etc.

The term “haloalkyl” as used herein refers to an alkyl as defined herein, wherein one or more hydrogen atoms are each replaced by a halo substituent. For example, a (CiCs)haloalkyl is a (Ci-Cejalkyl wherein one or more of the hydrogen atoms hâve been replaced by a halo substituent. Such a range includes one halo substituent on the alkyl group t to complété halogénation of the alkyl group.

The term “(Ci-n)haloalkyl” as used herein, wherein n is an integer, either alone or in combination with another radical, is intended to mean an alkyl radical having 1 to n carbon atoms as defined above wherein one or more hydrogen atoms are each replaced by a halo substituent. Examples of (Ci-n)haloalkyl, wherein n is 2 include, but are not limited to, chloromethyl, chloroethyl, dichloroethyl, bromomethyl, bromoethyl, dibromoethyl, fluoromethyl, difluoromethyl, trifluoromethyl, fluoroethyl and difluoroethyl.

The term “(Ci-n)alkyl” as used herein, wherein n is an integer, either alone or in combination with another radical, is intended to mean acyclic, straight or branched chain alkyl radicals containing from 1 to n carbon atoms. “(Ci-4)alkyl” includes, but is not limited to, methyl, ethyl, propyl (n-propyl), butyl (n-butyl), 1-methylethyl (/so-propyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (/so-butyl), and 1,1-dimethylethyl (tert-butyl or t-butyl). The abbreviation Me dénotés a methyl group; Et dénotés an ethyl group, Pr dénotés a propyl group, iPr dénotés a 1-methylethyl group, Bu dénotés a butyl group and tBu dénotés a 1,1dimethylethyl group.

The term “alkyl” refers to a hydrocarbon containing normal, secondary, or tertiary atoms. For example, an alkyl group can hâve 1 to4 carbon atoms (i.e, (Ci-C4)alkyl), 1 to 3 carbon atoms (i.e., (Ci-C3)alkyl), or 1 or 2 carbon atoms (i.e., (Ci-C2)alkyl). Examples of suitable alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH₃)2), 1-butyl (n-Bu, nbutyl, -CH2CH2CH2CH3), 2-methyl-1 -propyl Q-Bu, i-butyl, -CH₂CH(CH₃)2), 2-butyl (s-Bu, sbutyl, -CH(CH3)CH2CH3), and 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3),. “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 radicale 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.

“Alkenyl” is a straight or branched hydrocarbon containing normal, secondary or tertiary carbon atoms with at least one site of unsaturation, i.e. a carbon-carbon, sp² double bond. As examples, an alkenyl group can hâve 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),.

The term “(C₂-n)alkenyl”, as used herein, wherein n is an integer, either alone or in combination with another radical, is intended to mean an unsaturated, acyclic straight or branched chain radical containing two to n carbon atoms, at least two of which are bonded 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 specified otherwise, the term “(C₂-n)alkenyl” is understood to encompass individual stereoisomers where possible, including but not limited to (E) and (Z) isomers, and mixtures thereof. When a (C₂._n)alkenyl group is substituted, it is understood to be substituted on any carbon atom thereof which would otherwise bear a hydrogen atom, unless specified otherwise, such that the substitution would give rise to a chemically stable compound, such as are recognized by those skilled in the art.

“Alkynyl” is a straight or branched hydrocarbon containing normal, secondary or tertiary carbon atoms with at least one site of unsaturation, i.e. a carbon-carbon, sp triple bond. For example, an alkynyl group can hâve 2 to 4 carbon atoms (i.e., C2-C4 alkynyl) or 2 to 3 carbon atoms (i.e., C₂-C₃ alkyne,). Examples of suitable alkynyl groups include, but are not limited to, acetylenic (-C^CH), propargyl (-CH₂C=CH), and the like.

The term “(C₂._n)alkynyl”, as used herein, wherein n is an integer, either alone or in combination with another radical, is intended to mean an unsaturated, acyclic straight or branched chain radical containing two to n carbon atoms, at least two of which are bonded to each other by a triple bond. Examples of such radicals in which n is 4 include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and 1-butynyl. When a (C₂._n)alkynyl group is substituted, it is understood to be substituted on any carbon atom thereof which would otherwise bear a hydrogen atom, unless specified otherwise, such that the substitution would give rise to a chemically stable compound, such as are recognized by those skilled in the art.

The term cycloalkyl refers to a cyclic aliphatic group. The cycloallkyl groups herein may be referenced by the number of carbon atoms in their ring, such as “C3-C4 cycloalkyl” referring to a cycloalkyl ring with 3 or 4 carbon ring atoms or “C3-C6 cycloalkyl” indicating a cycloalkyl ring with 3, 4, 5, or 6 carbon ring atoms, i.e. a cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl ring.

The term “carbocycle” or “carbocyclyl” refers to a saturated (i.e., cycloalkyl) or partially unsaturated (e.g., cycloalkenyl, cycloalkadienyl, etc.) ring having the number of carbon atoms specified, such as 3 to 4 carbon atoms or 3 to 6 carbon atoms as a monocyclic ring system. In one embodiment the carbocycle is a monocycle comprising 3-6 ring carbons (i.e. (Cs-Cejcarbocycle). Non-limiting examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, and cyclohexa-1,3-dienyl rings.

Each carbocyclyl group may be substituted by 0, 1, 2, or 3 substituents independently selected from halogen, -OH, -CN, -NO2, -NH₂, -NH(Ci-Ce alkyl), -N(Ci-Ce alkyl)2, Ci-Ce alkyl, Ci-Cs alkoxy, and -CF3.

Pharmaceutical Formulations

Also provided herein is a pharmaceutical formulation comprising a pharmaceutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, and a pharmaceutically acceptable carrier or excipient. Also provided are separate pharmaceutical formulations, each comprising a pharmaceutically effective amount of a compound of Formula (II) or one of the spécifie compounds of the examples herein, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, and a pharmaceutically acceptable carrier or excipient.

The compounds herein are formulated with conventional carriers and excipients, which will be selected in accord with ordinary practice. Tablets will contain excipients, glidants, fillers, binders and the like. Aqueous formulations are prepared in stérile form, and when intended for delivery by other than oral administration generally will be isotonie. AH formulations will optionally contain excipients such as those set forth in the Handbook of

Pharmaceutical Excipients (1986). Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextran, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and the like. The pH of the formulations ranges from about 3 to about 11, but is ordinarily about 7 to 10.

While it is possible for the active ingrédients to be administered alone it may be préférable to présent them as pharmaceutical formulations. The formulations, both for veterinary and for human use, comprise at least one active ingrédient, as above defined, together with one or more acceptable carriers and optionally other therapeutic ingrédients, particularly those additional therapeutic ingrédients as discussed herein. The carrier(s) must be acceptable in the sense of being compatible with the other ingrédients of the formulation and physiologically innocuous to the récipient thereof.

The formulations include those suitable for the foregoing administration routes. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations generally are found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). Such methods include the step of bringing into association the active ingrédient with the carrier which constitutes one or more accessory ingrédients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingrédient with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations suitable for oral administration may be presented as discrète units such as capsules, cachets or tablets each containing a predetermined amount of the active ingrédient; as a powder or granules; as a solution or a suspension in an aqueous or nonaqueous liquid; or as an oil-in-water liquid émulsion or a water-in-oil liquid émulsion. The active ingrédient may also be administered as a bolus, electuary or paste.

A tablet is made by compression or molding, optionally with one or more accessory ingrédients. Compressed tablets may be prepared by compressing in a suitable machine the active ingrédient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingrédient moistened with an inert liquid diluent. The tablets may optionally be coated or scored and optionally are formulated so as to provide slow or controlled release of the active ingrédient therefrom.

For infections of the eye or other external tissues e.g. mouth and skin, the formulations are preferably applied as a topical ointment or cream containing the active ingredient(s) in an amount of, for example, 0.075 to 20% w/w (including active ïngredient(s) in a range between 0.1% and 20% in incréments of 0.1% w/w 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 in an ointment, the active ingrédients may be employed with either a paraffinic or a watermiscible ointment base. Alternatively, the active ingrédients may be formulated in a cream with an oil-in-water cream base.

If desired, the aqueous phase of the cream base may include, 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. The topical formulations may desirably include a compound which enhances absorption or pénétration of the active ingrédient through the skin or other affected areas. Examples of such dermal pénétration enhancers include dimethyl sulphoxide and related analogs.

The oily phase of the émulsions may be constituted from known ingrédients in a known manner. While the phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilie emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax, and the wax together with the oil and fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cr eam formulations.

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

The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties. The cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stéarate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stéarate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other minerai oils are used.

Pharmaceutical formulations herein comprise a combination together with one or more pharmaceutically acceptable carriers or excipients and optionally other therapeutic agents. Pharmaceutical formulations containing the active ingrédient may be in any form suitable for the intended method of administration. When used for oral use for example, tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, émulsions, hard or soft capsules, solutions, syrups or elixirs may be prepared. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents including sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable préparation. Tablets containing the active ingrédient in admixture with non-toxic pharmaceutically acceptable excipient which are suitable for manufacture of tablets are acceptable. These excipients may be, for example, inert diluents, such as calcium or sodium carbonate, lactose, calcium or sodium phosphate; granulating and disintegrating agents, such as maize starch, oralginic acid; binding agents, such as starch, gelatin or acacia; and lubricating agents, such as magnésium stéarate, stearic acid or talc. 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.

Formulations for oral use may be also presented as hard gelatin capsules where the active ingrédient is mixed with an inert solid diluent, for example calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingrédient is mixed with water or an oil medium, such as peanutoil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcelluose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing orwetting agents such as a naturally-occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stéarate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose or saccharin.

Oil suspensions may be formulated by suspending the active ingrédient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a minerai oil such as liquid paraffin. The oral suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palatable oral préparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

Dispersible powders and granules suitable for préparation of an aqueous suspension by the addition of water provide the active ingrédient in admixture 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 excipients, for example sweetening, flavoring and coloring agents, may also be présent.

The pharmaceutical compositions may also be in the form of oil-in-water émulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, a minerai oil, such as liquid paraffin, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally-occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The émulsion may also contain sweetening and flavoring agents. Syrups and élixirs may be formulated with sweetening agents, such as glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent.

The pharmaceutical compositions may be in the form of a stérile injectable or intravenous préparations, such as a stérile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which hâve been mentioned above. The stérile injectable or intravenous préparation may also be a stérile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butane-diol or prepared as a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonie sodium chloride solution. In addition, stérile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the préparation of injectables.

The amount of active ingrédient that may be combined with the carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a time-release formulation intended for oral administration to humans may contain approximately 1 to 1000 mg of active material compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95% of the total compositions (weight:weight). The pharmaceutical composition can be prepared to provide easily measurable amounts for administration. For example, an aqueous solution intended for intravenous infusion may contain from about 3 to 500 qg of the active ingrédient per milliliter of solution in order that infusion of a suitable volume at a rate of about 30 mL/hr can occur.

Formulations suitable for topical administration to the eye also include eye drops wherein the active ingrédient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingrédient. The active ingrédient is preferably présent in such formulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10%, and particularly about 1.5% w/w.

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

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

Formulations suitable for intrapulmonary or nasal administration hâve a particle size for example in the range of 0.1 to 500 microns, such as 0.5, 1, 30, 35 etc., which is administered by rapid inhalation through the nasal passage or by inhalation through the mouth so as to reach the alveolar sacs. Suitable formulations include aqueous or oiiy solutions of the active ingrédient. Formulations suitable for aérosol or dry powder administration may be prepared according to conventional methods and may be delivered with other therapeutic agents such as compounds heretofore used in the treatment or prophylaxis of Pneumovirinae infections as described below.

Another embodiments provides a novel, efficacious, safe, nonirritating and physiologically compatible inhalable composition comprising a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable sait thereof, suitable for treating Pneumovirinae infections and potentially associated bronchiolitis. Preferred pharmaceutically acceptable salts are inorganic acid salts including hydrochloride, hydrobromide, sulfate or phosphate salts as they may cause less pulmonary irritation. Preferably, the inhalable formulation is delivered to the endobronchial space in an aérosol comprising particles with a mass médian aerodynamic diameter (MMAD) between about 1 and about 5 pm. Preferably, the compound of Formula (I) or Formula (II) is formulated for aérosol delivery using a nebulizer, pressurized metered dose inhaler (pMDI), or dry powder inhaler (DPI).

Non-limiting examples of nebulizers include atomizing, jet, ultrasonic, pressurized, vibrating porous plate, or équivalent nebulizers including those nebulizers utilizing adaptive aérosol delivery technology (Denyer, J. Aérosol medicine Pulmonary Drug Delivery 2010, 23 Supp 1, S1-S10). A jet nebulizer utilizes air pressure to break a liquid solution into aérosol droplets. An ultrasonic nebulizer works by a piezoelectric crystal that shears a liquid into small aérosol droplets. A pressurized nebulization system forces solution under pressure through small pores to generate aérosol droplets. A vibrating porous plate device utilizes rapid vibration to shear a stream of liquid into appropriate droplet sizes.

In a preferred embodiment, the formulation for nebulization is delivered to the endobronchial space in an aérosol comprising particles with a MMAD predominantly between about 1 pm and about 5 pm using a nebulizer able to aerosolize the formulation of the compound of Formula (I) or Formula (II) into particles of the required MMAD. To be optimally therapeutically effective and to avoid upper respiratory and systemic side effects, the majority of aerosolized particles should not hâve a MMAD greater than about 5 pm. If an aérosol contains a large number of particles with a MMAD larger than 5 pm, the particles are deposited in the upper airways decreasing the amount of drug delivered to the site of inflammation and bronchoconstriction in the lower respiratory tract. If the MMAD of the aérosol is smaller than about 1 pm, then the particles hâve a tendency to remain suspended in the inhaled air and are subsequently exhaled during expiration.

When formulated and delivered according to the method herein, the aérosol formulation for nebulization delivers a therapeutically efficacious dose of the compound of Formula (I) or Formula (II) to the site of Pneumovirinae infection sufficient to treat the Pneumovirinae infection. The amount of drug administered must be adjusted to reflect the efficiency of the delivery of a therapeutically efficacious dose of the compound of Formula (I) or Formula (II). In a preferred embodiment, a combination of the aqueous aérosol formulation with the atomizing, jet, pressurized, vibrating porous plate, or ultrasonic nebulizer permits, depending on the nebulizer, about, at least, 20, to about 90%, typically about 70% delivery of the administered dose of the compound of Formula (I) or Formula (II) into the airways. In a preferred embodiment, 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.

In another embodiment, a compound of Formula (I) or Formula (II) or a pharmaceutically acceptable sait thereof, is delivered as a dry inhalable powder. The compounds are administered endobronchially as a dry powder formulation to efficacious deliver fine particles of compound into the endobronchial space using dry powder or metered dose inhalers. For delivery by DPI, the compound of Formula (I) or Formula (II) is processed into particles with, predominantly, MMAD between about 1 pm and about 5 pm by milling spray drying, critical fluid processing, or précipitation from solution. Media milling, jet milling and spray-drying devices and procedures capable of producing the particle sizes with a MMAD between about 1 pm and about 5 pm are well known in the art. In one embodiment, excipients are added to the compound of Formula (I) or Formula (II) before processing into particles of the required sizes. In another embodiment, excipients are blended with the particles of the required size to aid in dispersion of the drug particles, for example by using lactose as an excipient.

Particle size déterminations are made using devices well known in the art. For example a multi-stage Anderson cascade impactor or other suitable method such as those specifically cited within the US Pharmacopoeia Chapter 601 as characterizing devices for aérosols within metered-dose and dry powder inhalers.

In another preferred embodiment, a 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 devices. Non-limiting examples of dry powder inhalers and devices include those disclosed in US5,458,135; US5,740,794; US5775320; US5,785,049; US3,906,950;

US4,013,075; US4,069,819; US4,995,385; US5,522,385; US4,668,218; US4,667,668; US4,805,811 and US5,388,572. There are two major designs of dry powder inhalers. One design is a metering device in which a réservoir for the drug is place within the device and the patient adds a dose of the drug into the inhalation chamber. The second design is a factory-metered device in which each individual dose has been manufactured in a separate container. Both Systems dépend on the formulation of the drug into small particles of MMAD from 1 pm and about 5 pm and often involve co-formulation with larger excipient particles such as, but not limited to, lactose. Drug powder is placed in the inhalation chamber (either by device metering or by breakage of a factory-metered dosage) and the inspiratory flow of the patient accelerates the powder out of the device and into the oral cavity. Non-laminar flow characteristics of the powder path cause the excipient-drug aggregates to décomposé, and the mass of the large excipient particles causes their impaction at the back of the throat, while the smaller drug particles are deposited deep in the lungs. In preferred embodiments, a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable sait thereof, is delivered as a dry powder using either type of dry powder inhaler as described herein, wherein the MMAD of the dry powder, exclusive of any excipients, is predominantly in the range of 1 pm to about 5 pm.

In another embodiment, a 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 US5,261,538; US5,544,647; US5,622,163; US4,955,371; US3,565,070; US3,361306 and US6,116,234. In preferred embodiments, a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable sait thereof, is delivered as a dry powder using a metered dose inhaler wherein the MMAD of the dry powder, exclusive of any excipients, is predominantly in the range of about 1-5 pm.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingrédient such carriers as are known in the art to be appropriate.

Formulations suitable for parentéral administration include aqueous and nonaqueous stérile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutés which render the formulation isotonie with the blood of the intended récipient; and aqueous and non-aqueous stérile suspensions which may include suspending agents and thickening agents.

The formulations are 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 the stérile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from stérile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the active ingrédient.

It should be understood that in addition to the ingrédients 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.

Further provided are veterinary compositions comprising at least one active ingrédient as above defined together with a veterinary carrier therefor.

Veterinary carriers are materials useful for the purpose of administering the composition and may be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingrédient. These veterinary compositions may be administered orally, parenterally or by any other desired route.

Compounds herein are used to provide controlled release pharmaceutical formulations containing as active ingrédient one or more of the compounds (controlled release formulations) in which the release of the active ingrédient are controlled and regulated to allow less frequency dosing or to improve the pharmacokinetic or toxicity profile of a given active ingrédient.

Effective dose of active ingrédient dépends at least on the nature of the condition being treated, toxicity, whether the compound is being used prophylactically (lower doses) or against an active viral infection, the method of delivery, and the pharmaceutical formulation, and will be determined by the clinician using conventional dose escalation studies. It can be expected to be from about 0.0001 to about 100 mg/kg body weight per day; typically, from about 0.01 to about 10 mg/kg body weight per day; more typically, from about .01 to about 5 mg/kg body weight per day; most typically, from about .05 to about 0.5 mg/kg body weight per day. For example, the daily candidate dose for an adult human of approximately 70 kg body weight will range from 1 mg to 1000 mg, preferably between 5 mg and 500 mg, and may take the form of single or multiple doses.

Routes of Administration

One or more of the compounds (herein referred to as the active ingrédients) are administered by any route appropriate to the condition to be treated. Suitable routes include oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), vaginal and parentéral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and épidural), and the like. It will be appreciated that the preferred route may vary with for example the condition of the récipient. An advantage of the compounds herein is that they are orally bioavailable and can be dosed orally.

Combination Therapy

Compositions are also used in combination with other active ingrédients. For the treatment of Pneumovirinae virus infections, preferably, the other active therapeutic agent is active against Pneumovirinae virus infections, particularly respiratory syncytial virus infections. Non-limiting examples of these other active therapeutic agents are 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.

Many of the infections of the Pneumovirinae viruses are respiratory infections. Therefore, additional active therapeutics used to treat respiratory symptoms and sequelae of infection may be used in combination with the compounds of Formula (I) or Formula (II). The additional agents are preferably administered orally or by direct inhalation. For example, other preferred additional therapeutic agents in combination with the compounds of

Formula (I) or Formula (II) for the treatment of viral respiratory infections include, but are not limited to, bronchodilators and corticosteroids.

Glucocorticoids, which were first introduced as an asthma therapy in 1950 (Carryer, Journal of Allergy, 21, 282-287, 1950), remain the most potent and consistently effective therapy for this disease, although their mechanism of action is not yet fully understood (Morris, J. Allergy Clin. Immunol., 75 (1 Pt) 1-13, 1985). Unfortunately, oral glucocorticoid thérapies are associated with profound undesirable side effects such as truncal obesity, hypertension, glaucoma, glucose intolérance, accélération of cataract formation, bone minerai loss, and psychological effects, ail of which limit their use as long-term therapeutic agents (Goodman and Gilman, 10th édition, 2001). A solution to systemic side effects is to deliver steroid drugs directly to the site of inflammation. Inhaled corticosteroids (ICS) hâve been developed to mitigate the severe adverse effects of oral steroids. Non-limiting examples of corticosteroids that may be used in combinations with the compounds of Formula (I) or Formula (II) are dexamethasone, dexamethasone sodium phosphate, fluorometholone, fluorometholone acetate, loteprednol, loteprednol etabonate, hydrocortisone, prednisolone, fludrocortisones, triamcinolone, triamcinolone acetonide, betamethasone, beclomethasone diproprionate, méthylprednisolone, fluocinolone, fluocinolone acetonide, flunisolide, fluocortin-21-butylate, flumethasone, flumetasone pivalate, budesonide, halobetasol propionate, mometasone furoate, fluticasone propionate, ciclesonide; or a pharmaceutically acceptable salts thereof.

Other anti-inflamatory agents working through anti-inflamatory cascade mechanisms are also useful as additional therapeutic agents in combination with the compounds of Formula (I) or Formula (II) for the treatment of viral respiratory infections. Applying “antiinflammatory signal transduction modulators” (referred to in this text as AISTM), like phosphodiesterase inhibitors (e.g. PDE-4, PDE-5, or PDE-7 spécifie), transcription factor inhibitors (e.g. blocking NFkB through IKK inhibition), or kinase inhibitors (e.g. blocking P38 MAP, J N K, PI3K, EGFR or Syk) is a logical approach to switching off inflammation as these small molécules target a limited number of common intracellular pathways - those signal transduction pathways that are critical points for the anti-inflammatory therapeutic intervention (see review by P.J. Bames, 2006). These non-limiting additional therapeutic agents include: 5-(2,4-Difluoro-phenoxy)-1-isobutyl-1H-indazole-6-carboxylic acid (2dimethylamino-ethyl)-amide (P38 Map kinase inhibitor ARRY-797); 3-CyclopropylmethoxyN-(3,5-dichloro-pyridin-4-yl)-4-difluorormethoxy-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-[(methylsulfonyl)amino]-1dibenzofurancarboxamide (PDE-4 inhibitor Oglemilast); N-(3,5-Dichloro-pyridin-4-yl)-2-[1-(4fluorobenzyl)-5-hydroxy-1H-indol-3-yl]-2-oxo-acetamide (PDE-4 inhibitor AWD 12-281); 8Methoxy-2-trifluoromethyl-quinoline-5-carboxylic acid (3,5-dichloro-1-oxy-pyridin-4-yl)-amide (PDE-4 inhibitor Sch 351591); 4-[5-(4-Fluorophenyl)-2-(4-methanesulfinyl-phenyl)-1 Himidazol-4-yl]-pyridine (P38 inhibitor SB-203850); 4-[4-(4-Fluoro-phenyl)-1-(3-phenyl-propyl)5-pyridin-4-yl-1H-imidazol-2-yl]-but-3-yn-1-ol (P38 inhibitor RWJ-67657); 4-Cyano-4-(3cyclopentyloxy-4-methoxy-phenyl)-cyclohexanecarboxylic acid 2-diethylamino-ethyl ester (2diethyl-ethyl ester prodrug of Cilomilast, PDE-4 inhibitor); (3-Chloro-4-fluorophenyl)-[7methoxy-6-(3-morpholin-4-yl-propoxy)-quinazolin-4-yl]-amine (Gefitinib, EGFR inhibitor); and 4-(4-Methyl-piperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]benzamide (Imatinib, EGFR inhibitor).

Combinations comprising inhaled p2-adrenoreceptor agonist bronchodilators such as formoterol, albuterol or salmeterol with the compounds of Formula (I) or Formula (II) are also suitable, but non-limiting, combinations useful for the treatment of respiratory viral infections.

Combinations of inhaled 82-adrenoreceptor agonist bronchodilators such as formoterol or salmeterol with ICS’s are also used to treat both the bronchoconstriction and the inflammation (Symbicort® and Advair®, respectively). The combinations comprising these ICS and p2-adrenoreceptor agonist combinations along with the compounds of Formula (I) or Formula (II) are also suitable, but non-limiting, combinations useful for the treatment of respiratory viral infections.

For the treatment or prophylaxis of pulmonary broncho-constriction, anticholinergics are of potential use and, therefore, useful as an additional therapeutic agents in combination with the compounds of Formula (I) or Formula (II) for the treatment of viral respiratory infections. These anticholinergics include, but are not limited to, antagoniste of the muscarinic receptor (particularly of the M3 subtype) which hâve shown therapeutic efficacy in man for the control of cholinergic tone in COPD (Witek, 1999); 1-{4-Hydroxy-1-[3,3,3-tris(4-fluoro-phenyl)-propionyl]-pyrrolidine-2-carbonyl}-pyrrolidine-2-carboxylic acid (1-methylpiperidin-4-ylmethyl)-amide; 3-[3-(2-Diethylamino-acetoxy)-2-phenyl-propionyloxy]-8isopropyl-8-methyl-8-azonia-bicyclo[3.2.1]octane (lpratropium-N,N-diethylglycinate); 1Cyclohexyl-3,4-dihydro-1H-isoquinoline-2-carboxylic acid 1-aza-bicyclo[2.2.2]oct-3-yl ester (Solifenacin); 2-Hydroxymethyl-4-methanesulfinyl-2-phenyl-butyric acid 1-azabicyclo[2.2.2]oct-3-yl ester (Revatropate); 2-{1-[2-(2,3-Dihydro-benzofuran-5-yl)-ethyl]pyrrolidin-3-yl}-2,2-diphenyl-acetamide (Darifenacin); 4-Azepan-1-yl-2,2-diphenyl-butyramide (Buzepide); 7-[3-(2-Diethylamino-acetoxy)-2-phenyl-propionyloxy]-9-ethyl-9-methyl-3-oxa-9azonia-tricyclo[3.3.1.02,4]nonane (Oxitropium-N,N-diethylglycinate); 7-[2-(2-Diethylaminoacetoxy)-2,2-di-th iophen-2-yl-acetoxy]-9,9-d imethy l-3-oxa-9-azon iatricyclo[3.3.1.02,4]nonane (Tiotropium-N,N-diethylglycinate); Dimethylamino-acetic acid 2-(3diisopropylamino-1-phenyl-propyl)-4-methyl-phenyl ester (Tolterodine-N,Ndimethylglycinate); 3-[4,4-Bis-(4-fluoro-phenyl)-2-oxo-imidazolidin-1-yl]-1-methyl-1-(2-oxo-2pyridin-2-yl-ethyl)-pyrrolidinium; 1-[1-(3-Fluoro-benzyl)-piperidin-4-yl]-4,4-bis-(4-fluorophenyl)-imidazolidin-2-one; 1-Cyclooctyl-3-(3-methoxy-1-aza-bicyclo[2.2.2]oct-3-yl)-1phenyl-prop-2-yn-1-ol; 3-[2-(2-Diethylamino-acetoxy)-2,2-di-thiophen-2-yl-acetoxy]-1-(3phenoxy-propyl)-1-azonia-bicyclo[2.2.2]octane (Aclidinium-N,N-diethylglycinate); or (2Diethylamino-acetoxy)-di-thiophen-2-yl-acetic acid 1-methyl-1-(2-phenoxy-ethyl)-piperidin-4yl ester.

The compounds of Formula (I) or Formula (II) may also be combined with mucolytic agents to treat both the infection and symptoms of respiratory infections. A non-limiting example of a mucolytic agent is ambroxol. Similarly, the compounds of Formula (I) or Formula (II) may be combined with expectorants to treat both the infection and symptoms of respiratory infections. A non-limiting example of an expectorant is guaifenesin.

Nebulized hypertonie saline is used to improve immédiate and long-term clearance of small airways in patients with lung diseases (Kuzik, J. Pediatrics 2007, 266). The compounds of Formula (I) or Formula (II) may also be combined with nebulized hypertonie saline particularly when the Pneumovirinae virus infection is complicated with bronchiolitis. The combination of the compounds of Formula (I) or Formula (II) with hypertonie saline may also comprise any of the additional agents discussed above. In one embodiment, nebulized about 3% hypertonie saline is used.

It is also possible to combine any compound with one or more additional active therapeutic agents in a unitary dosage form for simultaneous or sequential administration to a patient. The combination therapy may be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations.

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

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

The combination therapy may provide “synergy” and “synergistic”, i.e. the effect achieved when the active ingrédients used together is greater than the sum of the effects that results from using the compounds separately. A synergistic effect may be attained when the active ingrédients are: (1) co-formulated and administered or delivered simultaneously in a combined formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect may be attained when the compounds are administered or delivered sequentially, e.g. in separate tablets, pills or capsules, or by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingrédient is administered sequentially, i.e. serially, whereas in combination therapy, effective dosages of two or more active ingrédients are administered together. A synergistic anti-viral effect dénotés an antiviral effect which is greater than the predicted purely additive effects of the individual compounds of the combination.

In still yet another embodiment, the présent application provides a method of treating

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 sait, solvaté, and/or ester thereof. Also provided are separate methods of treating Pneumovirinae virus infection in a human, each comprising administering to the human a therapeutically effective a pharmaceutically effective amount of a compound of Formula (II) or one of the spécifie compounds of the examples herein, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, and a pharmaceutically acceptable carrier or excipient.

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

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 solvaté of a compound of Formula (II) or one of the spécifie compounds of the examples herein, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof.

In still yet another embodiment, the présent application provides for a method of treating 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 sait, solvaté, and/or ester thereof.

In still yet another embodiment, the présent application provides for a method of treating 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 sait, solvaté, and/or ester thereof, and at least one additional active therapeutic agent.

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 spécifie compounds ofthe examples herein, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof.

Also 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 spécifie compounds of the examples herein, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, and at least one additional active therapeutic agent.

Also provided are separate methods of treating a human respiratory syncytial virus infection in a human in need thereof, wherein the human is also experiencing bronchiolitis, each method comprising administering to the human a therapeutically effective amount of a compound of Formula (I), Formula (II), or one of the spécifie compounds of the examples herein, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof.

Also provided are separate methods of treating a human respiratory syncytial virus infection in a human in need thereof, wherein the human is also experiencing pneumonia, each method comprising administering to the human a therapeutically effective amount of a compound of Formula (I), Formula (II), or one of the spécifie compounds of the examples herein, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof.

Also provided are separate methods of improving respiratory symptoms in a human experiencing a human respiratory syncytial virus infection, each method comprising administering to the human a therapeutically effective amount of a compound of Formula (I), Formula (II), or one of the spécifie compounds of the examples herein, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof.

Respiratory symptoms in a human experiencing a respiratory syncytial virus infection may include congested or runny nose, coughing, wheezing, sneezing, rapid breathing or difficulty breathing, apnea, bronchiolitis, and pneumonia.

Also provided is an embodiment comprising the use of a compound of Formula (I), or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, for the manufacture of a médicament for the treatment of a Pneumovirinae virus infection or a respiratory syncytial virus infection.

Also provided is an embodiment comprising the use of a compound of Formula (II) or one of the spécifie compounds of the examples herein, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, for the manufacture of a médicament for the treatment of a Pneumovirinae virus infection or a respiratory syncytial virus infection.

Also provided is a pharmaceutical formulation comprising a pharmaceutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable sait, solvaté, 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 spécifie compounds of the examples herein, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, and a pharmaceutically acceptable carrier or excipient.

Also provided is a pharmaceutical formulation comprising a pharmaceutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable sait, solvaté, 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 spécifie compounds of the examples herein, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, and a pharmaceutically acceptable carrier or excipient and a pharmaceutically effective amount of at least one additional active therapeutic agent.

Also provided are separate embodiments comprising a compound of Formula (I), Formula (II), or one of the spécifie compounds of the examples herein, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, for use in the treatment of a Pneumovirinae virus infection or a respiratory syncytial virus infection in a human.

Also provided are separate embodiments comprising a compound of Formula (I), Formula (II) or one of the spécifie compounds of the examples herein, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, for use as a médicament.

Also provided are separate embodiments comprising a method for manufacturing a médicament intended for treatment of a Pneumovirinae virus infection or a respiratory syncytial virus infection in a human, characterised in that a compound of Formula (I),

Formula (II), or one of the spécifie compounds of the examples herein, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, is used.

Also provided is a compound of Formula (I), or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, for the treatment of a Pneumovirinae virus infection or a respiratory syncytial virus infection in a human.

Also provided are separate embodiments comprising that a compound of Formula (II) or one of the spécifie compounds of the examples herein, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, for the treatment of a Pneumovirinae virus infection or a respiratory syncytial virus infection in a human.

Further provided is a compound as described in this spécification. Also provided is a pharmaceutical composition as described in this spécification. Also provided is a method of using a compound of Formula (I), as described in this spécification. Further provided is a method of making a compound of Formula (I), as described in this spécification.

Métabolites of the Compounds

Also falling within the scope herein are the in vivo metabolic products of the compounds described herein, to the extent such products are novel and unobvious over the prior art. Such products may resuit for example from the oxidation, réduction, hydrolysis, amidation, estérification and the like of the administered compound, primarily due to enzymatic processes. Accordingly, 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. Such products typically are identified by preparing a radiolabelled (e.g. ¹⁴C or ³H) compound, administering it parenterally in a détectable dose (e.g. greater than about 0.5 mg/kg) to an animal such as rat, mouse, guinea pig, monkey, or to man, allowing sufficient time for metabolism to occur (typically about 30 seconds to 30 hours) and isolating its conversion products from the urine, blood or other biological samples. These products are easily isolated since they are labeled (others are isolated by the use of antibodies capable of binding epitopes surviving in the métabolite). The métabolite structures are determined in conventional fashion, e.g. by MS or NMR analysis. In general, analysis of métabolites is done in the same way as conventional drug metabolism studies well-known to those skilled in the art. The conversion products, so long as they are not otherwise found in vivo, are useful in diagnostic assays for therapeutic dosing of the compounds even if they possess no HSV antiviral activity of their own.

Recipes and methods for determining stability of compounds in surrogate gastrointestinal sécrétions are known. Compounds are defined herein as stable in the gastrointestinal tract where less than about 50 mole percent of the protected groups are 5 deprotected in surrogate intestinal or gastric juice upon incubation for 1 hour at 37°C.

Simply because the compounds are stable to the gastrointestinal tract does not mean that they cannot be hydrolyzed in vivo. The prodrugs typically will be stable in the digestive system but may be substantially hydrolyzed to the parental drug in the digestive lumen, liver, lung or other metabolic organ, or within cells in general. As used herein, a prodrug is 10 understood to be a compound that is chemically designed to efficiently liberate the parent drug after overcoming biological barriers to oral delivery.

Abbreviations

Certain abbreviations and acronyms are used in describing the experimental details.

Although most of these would be understood by one skilled in the art, Table 1 contains a 15 list of many of these abbreviations and acronyms.

Table 1. List of abbreviations and acronyms.

Abbreviation	Meaning
Ac	acetate
ACN	acetonitrile
AIBN	azobisisobutyronitrile
Bn	benzyl
Bu	butyl
Bz	benzoyl
BzCI	benzoyl chloride
GDI	1,1 '-carbonyldiimidazole

DAST	diethylaminosulfur trifluoride
DCE	1,2-dichloroethane
DCM	dichloromethane
DMAP	4-dimethylamiopyridine
DMDO	dimethydioxirane
DMSO	dimethylsulfoxide
DMF	dimethylformamide
DMTrCI	4,4’-dimethoxytritylchloride
DMTr	4,4'-dimethoxytrityl
EDO	/V-(3-dimethylaminopropyl)-A/'-ethylcarbodiimide hydrochloride
Et	ethyl
Imid	imidazole
KOtBu	potassium tert-butoxide
LC	liquid chromatography
MCPBA	meta-chloroperbenzoic acid
Me	methyl
m/z	mass to charge ratio
MS or ms	mass spectrum
NIS	/V-iodosuccinimide
NMP	A/-methyl-2-pyrrolidone
Ph	phenyl

Ph3P	triphenylphosphine
PMB	para-methoxybenzyl
PMBCI	para-methoxybenzyl chloride
PhOC(S)CI	phenylchlorothionoformate
(PhO)3PMel	methyltriphenoxyphosphonium iodide
Pyr	pyridine
RT	room température
TBAF	tetrabutylammonium flouride
TBS	terf-butyldimethylsilyl
TBSCI	terï-Butyldimethylsilyl chloride
tmsn3	trimethylsilyl azide
TEA	triethylamine
TES	triethysilane
TFA	trifluoroacetic acid
THF	tetrahydrofuran
TMS	trimethylsilyl
TMSCI	trimethylsilyl chloride
Ts	4-toluenesulfonyl
TsOH	tosylic acid
δ	parts per million referenced to residual non-deuterated solvent peak

General Schemes

S1a

NIS

S1b

Scheme 1.

Scheme 1 shows a general synthesis of intermediates beginning with an iodination reaction (e.g. NIS) to generate nucleobase S1b.

hbf₄

NaNO₂

NIS

S2a S2b S2c

Scheme 2.

Scheme 2 shows a general synthesis of intermediates beginning with a fluorination reaction (e.g. HBF4, NaNÛ2) in a manner similar to that described in WO2012037038A1 to afford intermediate S2b. Intermediate S2b can then be iodinated (e.g. NIS) to generate nucleobase S2c.

BnÔ OBn

S3a

2) Et₃SiH BF₃-Et₂O

1) n-BuLi [-CH₂SiMe₂CI]₂

BnÔ ÔBn

2) HCO₂H, Pd/C

1) BzCI, Pyr; NH4OH

NHBz

HO ÔH

S3d

S3b S3c

1) DMTrCI, Pyr

2) TBSCI, Imid

3) TsOH

1)(PhO)₃PMel

2) KOfBu

2) lnBr₃ TMSCN

3) CsF

1) DMDO

NH₂Me

HÔ ÔH

S3h

Scheme 3.

Scheme 3 shows a general synthesis of compounds beginning with a lithium-halogen exchange (e.g. n-BuLi, [-CH2SiMe2CI]2) reaction with an appropriate nucleobase S3b, followed by addition to the lactone S3a. Réduction of the pendant T hydroxyl group under Lewis acidic conditions (e.g. BF₃Et₂O, EtsSiH) generates intermediate S3c. Intermediate S3c can first be protected (e.g. BzCI, Pyr; NH4OH) at the nitrogen functionality, and the benzyl groups can then be removed under reducing conditions (e.g. HCO2H, Pd/C, BCI3, BBr₃) to afford intermediate S3d. A sequence involving first sélective 5' hydroxyl protection (e.g. DMTrCI), then 2' and 3' hydroxyl protection (e.g. TBSCI), followed by sélective removal of the 5' hydroxyl protecting group under acidic conditions (e.g. TsOH) furnishes intermediate S3e. The 5' hydroxyl group can then be converted to the corresponding iodide (e.g. (PhO)₃PMel), which is then exposed to basic conditions (i.e. KOfBu) to effect an élimination reaction generating intermediate S3f. Oxidation of the olefin S3f (e.g. DMDO) followed by treatment with an appropriate nucleophile (e.g. TMSCN) under Lewis acidic conditions (e.g. lnBr₃) and removal ofthe hydroxyl protecting groups (e.g. CsF) affords intermediate S3g. Removal of the nitrogen protecting group (e.g. NH₂Me) yields the final compounds of the type S3h.

2) lnBr₃ TMSN₃

3) CsF

1) DMDO

Scheme 4.

Scheme 4 shows a general synthesis of compounds beginning with oxidation of the olefin S3f (e.g. DMDO) followed by treatment with an appropriate nucleophile (e.g. TMSN₃) under Lewis acidic conditions (e.g. lnBr₃) in a manner similar to that described in J. Med. Chem. 2007, 50, 5463-5470. Removal of the hydroxyl protecting groups (e.g. CsF) then affords intermediate S4a. Removal ofthe nitrogen protecting group (e.g. NH₂Me) yields the final compounds of the type S4b.

NH₂Me

Scheme 5.

Scheme 5 shows a general synthesis of compounds beginning with oxidation of the olefin S3f (e.g. DMDO) in the presence of the appropriate alcohol HOR^a followed by removal 5 of the hydroxyl protecting groups (e.g. CsF) to afford intermediate S5a. Removal of the nitrogen protecting group (e.g. NH₂Me) yields the final compounds of the type S5b.

S3f

Scheme 6.

Scheme 6 shows a general synthesis of compounds beginning with oxidation of the 10 olefin S3f (e.g. DMDO) followed by treatment with an appropriate nucleophile (e.g.

(HC^OsAI) in a manner similar to that described in Nucleosides, Nucléotides, and Nucleic Acids 2005, 24, 343-347. Removal of the hydroxyl protecting groups (e.g. CsF) then affords intermediate S6a. Removal of the nitrogen protecting group (e.g. NH₂Me) yields the final compounds of the type S6b. Elaboration of the final compound through hydrogénation conditions (e.g. H₂, Pd/C or Lindlar’s conditions) can selectively afford the final compounds of the type S6c and S6d respectively. Elaboration of the final compound S6d through cyclopropanation conditions (e.g. CH₂N₂) can yield the final compounds of the type S6e.

HÔ F

S7a

1)TMSCI, Pyr;

BzCI, Pyr;

NH₄OH

2) DMTrCI Pyr

HÔ F

S7b

NHBz

NHBz

2) CH₂O, NaOH

3) NaBH₄

1) EDCI*HCI Pyr, TFA, DMSO

S7f

1) DMTrCI, Et₃N

EDCI«HCI

S7g

Pyr, TFA DMSO

NHBz

[Ph₃PCH₂Br]⁺Br

KOtBu

S7h

2) TBSCI, Imid.

3) TsOH

Scheme 7.

Scheme 7 shows a general synthesis of compounds beginning with a synthetic sequence to protect the nitrogen (e.g. TMSCI, Pyr; BzCI, Pyr; NH₄OH) of intermediate S7a, 5 synthesized in a similar manner as described in WO2012037038A1. Sélective protection of the 5' hydroxyl group (e.g. DMTrCI) then generates intermediate S7b. Protection of the 2' hydroxyl group (e.g. TBSCI) followed by removal of the 5' hydroxyl group under acidic conditions (e.g. TsOH) furnishes intermediate S7e. Conversion of the 5' hydroxyl group to the aldéhyde under oxidative conditions (e.g. EDCIHCI, Pyr, TFA, DMSO) followed by condensation of the corresponding enolate with formaldéhyde and réduction (e.g. NaBH₄) yields intermediate S7f. Sequential sélective protection of the hydroxyl moieties with orthogonal protecting groups (e.g. DMTrCI and TBSCI) followed by removal of the more labile protecting group under acidic conditions (e.g. TsOH) then affords intermediate S7g. Conversion of the hydroxyl group to the aldéhyde under oxidative conditions (e.g. EDCI HCI, 15 Pyr, TFA, DMSO) generates intermediate S7h. Elaboration of the aldéhyde S7h to the haloolefin intermediate S7i can be effected under Wittig olefination conditions (e.g.

[Ph₃PCH₂Br]⁺Br, KOtBu). An élimination reaction under basic conditions (e.g. KOtBu) generates the alkyne, and removal of the hydroxyl protecting groups (e.g. TBAF) and nitrogen protecting group (e.g. NH₄OH) yields the final compounds of the type S7j.

1) νη₂οη·ηοι

2) CDI

3) NH₄OH

4) HF.Pyr

HÔ F

S8a

Scheme 8.

Scheme 8 shows a general synthesis of compounds beginning with oxime formation (e.g. NH2OH HCI), followed by conversion of the oxime to a nitrile group (e.g. GDI).

Removal of the nitrogen protecting group (e.g. NH4OH), and hydroxyl protecting groups (e.g. HF Pyr) then yields the final compounds of the type S8a.

2) TBAF

3) ΝΗ4ΟΗ

1)[Ph₃PCH₃]⁺BrKOtBu

HÔ F

S9a

Scheme 9.

Scheme 9 shows a general synthesis of compounds beginning with élaboration of the 10 aldéhyde S7h to the olefin with Wittig olefination conditions (e.g. [Ph3PCH₃]⁺Br, KOtBu).

Removal of the hydroxyl protecting groups (e.g. TBAF), and nitrogen protecting group (e.g.

NH4OH) yields the final compounds of the type S9a. Reducing conditions (e.g. H2, Pd/C) then can generate the final compounds of the type S9b, and cyclopropanation conditions (e.g. CH2N2) can generate the final compounds of the type S9c.

Scheme 10.

Scheme 10 shows a general synthesis of compounds beginning with an Appel reaction (e.g. Ph₃P, COU) to convert the hydroxyl group into a chloride. Removal of the hydroxyl protecting groups (e.g. TBAF), and nitrogen protecting group (e.g. NH₄OH) yields the final compounds of the type S10a.

Scheme 11.

Scheme 11 shows a general synthesis of compounds beginning with protection of the free hydroxyl group of intermediate S7g with a labile protecting group (e.g. PMBCI, K2CO3). Sélective removal of the 2' and 5' silyloxy protecting groups (e.g. TBAF), followed by reprotection with robust protecting groups (e.g. BnBr, NaH), and removal of the labile hydroxyl protecting group under acidic conditions (e.g. AcOH) affords intermediate S11a. Conversion ofthe hydroxyl group to the fluorine (e.g. DAST) followed by removal of the hydroxyl protecting groups (e.g. BBr₃), and nitrogen protecting group (e.g. NH₄OH) yields the final compounds of the type S11 b.

1) (PhO)₃PMel

2) KOfBu

Scheme 12.

Scheme 12 shows a general synthesis of compounds beginning with conversion of the 5' hydroxyl group to the corresponding iodide (e.g. (PhO)₃PMel), which is then treated with basic conditions (i.e. KOfBu) to effect an élimination reaction generating intermediate S12a. Oxidation of the olefin S12a (e.g. DMDO) followed by treatment with an appropriate nucleophile (e.g. TMSN3) under Lewis acidic conditions (e.g. SnCk) in a manner similar to that described in J. Med. Chem. 2007, 50, 5463-5470, and removal of the hydroxyl protecting groups (e.g. CsF) and nitrogen protecting group (e.g. NH₄OH) yields the final compounds of the type S12b.

Scheme 13.

Scheme 13 shows a general synthesis of compounds beginning with oxidation of the olefin S12a (e.g. DMDO) in the presence of the appropriate alcohol HOR^a and removal of the hydroxyl protecting groups (e.g. CsF). Removal of the nitrogen protecting group (e.g. NH₂Me) yields the final compounds of the type S13a.

2) (TMS)₃SiH, AIBN

1) PhOC(S)CI DMAP

S14a

1) TBAF

2) NH4OH

HÔ F S14b

Scheme 14.

Scheme 14 shows a general synthesis of compounds beginning with xanthate formation (e.g. PhOC(S)CI, DMAP) followed by a Barton-McCombie deoxygenation reaction (e.g. (TMSjsSiH, AIBN) to generate intermediate S14a. Removal of the hydroxyl protecting groups (e.g. TBAF), and nitrogen protecting group (e.g. NH4OH) yields the final compounds 15 of the type S14b.

,.»OAc

BnÔ OAc ²) NIS, Bu₄NI

3) BnBr, AgjO

S15a

1) K₂CO₃, MeOH

Scheme 15.

Scheme 15 shows a general synthesis of compounds beginning with intermediate S15a prepared in a manner similar to that described in Biosci. Biotech. Biochem. 1993, 57,

1433-1438. Removaî of the acetate protecting groups using hydrolytic 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. Lithiumhalogen exchange (e.g. n-BuLi, [-CH2SiMe2CI]2) with an appropriate nucleobase S3b and addition to the lactone S15b, followed by réduction of the T hydroxyl group under Lewis acidic conditions (e.g. BF₃-Et2O, EtSiH), and depratection (e.g. H₂, Pd black) yields the final compounds of the type SI 5c.

NHBz

TBSÔ ÔTBS

S3e

1) EDCI.HCI

Pyr, TFA, DMSO

2} CH₂O, NaOH

3) NaBHj

TBSÔ OTBS

S 16a

1) DMTrCI, Et₃N

2) TBSCI, imtd.

3) TsOH

NHBz

TBSO ÔTBS

1)Ph₃P, CCI₄

S16b

2) TBAF

3) NH4OH

S16c

Scheme 16.

Scheme 16 shows a general synthesis of compounds beginning with conversion of the 5' hydroxyl group to the aldéhyde under oxidative conditions (e.g. EDCIHCI, Pyr, TFA, DMSO) followed by condensation of the corresponding enolate with formaldéhyde and réduction (e.g. NaBH₄) to yields intermediate S16a. Sequential sélective protection of the hydroxyl moieties with orthogonal protecting groups (e.g. DMTrCI and TBSCI) followed by removaî of the more labile protecting group under acidic conditions (e.g. TsOH) then affords intermediate S16b. An Appel reaction (e.g. PhaP, CCI₄) then can convert the hydroxyl group into a chloride, and removaî of the hydroxyl protecting groups (e.g. TBAF), and nitrogen protecting group (e.g. NH₄OH) yields the final compounds of the type S16c.

NHBz

TBSÔ ÔTBS

S16b

2) TBAF

3) BnBr, Na H

4) AcOH

1)PMBCi, K₂CO₃

NHBz

BnÔ ÔBn

S17a

1) DAST

2) BBr₃

3) NH^OH nh₂

hÔ ÔH

S17b

Scheme 17.

Scheme 17 shows a general synthesis of compounds beginning with protection of the free hydroxyl group of intermediate S16b with a labile protecting group (e.g. PMBCI, K2CO3). Sélective removal of the 2’, 3', and 5' silyloxy protecting groups (e.g. TBAF), followed by reprotection of with robust protecting groups (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 the fluorine (e.g. DAST) followed by removal of the hydroxyl protecting groups (e.g. BBr₃), and nitrogen protecting group (e.g. NH4OH) yields the final compounds ofthe type S17b.

S18b

NCS

S18a

S18c

Selectfluor

Scheme 18.

Scheme 18 shows a general synthesis of compounds through appropriate electrophilic halogénation reactions of intermediate S18a to afford the final compounds of the type S18b (e.g. NCS), S18c (e.g. NIS), and S18d (e.g. Selectfluor).

no R³ Hd k³ _Hd k³

S18c S19a S19b

Scheme 19.

Scheme 19 shows a general synthesis of compounds beginning with a crosscoupling reaction (e.g. Zn(CN)₂, Pd(PtBu)₃) to yield the final compounds of the type S19a.

Compound S19a can then be elaborated through a hydrolysis reaction ofthe nitrile (e.g H2O2, NH4OH, H₂O) to afford compounds of the type S19b.

HÔ R^{3 s18c} S20a

R = H, TMS, Me, cyclopropyl, Ph

Scheme 20.

Scheme 20 shows a general synthesis of compounds beginning with a Sonogashira reaction (e.g. Cul, PdCl2(PPh₃)₂) to yield the final compounds ofthe type S20a.

nh₂

hô k³

S18c

Suzuki reaction

nd r³

S21a

R = H, Me, Ph

Scheme 21.

Scheme 21 shows a general synthesis of compounds beginning with a crosscoupling reaction (e.g. Pd(dppf)Ch, Cs2CO₃) to yield the final compounds ofthe type S21a

Hrf R³

S22a

HO-p'°

HO' 0-^° R.² ^2

P(O)CI₃; HO

[nBu₃NH]₂P₂O₇H₂ ^H° ° \,,O, j ^N N R¹ r⁴vj

HO R³

S22b

Scheme 22.

Scheme 22 shows a general synthesis of compounds involving synthesis of phosphorylated analogs of the type S22b.

S22a

R⁵O P LG i „ fBuMgCI

DMF, THF

S23a

R² NH₂

R’

HÔ

R³

S23b

Scheme 23.

Scheme 23 shows a general synthesis of compounds involving synthesis of phosphorylated analogs of the type S23b.

P LG

LG

S24a ) Base

2) Oxi dation

S 24b

Scheme 24.

Scheme 24 shows a general synthesis of compounds involving synthesis of phosphorylated analogs of the type S24b.

Expérimentais

1a

NIS

DMF

1b

Intermediate 1 b.

To a solution of intermediate 1a (50 mg, 373 mmol) in DMF (1 mL) was charged Niodosuccinimide (84 mg, 373 mmol) as a solid at RT. After 1.5 h, the reaction mixture was diluted 1M NaOH solution (10 mL), and the resulting siurry was stirred at RT. After 1 h, the 15 solids were collected by vacuum filtration and dried under reduced pressure to afford intermediate 1b.

Ή NMR (400 MHz, DMSO-d₆) δ 7.89 (s, 1H), 7.78 (br-s, 1H), 6.98 (d, J=4.4 Hz, 1H), 6.82 (d, J = 4.4 Hz, 1 H), 3.30 (br-s, 1H).

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 x 4.6 mm

Solvents: Acetonitrile with 0.1 % acetic acid, Water with 0.1 % acetic acid; Gradient: 0 min2.0 min 2-100% ACN, 2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN, 3.2 min-3.5 min 2% ACN at 2pl/min.

HPLC: t_r = 1.536 min; HPLC system: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at2 mL/min.

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

n-Butyllithium (2.5M in hexanes, 34.4 mL, 86.0 mmol) was added rapidly to a suspension of 7-iodopyrrolo[1,2-f][1,2,4]triazin-4-amine 1b (6.84 g, 26.3 mmol) and 1,2bis(chlorodimethylsilyl)ethane (5.66 g, 26.3 mmol) in THF (200 mL) at -78 °C under an argon atmosphère. Over the course of the addition the internai température 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 added rapidly via cannula. After 1 h, the reaction mixture was quenched v/ith acetic acid (15 mL), and the resulting mixture was allowed to warm to RT. The resulting mixture was diluted with ethyl acetate (800 mL) and was washed with saturated aqueous sodium bicarbonate solution (500 mL) and brine (500 mL). The organic layer was dried over anhydrous sodium sulfate, and was concentrated under reduced pressure. The crude residue was purified via SiO₂ column chromatography (220 g SiO₂ Combiflash HP Gold Column, 0-100% ethyl acetate/hexanes) to afford intermediate 1d.

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: Kinetex2.6p XB-C18 100A, 50 x4.6 mm

Solvents: Acetonitrile with 0.1 % acetic acid, Water with 0.1 % acetic acid; Gradient: 0 min1.5 min 2-100% ACN, 1.5 min-2.2 min 100% ACN, 2.2 min-2.4 min 100%-2% ACN, 2.4 min2.5 min 2% ACN.

HPLC: t_r = 3.442 min; HPLC system: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at 2 mL/min.

TLC: eluent: ethyl acetate, Rr= 0.5 (UV)

BF₃OEt

Et₃SiH

CH₂CI₂

1e

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.

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 added boron trifluoride diethyl etherate (1.59 ml, 12.9 mmol) slowly via syringe at 0 °C under an argon atmosphère. After 2 h, the reaction mixture was slowly diluted with saturated aqueous sodium bicarbonate solution (100 mL), and the resulting mixture was extracted with ethyl acetate (2 x 150 mL), was dried over anhydrous sodium sulfate, and was concentrated under reduced pressure. The crude residue was purified via S1O2 column chromatography (24 g S1O2 Combiflash HP Gold Column, 0-100% ethyl acetate/hexanes) to afford intermediate 1e.

¹H NMR (400 MHz, CDCI3) δ 7.88 (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).

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 x 4.6 mm Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% aceticacid; Gradient: 0 min-1.5 min 2100% ACN, 1.5 min-2.2 min 100% ACN, 2.2 min-2.4 min 100%-2% ACN, 2.4 min-2.5 min 2% ACN at 2pl/min.

HPLC: t_R = 3.596 min; HPLC system: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at2 mL/min.

TLC: eluent: ethyl acetate, Rf= 0.3 (UV)

Pyr; NH₄OH

BnO ÔBn 1f

BnÔ ÔBn 1e

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.

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 RT under an argon atmosphère. After 1 h, additional benzoyl chloride (1.69 ml, 14.68 mmol) was added slowly. After 19 h, water (20 mL) was added slowly and the reaction mixture became slightly cloudy. Ammonium Hydroxide (~10 mL) was then added slowly until the reaction mixture was basic at pH=10. After 1 h, water (150 mL) was added dropwise via addition tunnel and white solids slowly began to precipitate from the reaction mixture over the course of the addition. The resulting mixture was stirred for 24 h and the white solids were collected by vacuum filtration and were dried azeotropically from toluene to afford intermediate 1f.

¹H NMR (400 MHz, CDCI3) δ 8.23 (brs, 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), 4.634.44 (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).

LC/MS: îr = 1.91 min, MS m/z = 641.18 [M+1]; LC system: Thermo Accela 1250 UHPLC

MS system: Thermo LCQ Fleet; Column: Kinetex2.6p XB-C18 100A, 50 x 4.6 mm Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min-1.5 min 2100% ACN, 1.5 min-2.2 min 100% ACN, 2.2 min-2.4 min 100%-2% ACN, 2.4 min-2.5 min 2% ACN at2pl/min.

TLC: eluent: 50% ethyl acetate in hexanes, Rf= 0.6 (UV)

1g

Intermediate 1g - N-(7-((2S,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran2-yl)pyrrolo[1 _;2-f][1,2,4]triazin-4-yl)benzamide.

Ethanol (68.5 mL) and formic acid (51.7 mL, 1.37 mol) were added sequentially to a mixture of intermediate 1f (4.39 g, 6.85 mmol) and palladium on carbon (10% by wt, 2.2 g) at RT under an argon atmosphère. After 3 d, 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 x 20 mL) to afford intermediate 1g, which was used directly in the next step without further purification.

¹H NMR (400 MHz, CD₃OD) 0 8.15 (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).

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 x 4.6 mm

Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min1.5 min 2-100% ACN, 1.5 min-2.2 min 100% ACN, 2.2 min-2.4 min 100%-2% ACN, 2.4 min2.5 min 2% ACN at 2pl/min. ;

HPLC: t_R = 2.055 min; HPLC system: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at 2 mL/min.

NHBz NHBz /^Ύ^^ΝN

HO^_oW_n; DMTrCI^ DMTrO^o \ / Pyr \/

HO ÔH ^H(5 ÔH

1g

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

4,4'-Dimethoxytrityl chloride (2.23 g, 6.59 mmol) was added as a solid in one portion to a solution of intermediate 1g (2.44 g, 6.59 mmol) in pyridine (32.5 mL) at RT. After 5.5 h, the reaction mixture was diluted with ethyl acetate (300 mL) and the resulting mixture was washed with brine (3 x 200 mL). The organic layer was concentrated under reduced pressure, and the crude residue was purified via S1O2 column chromatography (80 g S1O2 Combiflash HP Gold Column, 0-100% ethyl acetate/hexanes) to afford intermediate 1h.

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 x 4.6 m;

Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% aceticacid; Gradient: 0 min1.5 min 2-100% ACN, 1.5 min-2.2 min 100% ACN, 2.2 min-2.4 min 100%-2% ACN, 2.4 min2.5 min 2% ACN at2pl/min.

HPLC: t_R = 4.270 min; HPLC System: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at 2 mL/min.

TLC: eluent: 50% ethyl acetate in hexanes, Rf= 0.15 (UV)

NHBz NHBz /V^N /^f^N

DMTrO—y _o ^TBSCI , DMTrO y o ^ⁿ^ n \ / Imid, DMF \ /

HÔ ÔH TBSÔ ÔTBS

1h 1Î

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

tert-Butyldimethylsilyl chloride (2.47 g, 16.4 mmol) was added to a solution of intermediate 1h (1.84 g, 2.74 mmol) and imidazole (2.23 g, 32.8 mmol) in Λ/,Λ/dimethylformamide (28.2 mL) at RT. After 17 h, saturated aqueous sodium bicarbonate solution (500 mL) was added slowly to the reaction mixture. The resulting mixture was extracted with ethyl acetate (500 mL), and the organic layer was washed with brine (2 x 400 mL), was dried over anhydrous sodium sulfate, and was concentrated under reduced pressure. The crude residue was purified via SiO₂ column chromatography (80 g SiO₂ Combiflash HP Gold Column, 0-100% ethyl acetate/hexanes) to afford intermediate 1i.

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 x 4.6 mm

Solvents: Acetonitrile with 0.1% aceticacid, Water with 0.1% aceticacid; Gradient: 0 min2.0 min 2-100% ACN, 2.0 min-3.55 min 100% ACN, 3.55 min-4.2 min 100%-2% ACN at 2pl/min

HPLC: t_R = 5.724 min; HPLC system: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at2 mL/min.

TLC: eluent: 50% ethyl acetate in hexanes, Rf= 0.75 (UV)

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)benzamicle.

A solution of p-toluenesulfonic acid monohydrate (509 mg, 2.67 mmol) in methanol (3.7 mL) was slowly added to a solution of intermediate 1i (2.41 g, 2.67 mmol) in dichloromethane (22.3 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 x 100 mL). The combined organic extracts were dried over anhydrous sodium sulfate, and were concentrated under reduced pressure. The crude residue was purified via SiO₂ column chromatography (120 g SiO₂ Combiflash HP Gold Column, 0-100% ethyl acetate/hexanes) to afford intermediate 1j.

¹H NMR (400 MHz, CDCI3) δ 8.72 (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, 1 H), 5.75 (d, J = 8.2 Hz, 1 H), 5.28 (dd, J = 8.1, 4.7 Hz, 1 H), 4.81 (d, J = 5.0 Hz, 1 H), 4.70 - 4.63 (m, 1 H), 4.44 (d, J = 12.3 Hz, 1 H), 4.24 (d, J = 12.4 Hz, 1 H), 1.48 (s, 9H), 1.30 (s, 9H), 0.65 (s, 3H), 0.64 (s, 3H), 0.41 (s, 3H), 0.00 (s, 3H).

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 x 4.6 mm

Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min2.0 min 2-100% ACN, 2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN, 3.2 min-3.5 min 2% ACN at 2pl/min.

HPLC: t_R = 5.622 min; HPLC system: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at 2 mL/min.

TLC: eluent: 50% ethyl acetate in hexanes, Rf= 0.55 (UV)

NHBz NHBz

Λΐ N ^H0 VoJ-'V ^N N^ \_J DMF \_J

TBSÔ OTBS TBSÔ OTBS

1j 1k

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.

Intermediate 1j (1.19 g, 1.99 mmol) was added to a solution of methyltriphenoxyphosphonium iodide (0.99 g, 2.19 mmol) in DMF (9.9 mL) at RT. 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 was washed with brine (3 x 100 mL). The organic layer was dried over anhydrous sodium sulfate, and was concentrated under reduced pressure. The crude residue was purified via S1O2 column chromatography (80 g S1O2 Combiflash HP Gold Column, 0-100% ethyl acetate/hexanes) to afford intermediate 1k.

¹H NMR (400 MHz, CDCb) δ 8.21 (brs, 1H), 7.61 (brt, J =7.2 Hz, 1H), 7.53 (brt, J =7.5 Hz, 3H), 7.05 (br s, 1 H), 5.44 (d, J = 4.5 Hz, 1 H), 4.52 (t, J = 4.3 Hz, 1 H), 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).

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 x 4.6 mm

Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min2.0 min 2-100% ACN, 2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN, 3.2 min-3.5 min 2% ACN at 2pl/min.

HPLC: t_R = 5.837 min; HPLC system: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at2 mL/min.

TLC: eluent: 20% ethyl acetate in hexanes, Rf= 0.45 (UV)

1k 11

Intermediate 11 - N-(7-((2S,3S,4S)-3,4-bis(tert-butyldimethylsilyloxy)-5methylenetetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide.

Potassium f-butoxide (700 mg, 6.24 mmol) was added to a solution of intermediate 1 k (1.77 g, 2.5 mmol) in pyridine (25 mL) at RT. After 2 h, the reaction mixture was diluted with saturated aqueous sodium bicarbonate solution (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), was dried over anhydrous sodium sulfate, and was concentrated under reduced pressure. The crude residue was purified via SiO2 column chromatography (40 g S1O2 Combiflash HP Gold Column, 0-100% ethyl acetate/hexanes) to afford intermediate 11.

LC/MS: îr = 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 x 4.6 mm

Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid

Gradient: 0 min-2.0 min 2-100% ACN, 2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN, 3.2 min-3.5 min 2% ACN at 2pl/min.

HPLC: îr = 5.750 min; HPLC system: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at 2 mL/min.

TLC: eluent: 50% ethyl acetate in hexanes, Rf= 0.20 (UV)

DMDO acetone

TBSÔ ÔTBS

TBSÔ ÔTBS

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)benzamicle.

DMDO (0.07M solution in acetone, 13.8 mL, 0.964 mmol) was added to a solution of intermediate 11 (560 mg, 0.964 mmol) in acetone (4.82 mL) at 0 °C. After 10 min, the reaction mixture was concentrated under reduced pressure was dried azeotropically with toluene (2x1 mL) to afford 1 m that was used immediately in the next step without further purification.

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 x 4.6 mm

Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid

Gradient: 0 min-2.0 min 2-100% ACN, 2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN, 3.2 min-3.5 min 2% ACN at 2pl/min.

TMSCN

TMSOlnBr₃ CH₂CI₂

TBSO OTBS

TBSÔ ÔTBS

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-4yl)benzamide.

To a solution of crude intermediate 1m (~592 mg, ~0.964 mmol) and trimethylsilyl cyanide (640 μΙ, 4.80 mmol) in dichloromethane (19.2 mL) was added indium (III) bromide (681 mg, 1.92 mmol) at 0 °C under an argon atmosphère. After 4.5 h, the reaction mixture was quenched with saturated aqueous sodium bicarbonate solution (6 mL) and was allowed to warm to RT. The resulting mixture was partitioned between dichloromethane (20 mL) and saturated aqueous sodium bicarbonate solution (20 mL). The phases were split and the aqueous layer was extracted with dichloromethane (20 mL). The combined organic layers were dried over anhydrous sodium sulfate and were concentrated under reduced pressure to afford intermediate 1n (1:1 diastereomeric mixture) (710 mg) that was used directly in the next step without further purification.

LC/MS: first eluting isomer t_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: Kinetex2.6p XB-C18 100A, 50 x4.6 mm

Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min2.0 min 2-100% ACN, 2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN, 3.2 min-3.5 min 2% ACN at 2pl/min.

CsF

DMF

1o

Intermediate 1o - N-(7-((2S,3R,4S)-5-cyano-3,4-dihydroxy-5(hydroxymethyl)tetrahydrofuran-2-yl)pyrro!o[1,2-f][1,2,4]triazin-4-yl)benzamice.

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

LC/MS: first eluting isomer t_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 x 4.6 mm

Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min2.0 min 2-100% ACN, 2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN, 3.2 min-3.5 min 2% ACN at2pl/min.

MeNH₂

MeOH

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.

Methylamine (40% in water, 0.3 mL) was added to a solution of crude intermediate

1o in methanol (1 mL) at RT. After 2.5 h, the reaction mixture was concentrated under reduced pressure, and was directly purified by preparatory HPLC (Phenominex Luna 5u C18 100Â 100 x 30 mm column, 5-15% acetonitrile/water gradient, 25 min). The fractions containing the desired product and the 4’ anomer were combined and were concentrated under reduced pressure. The 4’ anomers were then separated by preparatory HPLC (Phenominex Luna 5u C18 100Â 100 x 30 mm column, 5-15% acetonitrile/water gradient, 25 min). The fractions containing the desired product were combined and were lyophulized to afford Example 1.

¹H NMR (400 MHz, CD₃OD) δ 7.79 (s, 1 H), 6.85 (d, J = 4.5 Hz, 1 H), 6.76 (d, J = 4.5 Hz, 1 H), 5.45 (d, J = 5.9 Hz, 1 H), 4.59 (t, J = 5.7 Hz, 1 H), 4.40 (d, J = 5.6 Hz, 1 H), 3.88 (d, J = 12.0 Hz, 1H), 3.80 (d, J= 12.0 Hz, 1H).

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 x 4.6 mm

Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min1.5 min 2-100% ACN, 1.5 min-2.2 min 100% ACN, 2.2 min-2.4 min 100%-2% ACN, 2.4 min2.5 min 2% ACN at 2μl/min.

HPLC: t_R = 0.377 min; HPLC system: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at 2 mL/min.; HPLC: t_R = 6.643 min; HPLC system: Agilent 1100 sériés.; Column: Luna 5μ C18(2) 110A, 250 x 4.6 mm; Solvents: Acetonitrile, Water; Gradient: 5-15% ACN over 10 min at 2 mL/min

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

To an N₂ purged flask was added intermediate 2a, (2R,3R,4R,5S)-5-(4aminopyrrolo[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 3 times), 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 an N2 atmosphère for 1 h. Benzoyl chloride (675 pL, 5.82 mmol) was added dropwise and the reaction mixture was stirred for 1h. An additional portion of benzoyl chloride (100 pL) 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(_aq) (8 mL) was added in one portion and allowed to stir for 15 min at which point the bis-Bz product was converted to the desired product. The solvents were removed under reduced pressure, and then the residue was coevaporated with CH3OH. Intermediate 2b was isolated after purification by silica gel chromatography, using an eluent ramp of 50%-100% EtOAc in hexanes.

Ή NMR (400 MHz, DMSO-c/₆) δ 8.26 - 7.91 (m, 2H), 7.88 - 7.79 (m, 1 H), 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.Q Hz, 1 H), 4.21 - 4.04 (m, 1 H), 3.82 (dd, J = 8.0, 3.9 Hz, 1 H), 3.71 (ddd, J = 12.3, 5.6, 2.6 Hz, 1 H), 3.52 (ddd, J = 12.2, 5.7, 4.5 Hz, 1H).

¹⁹F NMR (376 MHz, DMSO-d₆) δ -196.33 (dt, J = 55.1, 22.5 Hz).

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 2.6p C18 100A, 50 x 3.00 mm

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid

Gradient: 0 min-1.4 min 2-100% ACN, 1.4 min-1.80 min 100% ACN, 1.8 min-1.85 min 100%-2% ACN, 1.85 min-2 min 2% ACN.

2b

DMTrCI, Pyr

2c

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

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 an N2 atmosphère. 4,4’Dimethoxytrityl chloride (1.71 g, 5.0 mmol) was added in one portion at room température and allowed to stir for 2 h. Ethanol (2 mL) was added and the resulting solution was stirred for 5 min. Solvents were removed under reduced pressure. The residue was purified by silica gel chromatography, using an eluent ramp of 0%-100% EtOAc in hexanes, to afford intermediate 2c.

¹H NMR (400 MHz, DMSO-d₆) δ 8.28 - 8.02 (m, 2H), 7.61 (t, J = 7.5 Hz, 1 H), 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).

¹⁹F NMR (376 MHz, DMSO-d₆) δ -195.58 (dt, J = 52.1, 24.7 Hz).

LC/MS: îr = 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 x 3.00 mm

Solvents: Acetonitrile with 0.1 % formic acid, Water with 0.1 % formic acid

Gradient: 0 min-1.4 min 2-100% ACN, 1.4 min-1.80 min 100% ACN, 1.8 min-1.85 min 100%-2% ACN, 1.85 min-2 min 2% ACN.

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.

To a solution of intermediate 2c (1.47 g, 2.18 mmol) in DMF (8 mL), prepared under an N₂ atmosphère, was added imidazole (251 mg, 3.70 mmol), followed by tert butylchlorodimethylsilane (492 mg, 3.27 mmol). The solution was allowed to stir at room température for 16 h. The solution was diluted with H2O (5 mL) and then the solvents were removed under reduced pressure. The residue was partitioned between EtOAc and H₂O. The layers were separated and the organic phase was then washed with brine. The organics were dried over Na2SO4, which was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude material was used as is in the next step.

The crude material was dissolved in CHCI3 (15 mL) and cooled to 0 °C. pToluenesulfonic acid hydrate (414 mg, 2.18 mmol) dissolved in CH3OH (6 mL) was added dropwise to the mixture and was allowed to stir for 15 min. The reaction was quenched with sat. NaHCO₃₍aq). The organics were washed with brine and dried over Na₂SO4, filtered and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography, using an eluent ramp of 0%-50% EtOAc in hexanes, to afford intermediate 2d.

¹H NMR (400 MHz, DMSO-d₆) δ 8.21 - 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, 1 H), 5.07 (dt, J = 54.6, 3.8 Hz, 1 H), 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).

¹⁹F NMR (376 MHz, DMSO-cfe) δ -198.05 (dt, J = 54.2, 19.8 Hz).

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: Kinetex2.6p C18 100A, 50 x 3.00 mm

Solvents: Acetonitrile with 0.1 % formic acid, Water with 0.1 % formic acid

Gradient: 0 min-1.4 min 2-100% ACN, 1.4 min-1.80 min 100% ACN, 1.8 min-1.85 min 100%-2% ACN, 1.85 min-2 min 2% ACN.

2d

1. DMSO, Toi, EDCI, Pyr, TFA

2. Formaldéhyde, NaOH Dioxane, H2O

3. NaBH₄, EtOH

NHBz

TBSÔ F

2e

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

To solution of intermediate 2d (856 mg, 1.75 mmol) in toluene (4 mL), and DMSO (6 mL), prepared under an N2 atmosphère, was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (504 mg, 2.63 mmol). To this mixture was added pyridine (150 pL), and TFA (70 pL) and the mixture was stirred at RT for 15 min. Additional EDCI (100 mg) and pyridine (100 pL) was added and the mixture was stirred for an additional 45 min. The reaction was quenched with H2O (10 mL) and CH2CI2 (10 mL). The organics were washed with brine and dried over Na₂SO4, filtered, and the solvent was removed under reduced pressure to afford the crude aldéhyde, which was used as is for the next step.

The crude aldéhyde was dissolved in dioxane (5 mL) and 37% formaldehyde(_aq) (925 pL) was added followed by 2N NaOH_(aq) (925 pL). After stirring at room température for 3 h, the reaction was quenched with AcOH, diluted with EtOAc, and washed with H₂O. The organics were dried over Na₂SO4, filtered, and the solvent was removed under reduced pressure to afford the crude aldol product, which was carried forward as is to the next step.

The crude aldol product was dissolved in EtOH (9 mL) under an N2 atmosphère and cooled to 0 °C. NaBH₄ (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 CH2CI2 and washed with a 1:1 solution of water and sat. NaHCO₃(_aq). The organic phase was dried over Na₂SO₄, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography, using an eluent ramp from 0%-100% EtOAc in hexanes, to afford intermediate 2e.

¹H NMR (400 MHz, DMSO-d₆) δ 8.20-7.99 (m, 2H), 7.51 (dt, J = 39.5, 7.5 Hz, 3H), 7.156.93 (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, 1 H), 4.48 (dd, J = 10.7, 4.8 Hz, 1 H), 4.34 (dd, J = 6.7, 4.9 Hz, 1 H), 3.62 (dd, 9=11.9, 4.9 Hz, 1 H), 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).

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 2.6p C18 100A, 50 x 3.00 mm

Solvents: Acetonitrile with 0.1 % formic acid, Water with 0.1 % formic acid

Gradient: 0 min-1.4 min 2-100% ACN, 1.4 min-1.80 min 100% ACN, 1.8 min-1.85 min 100%-2% ACN, 1.85 min-2 min 2% ACN.

NHBz

1. DMTrCI, TEA, DCM ^N __________________> ¹ BbO—

2. TBSCI, Imid, DMF

DMTrO—* \__l

TBSÔ F

2f

Intermediate 2f - N-(7-((2S,3S,4R,5R)-5-((bis(4methoxyphenyl)(phenyl)methoxy)methyl)-4-(tert-butyldimethylsilyloxy)-5-((tertbutyldimethylsilyloxy)methyl)-3-fluorotetrahydrofuran-2-yl)pyrrolo[1,2-f][1,2,4]trîazin4-yl)benzamide.

Intermediate 2e (370 mg, 0.717 mmol) was dissolved in CH2CI2 (10 mL) and TEA (200 pL) under an N2 atmosphère, and was then cooled to 0 °C. 4,4’-Dimethoxytrityl chloride 4,4’-Dimethoxytrityl chloride (0.364g, 1.07mmol) was added in one portion and the reaction mixture was allowed to stirfor 30 min. CH3OH (2 mL) was added and the solution was diluted with CH2CI2 and sat. NaHCO₃(aq). The organic layer was washed with brine, dried over Na2SO₄, filtered, and the solvents were removed under reduced pressure. The residue was purified by silica gel chromatography, using an eluent ramp of 5%-100% EtOAc in hexanes, to afford the crude product as a mixture of bis-DMTr and 4’β products. This mixture was carried forward without further purification.

To the crude product (574 mg, mixture) in DMF (3 mL), under an N₂ atmosphère, was added imidazole (143 mg, 2.10 mmol), followed by terf-butylchlorodimethylsilane (158 mg, 1.05 mmol). The solution was allowed to stir at room température for 2 h. The solution was diluted with CH3OH (1 mL) and EtOAc. The organics were washed with H2O and then with 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 ramp of 0%-50% EtOAc in hexanes, to afford intermediate 2f that contained some bis-DMTr material.

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 x 3.00 mm

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1 % formic acid

Gradient: 0 min-1.5 min 2-100% ACN, 1.5 min-2.8 min 100% ACN, 2.8 min-2.85 min 100%2% ACN, 2.85 min-3 min 2% ACN.

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

Intermediate 2f was dissolved in CHCh (5 mL) and was cooled to 0 °C. pToluenesulfonic acid hydrate (90 mg, 0.474 mmol), dissolved in CH3OH (4 mL) was added dropwise to the mixture and the reaction mixture was allowed to stir for 5 min. The reaction mixture was quenched with sat. NaHCO3(_aq). The organics were washed with brine, dried over Na₂SO4, filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography, using an eluent ramp of 0%-40% EtOAc in hexanes, to afford intermediate 2g.

¹H NMR (400 MHz, CDCI3) δ 8.09-7.88 (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).

¹⁹F NMR (376 MHz, CDCI3) δ -196 (m)

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 x 3.00 mm

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid

Gradient 0 min-1.5 min 2-100% ACN, 1.5 min-2.8 min 100% ACN, 2.8 min-2.85 min 100%2% ACN, 2.85 min-3 min 2% ACN.

1. DMSO, Toi, EDCI, Pyr, TFA

2. bromomethyl triphenyl phosphonium bromide, KOtBu, THF

2g

2h

Intermediate 2h - N-(7-((2S,3S,4R,5R)-5-((E)-2-bromovinyl)-4-(tertbutyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy)methyl)-3-fluorotetrahydrofuran2-yl)pyrrolo[1,2-f][1,2,4]triazin-4-yl)benzamide.

To solution of intermediate 2g (228 mg, 0.361 mmol) in toluene (0.75 mL), and DMSO (0.15 mL), was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (208 mg, 1.08 mmol) under an N₂ atmosphère. To this mixture was added pyridine (30 pL), and TFA (15 pL) and the mixture was stirred at RT for 30 min. The reaction was diluted with EtOAc and washed with H₂O followed by brine. The organics were dried over Na₂SO4, filtered, and solvent was removed under reduced pressure to afford the crude aldéhyde. This material was used as is for the next step.

To a suspension of bromomethyltriphenylphosphonium bromide (314 mg, 0.72 mmol) in THF (4 mL) at-40°C was added KOtBu (1.0M in THF, 1.08 mL, 1.08 mmol) and the reaction mixture was stirred for 2 h under an N₂ atmosphère. The crude aldéhyde 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 re-cooled to -40 °C and the reaction mixture was quenched with sat NH₄CI_(aq). The layers were separated and the organics were dried over Na₂SO₄, filtered, and solvent was removed under reduced pressure. The residue was purified by silica gel chromatography, using an eluent of 0%50% EtOAc in hexanes, to afford intermediate 2h.

¹H NMR (400 MHz, CDCI₃) δ 8.08 - 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, 1 H), 4.69 - 4.46 (m, 1 H), 3.97 (d, J = 11.4 Hz, 1 H), 3.53 (d, J = 11.4 Hz, 1H), 0.84 (d, J= 3.9 Hz, 18H), 0.13--0.10 (m, 12H).

¹⁹F NMR (376 MHz, CDCI₃) δ -190.60 (m).

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 x 3.00 mm

Solvents: Acetonitrile with 0.1 % formic acid, Water with 0.1 % formic acid

Gradient: 0 min-1.5 min 2-100% ACN, 1.5 min-2.8 min 100% ACN, 2.8 min-2.85 min 100%2% ACN, 2.85 min-3 min 2% ACN.

2h 2i

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

Intermediate 2h (204 mg, 0.289 mmol) was dissolved in THF (8 mL) under an N₂ atmosphère and cooled to -40°C. KOtBu (1.0M in THF, 1.08 mL, 1.08 mmol) was slowly added. The reaction was allowed to stir for 20 min as was quenched with sat. NH4CI(_aq). The solution was diluted with EtOAc and washed with brine. The organics were dried over Na2SO4, filtered, and solvent was removed under reduced pressure. The residue was purified by silica gel chromatography, using an eluent ramp of 0%-50% EtOAc in hexanes, to afford intermediate 2i.

¹H NMR (400 MHz, CDCb) δ 8.12 - 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).

¹⁹F NMR (376 MHz, CDCI₃) δ -193.10 (broad-s).

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 x 3.00 mm

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1 % formic acid

Gradient: 0 min-1.5 min 2-100% ACN, 1.5 min-2.8 min 100% ACN, 2.8 min-2.85 min 100%2% ACN, 2.85 min-3 min 2% ACN.

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.

To a solution of intermediate 2i (152 mg, 0.243 mmol) in THF (3.5 mL) under an N₂ atmosphère was added TBAF (1.0M in THF, 700 pL, 0.700 mmol) at room température and the mixture was allowed to stir for 30 min. Solvents were removed under reduced pressure.

The residue was purified by silica gel chromatography, using an eluent ramp of 40%-100% EtOAc in hexanes, to afford intermediate 2j.

LC/MS: îr = 0.88 min, MS m/z = 397.16 [M+1]; LC system: Thermo Accela 1250 UIHPLC

MS System: Thermo LCQ Fleet; Column: Kinetex 2.6μ C18 100A, 50 x 3.00 mm

Solvents: Acetonitrile with 0.1 % formic acid, Water with 0.1 % formic acid

Gradient: 0 min-1.5 min 2-100% ACN, 1.5 min-2.8 min 100% ACN, 2.8 min-2.85 min 100%2% ACN, 2.85 min-3 min 2% ACN.

NHBz NH₂

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

To a solution of intermediate 2j (71 mg, 0.179 mmol) in CH3OH (2 mL) was added con. NH₄OH₍aq) (0.7 mL) and the resulting solution was stirred at room température for 16 h. Solvents were removed under reduced pressure. The residue was purified by silica gel chromatography, using an eluent ramp of 0%-20% CH₃OH in CH2CI2, followed by reverse phase HPLC, using an eluent ramp of 0%-20% ACN in H2O, to yield example 2.

¹H NMR (400 MHz, CD3OD) δ 7.77 (s, 1 H), 6.90 - 6.70 (m, 2H), 5.62 (dd, J = 25.5, 2.6 Hz, 1 H), 5.18 (ddd, J = 56.0, 5.4, 2.7 Hz, 1 H), 4.57 (dd, J = 20.5, 5.4 Hz, 1 H), 3.93 - 3 59 (m, 2H), 3.02 (d, J = 0.7 Hz, 1H).

¹⁹F NMR (376 MHz, CD3OD) δ -193.76 (ddd, J = 56.0, 25.5, 20.4 Hz).

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: Kinetex2.6p C18 100A, 50x3.00 mm

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1 % formic acid

Gradient: 0 min-1.4 min 2-100% ACN, 1.4 min-1.80 min 100% ACN, 1.8 min-1.85 min 100%-2% ACN, 1.85 min-2 min 2% ACN.

HPLC: t_R = 3.112 min; HPLC system: Agilent 1100 sériés.

Column: Phenomenex Kinetex C18 2.6 pm 100A, 4.6 x 100 mm

Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA

Gradient: 0 min-8.0 min 2-98% ACN at 1.5 mL/min.

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

To a solution of intermediate 2g (1.13 g, 1.79 mmol) in DMSO (1 mL) and toluene (10 mL), prepared under an N₂ atmosphère, was added EDCI HCI (1.02 g, 5.36 mmol) and pyridine (149 pL, 1.92 mmol). TFA (74 pL, 0.97 mmol) was added drop-wise. After 1 hrthe reaction was checked by LC/MS. A single peak was observed, with a rétention time similar to the starting material’s, but with an M+1 peak equal to that expected for the product.

Another 50 pL of pyridine was added and the reaction was stirred for another 15 min. No change by LC/MS. The reaction was diluted with EtOAc and quenched with a 1:1 mixture of sat. NaHCO₃(aq.) and H₂O. The mixture was partitioned between EtOAc and more 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 taken up in CH₂CI₂, concentrated, and the resulting material was placed under high vacuum for 1 h. The product, intermediate 3a was used as is in the next reaction.

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 2.6p C18 100A, 50 x 3.00 mm

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1 % formic acid

Gradient: 0 min-1.5 min 2-100% ACN, 1.5 min-2.8 min 100% ACN, 2.8 min-2.85 min 100%2% ACN, 2.85 min-3 min 2% ACN.

honh₂.hci, Pyridine

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

To a solution of intermediate 3a (crude material from previous step, assumed 1.79 mmol) in pyridine (11 mL), prepared under an N₂ atmosphère, was added HONH₂ HCI in one portion at room température. The reaction was checked by LC/MS 5 minutes later; starting material was consumed. The reaction was checked again 25 minutes later. There was no change from the first time point. The reaction was concentrated and the residue was partitioned between EtOAc and H₂O. The organic layer was separated, washed with brine, dried over Na₂SÜ4 and filtered. The filtrate was concentrated, taken up in CH₂CI₂, concentrated again, and the residue was placed under high vacuum. The product intermediate 3b was used as is in the next reaction.

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 x 3.00 mm

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid

Gradient: 0 min-1.5 min 2-100% ACN, 1.5 min-2.8 min 100% ACN, 2.8 min-2.85 min 100%2% ACN, 2.85 min-3 min 2% ACN.

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

To a solution of intermediate 3b (crude material from the previous step, assumed 1.79 mmol) in ACN (16 mL) was added CDI (436 mg, 2.69 mmol) in a single portion. The reaction was run under an N₂ atmosphère. The reaction was checked by LC/MS after 20 minutes. Peaks with the mass of starting material and product are barely time resolved. The reaction was checked 1.5 h later. The UV peak corresponding to the starting material was nearly gone and the intensity of the mass peak was diminished. The reaction was diluted with CH2CI2 and quenched with a 1:1 mixture of sat. NaHCO3(_aq.) and H₂O. The layers were separated, the aqueous was back extracted with CH2CI2 and the combined organics layers were extracted with a 1:1 mixture of brine and H2O, dried over Na2SO4 and filtered. The filtrate was concentrated and intermediate 3c was isolated by silica gel column chromatography using the following solvent ramp: 0% EtOAc in hexanes ramping to 20% EtOAc in hexanes, pause at 20% EtOAc in hexanes and then ramping to 40% EtOAc in hexanes.

¹H NMR (400 MHz, DMSO-d₆) δ 8.31 (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).

¹⁹F NMR (376 MHz, DMSO-cfe) δ -194.658 (dt, J = 53, 21.4 Hz).

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 x 3.00 mm

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid

Gradient: 0 min-1.5 min 2-100% ACN, 1.5 min-2.8 min 100% ACN, 2.8 min-2.85 min 100%2% ACN, 2.85 min-3 min 2% ACN.

Intermediate 3d - (2R,3R,4S,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-3-(tertbutyldimethylsilyloxy)-2-((tert-butyldimethylsilyloxy)methyI)-4-fluorotetrahydrofuran2-carbonitrile.

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 ΝΗ₄ΟΗ(_3η) (3.74 mL). The cold bath was removed and the resulting heterogeneous solution was stirred ovemight. The next day the reaction was incomplète as determined by LC/MS. Additional concentrated NH₄OH(_aq) (4 mL) and 2-MeTHF (12 mL) was added. The reaction became homogenous but after 20 minutes there was no additional reaction progress. 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) to turn the solution homogenous and monophasic. The reaction was stirred at room température for 22 h. The reaction was nearly complété (about 5% of the starting material remains). The reaction was diluted with CH2CI2 and H2O. The layers were separated, and the aqueous layer was diluted with saturated NaHCO3(_aq) and extracted with CH2CI2. The aqueous layer was neutralized with 2N HCl and then extracted with CH₂CI₂. 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 ramp: 0% EtOAc in hexanes ramping to 50% EtOAc in hexanes, pause at 50% EtOAc in hexanes and then ramping to 100% EtOAc in hexanes.

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 x 3.00 mm

Solvents: Acetonitrile with 0.1 % formic acid, Water with 0.1 % formic acid

Gradient: 0 min-1.4 min 2-100% ACN, 1.4 min-1.8 min 100% ACN, 1.8 min-1.85 min 100%2% ACN, 1.85 min-2 min 2% ACN.

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

To a solution of intermediate 3d (100 mg, 0.191 mmol) in THF (2 mL), in a polypropylène tube, was added 70% HF Pyridine in pyridine (60 pL, 0.478 mmol) at 0 °C, under an N₂ atmosphère. The reaction was checked after 20 minutes; there was no reaction, so additional 70% HF Pyridine in pyridine (150 pL) was added and the cold bath was removed. After 1 hour and 50 minutes additional 70% HF Pyridine in pyridine (150 pL) was added. After another 2 hours additional 70% HF Pyridine in pyridine (300 pL) was added. After another 2 hours and 15 minutes additional 70% HF Pyridine in pyridine (1 mL) was added. The reaction tums clear and homogenous upon this final addition of 70% HF Pyridine in pyridine. The reaction was stirred overnight. The reaction was complété the next day. The reaction was cooled in an ice bath and quenched with H₂O and a small amount of saturated NaHCO₃₍aq). The mixture was concentrated and the residue was taken up in DMSO. Remaining insoluble material was removed by filtration and the filtrate was semipurified by HPLC. The isolated material was dissolved in DMF and purified by HPLC. Example 3 was isolated, with 0.5% as a TFA sait.

¹H NMR (400 MHz, DMF-d₇) δ 7.92 (s, 1 H), 6.99 (d, J=4.4 Hz, 1H), 6.89 (d, J = 4 9Hz, 1H), 6.64 (d, J = 6 Hz, 1 H), 5.92 (t, J = 6.4 Hz, 1 H), 5.83 (dd, J = 25.2, 2 Hz, 1 H), 5.40 (ddd, J = 54.8, 4.8, 2.4 Hz, 1 H), 4.75 (dt, J = 22, 5.2 Hz, 1 H), 4.02 (dd, J = 12, 6.4Hz, 1 H), 3.87 (dd, J = 12, 6.4 Hz, 1H).

¹⁹F NMR (376 MHz, DMF-d₇) δ -74.92 (s), -193.726 (dt, J = 54.5, 23.3 Hz).

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 x 3.00 mm

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1 % formic acid

Gradient: 0 min-1.4 min 2-100% ACN, 1.4 min-1.8 min 100% ACN, 1.8 min-1.85 min 100%2% ACN, 1.85 min-2 min 2% ACN.

HPLC: îr = 3.220 min; HPLC System: Agilent 1100 sériés.; Column: Phenomenex Kinetex C18 2.6 pm 100A, 4.6 x 100 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA

Gradient: 0 min-8.0 min 2-98% ACN at 1.5 mL/min.

4a 4b

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

Into a 10-L 4-necked round-bottom flask, was placed a solution of intermediate 4a, (3aR,5S,6S,6aR)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydrofuro[3,2d][1,3]dioxol-6-ol (500 g, 1.90 mol) in dichloromethane/water (2.7 L / 2.3 L) at room température. To this was added sodium carbonate (290 g, 3.42 mol). Addition of potassium carbonate (451 g, 3.24 mol) was next. This was followed by the addition of 2,2,6,6tetramethylpiperidinooxy (TEMPO, 15.2 g, 96.31 mmol). To the mixture was added tetrabutylammonium bromide (31 g, 95.20 mmol). To the above was added Nbromosuccinimide (514 g, 2.86 mol), in portions at 35 °C. The resulting solution was allowed to react, with stirring, for 2 h at room température. The resulting solution was extracted with

2x1 L of dichloromethane and the organic layers combined. The resulting mixture was washed with 1 x 1.5 L of water. The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. This resulted in (crude) intermediate 4b.

4b

NaBH₄

MeOH

4c

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

Into a 2-L 4-necked round-bottom flask, was placed a solution of intermediate 4b (370 g, 1.29 mol) in methanol (1300 mL). To the above was added sodium borohydride (26.4 g, 706.38 mmol), in portions at room température. The resulting solution was allowed to react, with stirring, for 2 h at room température. The resulting mixture was conœntrated under vacuum. The reaction was then quenched by the addition of 1000 mL of 5% aqueous ammonium chloride solution. The resulting solution was extracted with 3 x 500 mL of dichloromethane and the organic layers 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 re-crystallization from petroleum ether. This resulted in intermediate 4c.

Intermediate 4d.

Into a 5000-mL 4-necked round-bottom flask, was placed a solution of intermediate 4c (350 g, 1.34 mol) in dichloromethane (700 mL). To this was added tetrabutylammonium bromide (476.8 g, 1.48 mol). To the mixture was added 50% sodium hydroxide/water (700 g). To the above was added 2-(bromomethyl) naphthalene (340 g, 1.54 mol) in several batches. The resulting solution was allowed to react, with stirring, for 4 h at room température. The reaction was then quenched by the addition of 1800 mL of dichloromethane/water (1:1). The resulting solution was extracted with 2x1 L of dichloromethane and the organic layers 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 re-crystallization from petroleum ether. This resulted in intermediate 4d.

4d

AcOH/ H₂O

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

Into a 5-L 4-necked round-bottom flask, was placed intermediate 4d (500 g, 1.25 mol). To this was added acetic acid (1.8 L). To the mixture was added water (600 mL). The resulting solution was allowed to react, with stirring, overnight at room température. The solids were filtered out. The resulting solution was extracted with 3x1 L of petroleum ether and the aqueous layers combined. The resulting solution was diluted with 2 L of ethyl acetate. The resulting mixture was washed with 2 x 2 L of sodium chloride(_aq). The pH value of the solution was adjusted to 8 with sodium carbonate (50 %). The resulting solution was extracted with 2x1 L of ethyl acetate and the organic layers combined and concentrated under vacuum. This resulted in intermediate 4e.

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

Into a 10 L 4-necked roundbottom flask, was placed a solution of intermediate 4e (300 g, 833.33 mmol) in 1,4-dioxane (2100 mL) atroom température. This was followed by the addition of a solution of sodium periodate (250 g) in water (4000 mL) at room température in 0.5 h. The resulting solution was allowed to react, with stirring, for 0.5 hr at room température. The solids were filtered out. The resulting solution was extracted with 3 x 1000 mL of ethyl acetate and the organic layers combined. The resulting mixture was washed with 2 x 1000 mL of sodium chloride (_aq). The resulting mixture was concentrated under vacuum. This resulted in intermediate 4f.

HCHO

NaOH

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

Into a 10-L 4-necked round-bottom flask, was placed a solution of intermediate 4f (250 g, 761.36 mmol) in water/tetrahydrofuran (1250/1250 mL) at room température. To the above was added 2N sodiun hydroxide(aq) (1500 mL) dropwise with stirring at 0-15 °C. To the mixture was added formaldéhyde (620 mL). The resulting solution was allowed to react, with stirring, overnight at room température. The resulting solution was extracted with 2 x 2000 mL of ethyl acetate. The resulting mixture was washed with 2 x 2000 mL of sodium chloride(aq). The organic layers combined and dried over anhydrous sodium sulfate. The residue was applied onto a silica gel column with petroleum ether: ethyl acetate (2/1). The crude product was re-crystallized from ethyl acetate:ethanol in the ratio of 1g/(1 mL:1 mL). This resulted in intermediate 4g.

Intermediate 4h - ((3aR,5R,6S,6aR)-5-((tert-butyldiphenylsilyloxy)methyl)-2,2-dimethyl6-(naphthalen-2-ylmethoxy)tetrahydrofuro[3,2-d][1,3]dioxol-5-yl)methanol.

Into a 5-L 4-necked round-bottom flask purged and maintained with an inert atmosphère of nitrogen, was placed a solution of intermediate 4g (125 g, 346.84 mmol) in dichloromethane (2500 mL) at room température. To the mixture was added triethylamine (157.5 mL) at room température. To the above was added tert-butyldiphenylsilyl chloride (157.5 mL) dropwise with stirring at 0-10 °C. The resulting solution was allowed to react, with stirring, overnight at room température. The reaction was then quenched by the addition of

37.5 mL of methanol. The resulting mixture was washed with 2 x 500 mL of 5% hydrogen chloride(_aq) and 2 x 500 mL of sodium bicarbonate(_aq). The resulting mixture was washed with 2 x 500 mL of 1N sodium hydroxide(_aq). The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. The crude productwas purified by re-crystallization from dichloromethane/hexane. This resulted in intermediate 4h.

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

Into a 1000-mL 3-necked round-bottom flask purged and maintained with an inert atmosphère of nitrogen, was placed a solution of intermediate 4h (20 g, 31.73 mmol) in toluene (320 mL), triphenylphosphine (35 g, 132.11 mmol), imidazole (8.96 g, 132.26 mmol). This was followed by the addition of iodine (16.95 g, 66.8 mmol) in several batches at 60 °C. The resulting solution was stirred overnight at 80 °C in an oil bath. The reaction mixture was cooled to room température with a water/ice bath. The resulting solution was diluted with 1000 mL of ethyl acetate. The resulting mixture was washed with 2 x 300 mL of sodium thiosulfate(a_q). The resulting mixture was washed with 1 x 300 mL of sodium chloride(_aqj. The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied onto a silica gel column with ethyl acetate/petroleum ether (1:10). This resulted in intermediate 4L

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

Into a 2000-mL round-bottom flask purged and maintained with an inert atmosphère of nitrogen, was placed a solution of intermediate 4i (66 g, 88.47 mmol) in ethanol/ethyl acetate (600/600 mL), triethylamine (20.7 g, 202.52 mmol), palladium on carbon (10% wt, 24.8 g, 23.30 mmol). The resulting solution was stirred for 3 h at 40 °C. The solids were filtered out. The resulting mixture was concentrated under vacuum. The resulting solution was diluted with 1500 mL of ethyl acetate. The resulting mixture was washed with 1 x 500 mL of sodium chloride(_aq). The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. This resulted in intermediate 4j.

Intermediate 4k - (3aR,5R,6S,6aR)-5-((tert-butyldiphenylsilyloxy)methyl)-2,2,5trimethyltetrahydrofuro[3,2-d][1,3]dioxol-6-ol.

Into a 500-mL round-bottom flask, was placed 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-1,4benzoquinone (DDQ, 780 mg, 3.40 mmol). The resulting solution was stirred for 1 h at room température. The resulting solution was diluted with 50 mL of dichloromethane. The resulting mixture was washed with 1 x 30 mL of water and 2 x 30 mL of sodium bicarbonate(_aq). The resulting mixture was washed with 1 x 30 mL of sodium chloride_(aq). The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied onto a silica gel column with ethyl acetate/petroleum ether (1:20~1:10). This resulted in intermediate 4k.

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

Into a 50-mL round-bottom flask, was placed a solution of intermediate 4k (520 mg, 1.12 mmol) in tetrahydrofuran (9 mL), Tetrabutylammonium fluoride (369 mg, 1.40 mmol).

The resulting solution was stirred for 2 h at room température. The resulting mixture was concentrated under vacuum. The residue was applied onto a silica gel column with dichloromethane/methanol (100:1). This resulted in intermediate 4I.

¹H NMR (300 MHz, DMSO-d₆): δ 5.64 (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).

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

Into a 50-mL 3-necked round-bottom flask purged and maintained with an inert atmosphère of nitrogen, was placed a solution of intermediate 4I (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 portions at 0 °C. The resulting solution was stirred for 30 min at 0 °C. The resulting solution was allowed to react, with stirring, for an additional 30 min at room température. To this was added benzyl bromide (452 mg, 2.62 mmol) dropwise with stirring at 0 °C. The resulting solution was allowed to react, with stirring, for an additional 3 h at room température. The reaction was then quenched by the addition of 30 mL of ammonium chloride(aq). The resulting solution was extracted with 50 mL of dichloromethane and the organic layers were combined and dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied onto a silica gel column with ethyl acetate/petroleum ether (1:30-1:20). This resulted in intermediate 4m.

4m h₂so₄

AcOH, Ac₂O

.JDAc

Bnd t)Ac

4n

Intermediate 4n - (2R,3R,4S,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-5methyltetrahydrofuran-2,3-diyl diacetate.

Into a 1000-mL round-bottom flask, was placed intermediate 4m (Also prepared according to Biosci. Biotech. Biochem. 1993, 57, 1433-1438, 45 g, 111.19 mmol), aceticacid (270 mL), acetic anhydride (90 mL), sulfuric acid (45 d). The resulting solution was stirred for 30 min at room température. The reaction was then quenched by the addition of 1000 mL of water/ice. The resulting solution was diluted with 3000 mL of ethyl acetate. The resulting mixture was washed with 2 x 1000 mL of water and 4 x 1000 mL of sodium bicarbonate^). The resulting mixture was washed with 2 x 1000 mL of sodium chloride(_aq). The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied onto a silica gel column with ethyl acetate/petroleum ether (1:30-1:20). This resulted in intermediate 4n.

¹H NMR (300 MHz, CDCI₃): δ 7.28 - 7.38 (m, 10H), 6.13 (s, 1 H), 5.37 (d, J = 4.8Hz, 1 H), 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+N a]

1)K₂CO₃, MeOH BnO'^'°'r^iO

Bnd ÔAc ²) ^NIS. Bu₄NI, DCM BnÔ oh

4n 4o

Intermediate 4o - (3R,4S,5R)-4-(benzyloxy)-5-(benzyloxymethyl)-3-hydroxy-5methyldihydrofuran-2(3H)-one.

Intermediate 4n (1.3 g, 3 mmol) was dissolved in anhydrous MeOH (15 mL). Potassium carbonate powder (456 mg, 3.3 mmol) was added, and the reaction mixture was stirred for 1 h. The reaction mixture was then concentrated under reduced pressure. Added acetonitrile and stirred for 5 minutes. Filtered off insoluble and washed with acetonitrile. Concentrated filtrate under reduced pressure. Dissolved resulting material in anhydrous DCM (20 mL). Added tetrabutylammonium iodine (1.66 g, 4.5 mmol) and /V-iodo-succinimide (NIS, 1.69 g, 2.5 mmol). Stirred the reaction mixture in the darkfor 16 h. Added more NIS (0.85 g, 1.25 mmol) and stirred for 4 hours. Added more NIS (0.85 g, 1.25 mmol) and stirred for 2 days in the dark. Diluted reaction with EtOAc and washed with aqueous sodium thiosulfate solution twice and then with saturated aqueous sodium chloride solution. Dried organic portion over anhydrous sodium sulfate and concentrated under reduced pressure. Purified with silica gel column (0-30% EtOAc in hexanes) to afford intermediate 4o.

¹H NMR (400 MHz, CDCI₃): δ 7.35-7.22 (m, 10H), 4.82 (bs, 1H), 4.75-4.66 (m, 2H), 4.55 _ 4.44 (_m, 2H), 4.13 (d, J = 8Hz, 1 H), 3.70-3.45 (m, 2H), 1.38 (s, 3H).

LC/MS: t_R = 2.58 min, MS m/z = 342.9 [M+1], 360.0 [M+H₂O]; LC/MS system: Thermo LCQ Advantage; Phenomenex Gemini, Cis, 5u, 11 OA, 30x4.6 mm; Buffer A: 0.1% Acetic acid in Water; Buffer B: 0.1% Acetic acid in Acetonitrile

5-100% Buffer B in 2.5 mins then 100% for 0.9min @ 2 mL/min.

HPLC: t_R = 3.78 min; HPLC system: Agilent 1100; Phenomenex Gemini, Cis, 5u, 110A, 50 x 4.6 mm; Buffer A: 0.05% TFA in Water; Buffer B: 0.05% TFA in Acetonitrile; 2-98% Buffer B in 5 minutes @ 2mL/min.

BnO'^>^y^{o a}92O, BnBr BnO'^^T⁰ BnÔ ÔH EtOAc BnÔ 5_Bn

4o 4p

Intermediate 4p - (3R,4S,5R)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)-5methyldihydrofuran-2(3H)-one.

Dissolved intermediate 4o (955 mg, 2.79 mmol) in EtOAc (10 mL). Added benzyl bromide (400 pL, 3.35 mmol) and silver(l) oxide (712 mg, 3.07 mmol). Stirred at60 °C under N₂(g) in the dark for 3 h. Added more benzyl bromide (400 pL, 3.35 mmol) and stirred at 60 °C under N₂(g) in the dark for 16 h. Added more silver(l) oxide (350 mg, 1.5 mmol) and stirred at 60 °C under N₂(g) in the dark for 8 h. Cooled to room température. Filtered off solids and washed with EtOAc. Concentrated filtrate under reduced pressure to give an oil. Added hexanes and stirred for 2 h to give solid. Collected solid and washed with hexanes. Dried solid under high vacuum to afford intermediate 4p.

¹H NMR (400 MHz, CDCI3): δ 7.35 - 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).

LC/MS: t_R = 2.91 min, MS m/z = 433.1 [M+1], 450.1 [M+H₂O]; LC/MS system: Thermo LCQ Advantage; Phenomenex Gemini, Cia, 5u, 110A, 30x4.6 mm; Buffer A: 0.1 % Acetic acid in Water; Buffer B: 0.1% Acetic acid in Acetonitrile; 5-100% Buffer B in 2.5 mins then 100% for 0.9min @ 2 mL/min.

HPLC: t_R = 4.54 min; HPLC System: Agilent 1100; Phenomenex Gemini, Cis, 5u, 110A, 50 x

4.6 mm; Buffer A: 0.05% TFA in Water; Buffer B: 0.05% TFA in Acetonitrile

2-98% Buffer B in 5 minutes @ 2mL/min.

Bnd ÔBn

4p 1b

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

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 under Ar(g) at -70 °C. Added n-butyllitium (2.5M solution in hexanes, 684 pL, 1.71 mmol) dropwise to the reaction mixture while maintaining internai température below -65 °C. Allowed the reaction to warm to -40 °C and keptfor 15 min. A solution of intermediate 4p (224 mg, 0.518 mmol) in THF (10 mL) precooled to -70 °C was then added to the reaction mixture under Ar(g). The resulting solution was stirred for 2 h at -40 °C. The reaction mixture was then poured into a stirring mixture of EtOAc and citric acid_(aq). Stirred for 5 min. Collected organic layer and washed with saturated NaCI_(aq). Dried organic layer over anhydrous Na₂SO₄ and concentrated under reduced pressure. Purified with prep HPLC to afford intermediate 4q.

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, Cis, 5u, 110A, 30 x 4.6 mm; Buffer A: 0.1 % Acetic acid in Water; Buffer B: 0.1% Acetic acid in Acetonitrile; 5-100% Buffer B in 2.5 mins then 100% for 0.9min @ 2 mL/min.

HPLC: t_R = 3.22 min; HPLC system: Agilent 1100; Phenomenex Gemini, Ci₈, 5u, 110A, 50 x 4.6 mm; Buffer A: 0.05% TFA in Water; Buffer B: 0.05% TFA in Acetonitrile; 2-98% Buffer B in 5 minutes @ 2ml_/min.

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

Intermediate 4q (81 mg, 0.143 mmol) was dissolved in anhydrous DCM (15 mL) and stirred under N₂(g) in an ice bath. Added triethylsilane (114 pL, 0.715 mmol) in one portion. Added boron trifluoride diethyl etherate (27 pL, 0.215 mmol) dropwise. Stirred for 15 min and then removed ice bath. Stirred for 60 min. Added triethylamine (100 pL, 0.715 mmol) and concentrated under reduced pressure. Dissolved in EtOAc and washed with saturated NaHCO3(aq) (2x) and then saturated with NaCI(_aq). Dried organic over anhydrous Na₂SO4 and concentrated under reduced pressure. Purified with silica gel column (0-80% EtOAc in hexanes) to afford intermediate 4r.

¹H NMR (400 MHz, CDCI₃): δ 7.71 (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 = 2.75min, MS m/z= 551.4 [M+1]; LC/MS system: Thermo LCQ Advantage

Phenomenex Gemini, Cis, 5u, 110A, 30x4.6 mm; Buffer A: 0.1% Acetic acid in Water; Buffer B: 0.1% Acetic acid in Acetonitrile; 5-100% Buffer B in 2.5 mins then 100% for 0.9min @ 2 mL/min.

HPLC: t_R = 3.57min; HPLC system: Agilent 1100; Phenomenex Gemini, C18, 5u, 110A, 50 x 4.6 mm; Buffer A: 0.05% TFA in Water; Buffer B: 0.05% TFA in Acetonitrile; 2-98% Buffer B in 5 minutes @ 2mL/min.

4r 4

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

Intermediate 4r (23 mg, 0.042 mmol) was dissolved into a formic acid/MeOH solution (1:9, 10 mL). Added palladium black and stirred at 60 °C for 90 min. Cooled to room température and filtered through Celite. Concentrated filtrate under reduced pressure. Purified with prep HPLC. Concentrated under reduced pressure. Dissolved in NaHCO3(_aq) and purified with HPLC under neutral condition to afford example 4.

Prep HPLC system: Gilson 215 Liquid Handler; Phenomenex Gemini, Cis 4u, 100 x 30.0 mm

Buffer A: 0.1% TFA in Water ; Buffer B: 0.1% TFA in Acetonitrile; 5-100% Buffer B in 13 minutes @ 20 mL/min.

¹H NMR (400 MHz, CDCI₃): δ 8.01 (s, 1 H), 7.41 (d, J = 4.8 Hz, 1 H), 7.02 (d, J = 4.8 Hz, 1 H), 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).

LC/MS: t_R = 0.30min, MS m/z = 281.3 [M+1], 279.0 [M-1]; LC/MS System: Thermo LCQ Advantage; Phenomenex Gemini, Cis, 5u, 110A, 30x4.6 mm; Buffer A: 0.1% Acetic acid in Water; Buffer B: 0.1% Acetic acid in Acetonitrile; 5-100% Buffer B in 2.5 mins then 100% for 0.9min @ 2 mL/min.

HPLC: t_R = 0.42 min; HPLC system: Agilent 1100; Phenomenex Gemini, Cw, 5u, 110A, 50 x 4.6 mm; Buffer A: 0.05% TFA in Water; Buffer B: 0.05% TFA in Acetonitrile; 2-98% Buffer B in 5 minutes @ 2mL/min.

tmsn₃ lnBr₃ ch₂ci₂

TBSÔ ÔTBS

TBSÔ ÔTBS

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-4yl)benzamide.

To a solution of crude intermediate 1m, N-(7-((2S,3S,4S)-3,4-bis(tertbutyldimethylsilyloxy)-5-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrrolo[1,2

f][1,2,4]triazin-4-yl)benzamide, (-110 mg, -0.18 mmol) and azidotrimethysilane (242 μΙ, 1.84 mmol) in dichloromethane (1.5 mL) was added indium (III) bromide (130 mg, 0.369 mmol) at RT under an argon atmosphère. After 1 h, the reaction mixture was quenched with saturated aqueous sodium bicarbonate solution (1 mL). The resulting mixture was partitioned between dichloromethane (20 mL) and saturated aqueous sodium bicarbonate solution (20 mL). The phases were split and the aqueous layer was extracted with dichloromethane (20 mL). The combined organic layers were dried over anhydrous sodium sulfate and were concentrated under reduced pressure to afford intermediate 5a that was used directly in the next step without further purification.

LC/MS: îr = 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 x 4.6 mm

Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min2.0 min 2-100% ACN, 2.0 min-3.55 min 100% ACN, 3.55 min-4.2 min 100%-2% ACN at 2pl/min.

5a 5b

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.

To a solution of crude intermediate 5a (-120 mg, -0.168 mmol) in DMF (5 mL) was added césium fluoride (256 mg, 1.68 mmol) at RT. After 25 h, the reaction mixture was diluted with brine (100 mL), and the resulting mixture was extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried over anhydrous sodium sulfate and were concentrated under reduced pressure to afford intermediate 5b that was used directly in the next step without further purification.

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 x 4.6 mm Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min-2.0 min 2100% ACN, 2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN, 3.2 min-3.5 min 2% ACN at 2pl/min.

HPLC: îr = 2.46 min’ HPLC System: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at 2 mL/min.

nh₄oh

MeOH

5b

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.

Concentrated ammonium hydroxide (1 mL) was added to a solution of crude intermediate 5b in methanol (1 mL) at RT. After 2 d, the reaction mixture was concentrated under reduced pressure, and was directly purified by preparatory HPLC (Phenominex Synergi 4u Hydro-RR 80Â 150 x 30 mm column, 5-100% acetonitrile/water gradient). The fractions containing the desired were combined and were concentrated under reduced pressure. The residue repurified via SiO2 column chromatography (4 g SiO2 Combiflash HP Gold Column, 0-20% methanol/dichloromethane) to afford example 5.

Ή NMR (400 MHz, CD₃OD) δ 7.79 (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, 1 H), 4.63 (t, J = 5.8 Hz, 1 H), 4.37 (d, J = 5.7 Hz, 1 H), 3.69 (d, J = 12.0 Hz, 1H), 3.59 (d, J = 12.0 Hz, 1H).

LC/MS: t_R = 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 x4.6 mm; Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min-1.5 min 2100% ACN, 1.5 min-2.2 min 100% ACN, 2.2 min-2.4 min 100%-2% ACN, 2.4 min-2.5 min 2% ACN at2pl/min.

HPLC: t_R = 1.287 min; HPLC System: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at 2 mL/min.;

TLC: eluent: 20% methanol in dichloromethane, Rf= 0.4 (UV)

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

Example 3 (15.0 mg, 0.05 mmol) was dried in a flask under vacuum ovemight. Trimethylphosphate (0.5 mL) and 1,8-bis(dimethylamino)naphthalene (25 mg, 0.12 mmol) were added to the flask and the solution was allowed to stir under N₂ cooled by an ice/water bath. Distilled phosphorus oxychloride (10 pL, 0.11 mmol) was added and the reaction was allowed to stir for 4 h with cooling. Tributylamine (0.1 ml, 0.42 mmol) and tributylammonium pyrophospate (0.8 mL of a 0.5 M solution in DMF, 0.4 mmol) were added and the reaction was allowed to stir for an additional 45 min with cooling. The reaction was quenched with triethylammonium bicarbonate (0.5 M, 5 mL). The solvents were removed by rotary évaporation 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 0-1 M triethylammonium bicarbonate. The 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 Rétention time: 12.015 min; Column: DNAPac PA-100 4 x250 mm SN

Solvent A: milliQ water; Solvent B: 0.5 M tetraethylammonium bromide; Solvent gradient program: equilibrate using 100%A for 10 min, then ramp 0-80% B over 14min.; Flow: 1 mL/min.

Example 7 (also TP2) - ((2R,3R_J4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-2ethynyl-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate.

Example 2 (16.0 mg, 0.055 mmol) was dried in a flask under vacuum ovemight. Trimethylphosphate (0.5 mL) and 1,8-bis(dimethylamino)naphthalene (28 mg, 0.13 mmol) were added to the flask and the solution was allowed to stir under N₂ cooled by an ice/water bath. Distilled phosphorus oxychloride (11 pL, 0.12 mmol) was added and the reaction was allowed to stir for 4 h with cooling. 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 allowed to stir for an additional 45 min with cooling. The reaction was quenched with triethylammonium bicarbonate (0.5 M, 5 mL). The solvents were removed by rotary évaporation 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 0-1 M triethylammonium bicarbonate. The 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.

MS m/z = 531.0 [M-1]

Ion Exchange HPLC Rétention time: 19.829 min; Column: DNAPac PA-100 4 x 250 mm SN

Solvent A: milliQ water; Solvent B: 0.5 M tetraethylammonium bromide; Solvent gradient program: equilibrate using 100%A for 10 min, then ramp 0-80% B over 14 min.; Flow: 1 mL/min

Example 8 (TP3) - ((2R,3S,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yi)-2-cyano3,4-dihydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate.

To a solution of example 1 (5.0 mg, 0.017 mmol) in PO(OMe)₃ (0.6 mL) at 0 °C was added POCI₃ (45 mg, 0.29 mmol). The reaction mixture was stirred at 0 °C for 10 h, at which point lon-exchange HPLC showed approximately 50% conversion. A solution of pyrophosphate tributylamine salts (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 h, and ion-exchange HPLC showed the reaction was completed. The reaction was quenched with triethylammonium bicarbonate buffer (1 M, 5 mL). The reaction mixture was stirred at RT for 0.5 h, then concentrated and co-evaporated with water twice. The residue was dissolved in H₂O (5 mL) and loaded to a ion-exchange column, eluted with H2O, then 5-35% triethylammonium bicarbonate buffer (1M)-H₂O. The product fractions were combined, concentrated and co-evaporated with H₂O. The residue was purified by ion-exchange column again to give crude material. ³¹P NMR showed this material contained impurities, so the material was repurified with C-18 column, eluted with 0-15% ACN-H2O containing 0.05% TEA, and the fractions containing product were combined and concentrated to give 3.6 mg material, which contained only 1.5 equiv of TEA as indicated by ¹H NMR analysis. The material was dissolved in H2O (1 mL) and triethylammonium bicarbonate buffer (1 M, 0.1 mL) was added. The resulting mixture was concentrated under reduced pressure and coevaporated with H₂O twice under reduced pressure to give example 8 (TP3), as a tetra-TEA sait.

¹H NMR (400 MHz, D₂O): δ 7.78 (s, 1 H), 6.85 (d, J = 2.4 Hz, 1 H), 6.82 (d, J = 2.4 Hz, 1 H), 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).

³¹P 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]

Example 9 (TP4) - ((2R,3S_J4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-azido3,4-dihydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate.

To a solution of example 5 (6.0 mg, 0.019 mmol) in PO(OMe)3 (0.6 mL) at 0 °C was added POCI3 (45 mg, 0.29 mmol). The reaction mixture was stirred at 0 °C for 10 h, at which point lon-exchange HPLC showed approximately 50% conversion. A solution of pyrophosphate tributylamine salts (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 h. The reaction was quenched with triethylammonium bicarbonate buffer (1 M, 5 mL). The reaction mixture was stirred at RT for 0.5 h, then concentrated and co-evaporated with water twice. The residue was dissolved in H₂O (5 mL) and loaded to a ion-exchange column, eluted with H₂O, then 5-35% triethylammonium bicarbonate buffer (1M)-H₂O. The product fractions were combined, concentrated and co-evaporated with H₂O. The residue was purified by ionexchange column again to give crude material. ³¹P NMR showed this material contained impurities, so the material was repurified with ion-exchange column again to give crude material. The material was treated with NaHCOs (10 mg) and the mixture was concentrated under reduced pressure. The solid residue was dissolved in 0.5 mL of H₂O and 40 pL of NaOH (1N) was added. The resulting mixture was purified with C-18 column, eluted with H₂O, and the fractions containing product were combined and concentrated under reduced pressure to afford Example 9 (TP4) as the tetra-sodium sait.

¹H NMR (400 MHz, D₂O): δ 7.76 (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).

³¹P 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]

Y I-IQ TEA, CH₂CI₂ o

-'ΌΙ O

PD1a

Intermediate PD1a - S-2-hydroxyethyl 2,2-dimethylpropanethioate.

To a solution of 2-thioethanol (3.50 mL, 50.0 mmol) and triethylamine (7.02 mL, 50.0 mmol) in CH₂Cl2, that has been cooled to -78 °C was added pivalyl chloride (6.15 mL, 50.0 mmol) dropwise over 30 min. The reaction was allowed to warm slowly to room température and progression was monitored by TLC. After 30 min, the reaction was determined to be complété and was quenched with water. The layers were separated and the aqueous was washed with CH2CI2. The organics were combined and dried over sodium sulfate. The solids were filtered and the solvent was removed under reduced pressure. The crude was purified by silica gel chromatography 0- 50% EtOAc/hexanes to afford intermediate PD1a.

¹H NMR (400 MHz, DMSO-d₆) δ 4.89 (t, J = 5.5 Hz, 1 H), 3.49 - 3.36 (m, 2H), 2.86 (t, J = 6.7 Hz, 2H), 1.14 (s, 9H).

Take up phosphorous oxychloride (281 pL, 3.08 mmol) in CH2CI2 (5 mL) and cool the solution to -78 °C. Take up the thioester PD1a (1.00 g, 6.17 mmol) in CH2Cl2(5 mL) and add slowly to the POCI3 solution. Next add TEA (891 pL, 6.16 mmol) dropwise and allow to stir cold for 30 min. Then warm to room température and allow to stir for 2 h. Add the pnitrophenol (428 mg, 3.08 mmol) in one portion followed by a slow addition of TEA (449 pL, 3.08 mmol). Stir at room température for 30 min. TLC (70:30 Hexanes/EtOAC) showed only one spot, but LC/MS had two peaks (product and bis-p-nitrophenolate). The solution was diluted with ether and the solids were 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 afford intermediate PD1b, S,S'-2,2'-((4-nitrophenoxy)phosphoryl)bis(oxy)bis(ethane-2,1-diyl) bis(2,2dimethylpropanethioate).

¹H NMR (400 MHz, CDCI₃) δ 8.29 - 8.21 (m, 2H), 7.46 - 7.36 (m, 2H), 4.23 (br q, J = 7.7 Hz, 4H), 3.16 (brt, J =5.7 Hz, 4H), 1.23 (s, 18H).

³¹P NMR (162 MHz, CDCI3) δ -7.72 (s).

tBuMgCI

PD1b

THF

NMP

PD1

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

Example 1 (6.0 mg, 0.02 mmol) was dissolved in NMP (0.1 mL), and THF (0.2 mL) was added. te/Y-Butyl magnésium chloride (1.0 M solution in THF, 0.031 mL, 0.031 mmol) was then added at RT under an argon atmosphère. After 10 min, a solution of intermediate PD1b (15.7 mg, 0.031 mmol) in THF (0.1 mL) was added and the resulting mixture was warmed to 50 °C. After 5 h, the resulting residue was purified by preparatory HPLC (Phenominex Synergi 4u Hydro-RR 80Â 150 x 30 mm column, 40-100% acetonitrile/water gradient). The fractions containing the desired product were combined and were lyophilized to afford example 10 (PD1).

¹H NMR (400 MHz, CDCh) δ 7.82 (s, 1 H), 6.69 (d, J = 4.5 Hz, 1 H), 6.64 (d, J = 4.5 Hz, 1 H), 5.56 (d, J = 3.4 Hz, 1H), 4.61 (brs, 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).

³¹P NMR (162 MHz, CDCI3) δ -2.34 (s).

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 x4.6 mm

Solvents: Acetonitrile with 0.1% aceticacid, Water with 0.1% aceticacid; Gradient: 0 min2.0 min 2-100% ACN, 2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN, 3.2 min-3.5 min 2% ACN at 2pl/min.

HPLC: t_R = 3.204 min; HPLC system: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA

Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at 2 mL/min.

tBuMgCI

PD1b

THF

NMP

HO F

PD2

Example 11 (PD2) - S,S^,-2,2'-((((2R_J3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7yl)-2-cyano-4-fluoro-3-hydroxytetrahydrofuran-2yl)methoxy)phosphoryl)bis(oxy)bis(ethane-2,1-diyl) bis(2,2-dimethylpropanethioate).

Example 3 (10.5 mg, 0.036 mmol) was dissolved in NMP (0.1 mL), and THF (0.1 mL) was added. fert-Butyl magnésium chloride (1.0 M solution in THF, 0.054 mL, 0.054 mmol) was then added at RT under an argon atmosphère. 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 preparatory HPLC (Phenominex Synergi 4u Hydro-RR 80Â 150 x 30 mm column, 40-100% acetonitrile/water gradient). The fractions containing the desired product were combined and were lyophilized to afford example 11 (PD2).

¹H NMR (400 MHz, CDCI₃) δ 7.94 (s, 1 H), 6.75 (d, J = 4.5 Hz, 1 H), 6.67 (d, J = 4.5 Hz, 1 H), 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).

³¹P NMR (162 MHz, CDCI3) δ -2.10 (s).

¹⁹F NMR (376 MHz, CDCI3) δ -191.64 (ddd, J = 55.0, 27.8, 21.3 Hz).

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: Kinetex2.6p XB-C18 100A, 50 x4.6 mm; Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min-2.0 min 2100% ACN, 2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN, 3.2 min-3.5 min 2% ACN at 2pl/min.

HPLC: t_R = 3.385 min; HPLC system: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at 2 mL/min.

Cl

PD3a

1) DCM, TEA

2) p-NO₂PhOH, TEA

Intermediate PD3c - (2S)-2-ethylbutyl 2-((4-nitrophenoxy)(phenoxy) phosphorylamino)propanoate.

Dissolved phenyl dichlorophosphate PD3a (1.5 mL, 10 mmol) in 30 mL anhydrous DCM and stirred under N₂(g) in an ice bath. Added amino ester HCl sait PD3b, (S)-2ethylbutyl 2-aminopropanoate hydrochloride, (prepared according to Eur. J. Med. Chem. 2009,44, 3765-3770, 2.1 g, 10 mmol) in one portion. Added TEA (3 mL, 22 mmol) dropwise. Stirred for 1 h at 0 °C. Added p-nitrophenol (1.4 g, 10 mmol) in one portion and TEA (1.5 mL, 11 mmol). The reaction mixture was then stirred at room température for 16 h. Diluted with DCM and washed with saturated NaHCO₃(_aq). Dried organic over anhydrous Na₂SO₄ and concentrated under reduced pressure. Purified with silica gel column (0-15% EitOAc in hexanes) to afford intermediate PD3c.

¹H NMR (400 MHz, CDCI₃) δ 8.23 (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).

³¹P NMR (162 MHz, CDCI₃) δ -3.04 (s), -3.10 (s).

LC/MS: t_R = 2.87min, MS m/z = 451.1 [M+1], 449.0 [M-1]; LC/MS system: Thermo LCQ Advantage; Phenomenex Gemini, Cis, 5u, 110A, 30x4.6 mm; Buffer A: 0.1% Acetic acid in Water; Buffer B: 0.1% Acetic acid in Acetonitrile; 5-100% Buffer B in 2.5 mins then 100% for 0.9min @ 2 mL/min.

HPLC: t_R = 4.40min; HPLC system: Agilent 1100; Phenomenex Gemini, Ci₈, 5u, 110A, 50 x 4.6 mm; Buffer A: 0.05% TFA in Water; Buffer B: 0.05% TFA in Acetonitrile; 2-98% Buffer B in 5 minutes @ 2mL/min.

Example 12 fBuMgCI

DMF, THF

HÔ F

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-2yl)methoxy)(phenoxy)phosphorylamino)propanoate.

Dissolved example 3 (15 mg, 0.051 mmol) in anhydrous DMF (1 mL) and stirred under N₂(g). Dissolved p-nitrophenylphosphoamidate PD3c (35 mg, 0.077 mmol) in anhydrous DMF (0.5 mL) and added to the reaction mixture in one portion. Added fBuMgCI in THF (1M in THF, 77 pL, 0.077 mmol) dropwise. Stirred for 2 h. Added more pnitrophenylphosphoamidate (35 mg in 0.5 mL anhydrous DMF) and more fBuMgCI (1M in THF, 50 pL, 0.050 mmol). Stirred for 2 h. Added more p-nitrophenylphosphoamidate (35 mg in 0.5 mL anhydrous DMF) and more fBuMgCI solution (1M in THF, 50 pL, 0.050 mmol). Stirred for 16 hours. Diluted with EtOAc and washed with saturated NaHCOaoq) (^3x)· Washed with saturated NaCI_(aq) and dried organic over anhydrous Na₂SO₄. Concentrated under reduced pressure. Purified with silica gel column (0-5% MeOH in DCM). Combined fractions and concentrated under reduced pressure. Purified with preparatory HPLC with TFA as modifier to afford example 12 (PD3).

Prep HPLC system: Gilson 215 Liquid Handler; Phenomenex Gemini, Ci₈ 4u, 100 x 30.0 mm

Buffer A: 0.1% TFA in Water ; Buffer B: 0.1% TFA in Acetonitrile; 5-100% Buffer B in 13 minutes @ 20 mL/min.

¹H NMR (400 MHz, CDCI₃) δ 7.89 (s, 1H), 7.31-7.13 (m, 6H), 6.80-6.75 (m, 1H), 5.805.70 (m, 1H), 5.35-5.20 (m, 1 H), 4.80 - 4.62 (m, 1 H), 4.60 - 4.45 (m, 2H), 4.35 - 4.10 (m, 1 H), 4.06 - 3.96 (m, 3H), 1.49 - 1.28 (m, 8H), 0.90 - 0.82 (m, 6H).

³¹P NMR (162 MHz, CDCI3) δ 2.36 (s), 2.22 (s).

HPLC: t_R = 3.00min; HPLC system: Agilent 1100; Phenomenex Gemini, Cis, 5u, 110A, 50 x 4.6 mm; Buffer A: 0.05% TFA in Water; Buffer B: 0.05% TFA in Acetonitrile; 2-98% Buffer B in 5 minutes @ 2mL/min.

LC/MS: t_R = 2.39min, MS m/z = 605.1 [M+1], 603.0 [M-1]; LC/MS system: Thermo LCQ Advantage; Phenomenex Gemini, Cis, 5u, 11 OA, 30x4.6 mm; Buffer A: 0.1% Acetic acid in Water; Buffer B: 0.1% Acetic acid in Acetonitrile; 5-100% Buffer B in 2.5 mins then 100% for 0.9min @ 2 mL/min.

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

Intermediate 2g, N-(7-((2S,3S,4R,5R)-4-(tert-butyldimethylsilyloxy)-5-((tertbutyldimethylsilyloxy)methyl)-3-fluoro-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrrolo[1,2f][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). Added EDO (100 mg, 0.52 mmol) and then TFA-Pyridine (34 mg, 0.18 mmol). Stirred for 1 h. Added more EDCI (100 mg, 0.52 mmol) and stirred for 1 h. Monitoring by LC/MS showed starting material alcohol remained. Added more EDCI (100 mg, 0.52 mmol) and stirred for 1 h. Monitoring by LC/MS showed that the reaction reached full conversion. Diluted with ethyl acetate and washed with saturated NaHCO3(_aq) (2x) and then saturated NaCI(_aq). Dried organic layer over anhydrous Na₂SO₄ and concentrated under reduced pressure. Purified with silica gel column (0-20% EtOAc in hexanes). Combined fractions and concentrated under reduced pressure to give aldéhyde as solid. Suspended Methyl triphenylphosphonium bromide (500 mg, 1.40 mmol) in 10mL anhydrous THF and stirred at-78 °C under Ar_(g). Added 2.5 M n-butyllithium solution in hexane (560 pL, 1.40 mmol) dropwise. Stirred reaction mixture in an ice bath for 1 h to give a yellow mixture. Dissolved above prepared aldéhyde in 5 mL of anhydrous THF and added to reaction dropwise. Removed ice bath and let reaction warmed to RT. Stirred for 3 h at RT. Added saturated aqueous NH₄CI solution and extracted with ethyl acetate. Washed organic extract with saturated NaHCO3(_aq> and then saturated NaCI(_aq). Dried organic over anhydrous Na₂SO₄ and concentrated under reduced pressure. Purified with silica gel column (0-20% EtOAc in hexanes) to afford intermediate 6a.

¹H NMR (400 MHz, DMSO-c/6) δ 8.22 (br s, 1 H), 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).

¹⁹F NMR (376 MHz, DMSO-c/6) δ-191.86 (d, J = 56.8 Hz).

MS m/z = 627.3 [M+1],

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

Intermediate 6a (146 mg, 0.23 mmol) was dissolved into THF (10 mL) and the resulting solution was stirred in an ice bath. Added 1M TBAF solution in THF (700 pL, 0.70 mmol) and stirred for 2 h. Diluted with EtOAc and washed with saturated NaCI_(aq) (5x). Dried organic layer over anhydrous Na₂SO₄ and concentrated under reduced pressure. Dissolved in 7 M ammonia in MeOH (7 mL) and stirred for 18 h. Concentrated reaction under reduced pressure. Purified with Cw preparatory HPLC with TFA as modifier. Combined fractions and concentrated under reduced pressure. Dissolved in NaHCO3(a_q) and repurified with preparatory HPLC under neutral condition. Combined fractions and freeze-dried te afford example 13.

¹H NMR (400 MHz, D₂O) δ 7.54 (s, 1 H), 6.62 - 6.49 (m, 2H), 5.98 - 5.79 (m, 1 H), 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, 1 H), 3.62 - 3.43 (m, 2H).

¹⁹F NMR (376 MHz, D₂O) δ -193.23 (dd, J= 54.7, 44.2 Hz).

MS m/z= 295.2 [M+1]

Example 14 - (2R,3R,4R,5S)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazin-7-yl)-2-ethyl-4-fluoro2-(hydroxymethyl)tetrahydrofuran-3-ol

Example 13 (5 mg, 0.017 mmol) was dissolved into methanol (2 mL). 10% Pd/C Degussa Catalyst (2 mg) was then added and the resulting mixture was stirred under atmosphère of hydrogen gas. After 40 min, the resulting mixture was filtered to remove

Pd/C and the filtrate was concentrated under reduced pressure. The residue was dissolved in water and freeze-dried to afford example 14.

¹H NMR (400 MHz, D₂O) δ 7.67 (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).

¹⁹F NMR (376 MHz, D₂O) δ -197.62 (ddd, J = 54.5, 20.6, 15.0 Hz).

MS m/z= 297.3 [M+1].

tBuMgCI

PD1b

THF

NMP

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

Example 13 (5 mg, 0.017 mmol) was dissolved into anhydrous DMF (0.5 mL). Added p-nitro-phenonate (13 mg, 0.026 mmol) in one portion. Added 1M t-butylmagnesiurn chloride in THF (25 pL, 0.026 mmol) dropwise. Stirred for 1 h. Warmed to 50 °C and stirred for 2 h. Added more p-nitro-phenonate (13 mg, 0.026 mmol) and stirred for 2 h. Added more 1M tbutylmagnesium chloride in THF (25 pL, 0.026 mmol) and stirred for 16 h at 50 °C. Cooled to RT. The resulting mixture was purified directly by preparatory HPLC column and eluted with linear gradient 0-100% ACN in water to afford example 15 (PD4).

¹H NMR (400 MHz, CD₃OD) δ 7.84 (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).

¹⁹F NMR (376 MHz, CD3OD) δ 192.99 (td, J = 55.7, 23.6 Hz).

MS m/z = 663.0 [M+1],

PD3c fBuMgCI

NMP, THF

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

Example 5 (5 mg, 0.016 mmol) was dissolved in anhydrous /V-methyl-2-pyriOlidone (0.2 mL) and THF (0.1 mL) was added under an argon atmosphère. tert-Butyl maçinesium chloride (1M in THF, 24 pL, 0.024 mmol) was then added at RT, and white solids precipitated. After 5 min, a solution of p-nitrophenylphosphoamidate PD3c (15 mg, 0.032 mmol) in THF (0.1 mL) was added to the reaction mixture in one portion, and the resulting mixture was heated to 50 °C. After 3.5 h, the reaction mixture was allowed to cool to RT and was stirred for 18 h. p-Nitrophenylphosphoamidate PD3c (50 mg, 0.111 mmol) and tertbutyl magnésium chloride (1M in THF, 24 pL, 0.024 mmol) were then added and the reaction mixture was stirred for an additional 5 d. The resulting residue was then purified directly by preparatory HPLC (Phenominex Synergi 4u Hydro-RR 80Â 150 x 30 mm column, 40-100% acetonitrile/water gradient). The fractions containing the desired product were combined and were lyophilized to afford example 16 (PD5) (2:1 diastereomeric mixture).

¹H NMR (400 MHz, CDCI₃) δ 7.88 (br s, 1 H), 7.33 - 7.22 (br m, 2H), 7.22 - 7.10 (br m, 3H), 6.69 (brd, J =4.4 Hz, 1H), 6.61 (brd, 9 = 4.5 Hz, 1H), 5.64-5.56 (m, 1H), 4.54 (d, 9=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, 9=6.2 Hz, 1 H), 1.40 - 1.24 (m, 7H), 0.86 (t, J = 7.4 Hz, 6H).

³¹P NMR (162 MHz, CDCI3) δ 2.68 (s), 2.56 (s).

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 x 4.6 mm; Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min-2.0 min 2100% ACN, 2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN, 3.2 min-3.5 min 2% ACN at 2pl/min.

HPLC: t_R = 3.010 min; HPLC System: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at 2 mL/min.

Example 17 (TP5) - ((2R,3R,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-4-fluoro3-hydroxy-2-vinyltetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate.

To a solution of example 13 (6.0 mg, 0.020 mmol) in PO(OMe)3 (0.6 mL) at 0 °C was added POCI3 (50 mg, 0.32 mmol). The reaction mixture was stirred at 0 °C for 6 h, at which point lon-exchange HPLC showed approximately 90% conversion. A solution of pyrophosphate tributylamine salts (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 RT for 0.5 h, then concentrated and co-evaporated with water twice.

The residue was dissolved in H₂O (5 mL) and loaded to a ion-exchange column, eluted with H₂O, then 5-35% triethylammonium bicarbonate buffer (1M)-H₂O. The product fractions were combined, concentrated and co-evaporated with H₂O. The solid residue was dissolved in 3 mL of H₂O and 100 pL of NaOH (1 N) was added. The resulting mixture was purified with C-18 column, eluted with H₂O, and the fractions containing product were combined and concentrated under reduced pressure to afford Example 17 (TP5) as the tetra-sodium sait.

¹H NMR (400 MHz, D₂O): δ 7.74 (s, 1 H), 6.89 (d, J = 4.4 Hz, 1 H), 6.81 (d, J = 4.6 Hz, 1 H), 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).

³¹P 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).

¹⁹F NMR (376 MHz, CDCI₃) δ -194.58 (dt, J = 55.0, 23.8 Hz).

MS m/z = 533.0[M-1], 535.0 [M+1],

Example 18 (TP6) - ((2R,3R_J4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-ethyl-4fluoro-3-hydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate.

To a solution of example 14 (5.0 mg, 0.017 mmol) in PO(OMe)3 (0.6 mL) at 0 °C was added POCI3 (45 mg, 0.30 mmol). The reaction mixture was stirred at 0 °C for 6 h, at which point lon-exchange HPLC showed approximately 90% conversion. A solution of pyrophosphate tributylamine salts (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 RT for 0.5 h, then concentrated and co-evaporated with water twice. The residue was dissolved in H₂O (5 mL) and loaded to a ion-exchange column, eluted with

H₂O, then 5-35% triethylammonium bicarbonate buffer (1M)-H₂O. The product fractions were combined, concentrated and co-evaporated with H₂O. The solid residue was dissolved in 3 mL of H₂O and 100 pL of NaOH (1N) was added. The resulting mixture was purified with C-18 column, eluted with H₂O, and the fractions containing product were combined and concentrated under reduced pressure to afford 18 (TP6) as the tetra-sodium sait.

¹H NMR (400 MHz, D₂O): δ 7.73 (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, 1 H), 5.23 (dt, J = 55.2, 4.2 Hz, 1 H), 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).

¹⁹F NMR (376 MHz, CDCI₃) δ -194.77 (dt, J = 55.2, 21.2 Hz).

MS m/z = 535.1 [M-1], 536.9.0 [M+1],

2b

OPh

DMF

8a

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.

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 allowed to stir at room température for 5 min where complété conversion to product was observed by LCMS. The reaction was quenched with methanol and solvents were removed under reduced pressure. The crude material was partitioned between EtOAc and H₂O. The organics were separated and washed with brine. The resulting material was dried over Na₂SO₄, filtered and solvent removed under reduced pressure. The crude material was purified by silica gel chromatography (20 - 100 % EtOAc/Hexanes) to afford intermediate 8a.

¹H NMR (400 MHz, DMSO-cfe) δ 8.17 (m, 3H), 7.63 (t, J = 7.4 Hz, 1 H), 7.53 (t, J = 7.6 Hz, 2H), 7.17 - 6.96 (m, 2H), 5.74 (s, 1 H), 5.60 (d, J = 24.9 Hz, 1 H), 5.19 (ddd, J = 54.6, 4.5, 2.1 Hz, 1 H), 4.09 - 3.92 (m, 1 H), 3.72 (t, J = 6.4 Hz, 1 H), 3.63 (dd, J = 11.0, 3.4 Hz, 1 H), 3.44 (dd, J= 11.0, 5.9 Hz, 1H).

¹⁹F NMR (376 MHz, DMSO-cfe) δ -194.23 (m).

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 x 3.00 mm

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid

Gradient: 0 min-1.4 min 2-100% ACN, 1.4 min-1.80 min 100% ACN, 1.8 min-1.85 min 100%-2% ACN, 1.85 min-2 min 2% ACN.

^8a 8b

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

Intermediate 8a (80 mg, 0.166 mmol) was dissolved into 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 température and the solvent was removed under reduced pressure. The crude material was purified by silica gel chromatograpghy (0-70% EtOAc/Hex) to afford intermediate 8b.

¹H NMR (400 MHz, DMSO-cfe) δ 8.34-8.05 (m, 3H), 7.63 (t, J =7 A 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).

¹⁹F NMR (376 MHz, DMSO-cfe) δ -198.14 (ddd, J = 53.9, 24.7, 20.9 Hz).

LC/MS: t_R = 1.05 min, MS m/z= 355.15 [M+1]

100

LC system: Thermo Accela 1250 UHPLC

MS System: Thermo LCQ Fleet; Column: Kinetex2.6p C18 100A, 50 x 3.00 mm

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1 % formic acid

Gradient: 0 min-1.4 min 2-100% ACN, 1.4 min-1.80 min 100% ACN, 1.8 min-1.85 min 100%-2% ACN, 1.85 min-2 min 2% ACN.

HÔ F

8b

BnNMe₃CI, NaN₃₁ I2

NMM

Intermediate 8c - (2S,3R,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-a2iido-4fluoro-2-(iodomethyl)tetrahydrofuran-3-ol.

Benzyltrimethylammonium chloride (55 mg, 0.296 mmol) and sodium azide (19.3 mg, 0.296 mmol) were dissolved into ACN (1 mL). The resulting mixture was stirred at RT overnight and was then filtered and added via syringe to a solution of intermediate 8b (50 mg, 0.141 mmol) in THF (1 mL). /V-methylmorpholine (0.078 mL, 0.706 mmol) was then added followed by the dropwise addition of a solution of iodine (65 mg, 0.25 mmol) in THF (1 mL). After 15 min, ΛΖ-acetyl cysteine was added until the évolution of gas was no longer observable. Then saturated aqueous sodium thiosulfate was added until the solution was light yellow. The crude mixture was partitioned between EtOAc and H2O. The phases were split 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.

¹H NMR (400 MHz, DMSO-d₆) δ 8.31-8.05 (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, 1 H), 5.55 - 5.31 (m, 1 H), 4.62 (dt, J = 21.9, 5.9 Hz, 1 H), 3.78 - 3.56 (m, 2H).

¹⁹F NMR (376 MHz, DMSO-d₆) δ -194.44 (dt, J = 54.7, 22.8 Hz).

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 x 3.00 mm

101

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid

Gradient: 0 min-1.4 min 2-100% ACN, 1.4 min-1.80 min 100% ACN, 1.8 min-1.85 min 100%-2% ACN, 1.85 min-2 min 2% ACN.

8c

AûgO

DMAP

THF

8d

Intermediate 8d - (2S,3R,4S,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-az;ido-4fluoro-2-(iodomethyl)tetrahydrofuran-3-yl acetate.

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

¹H NMR (400 MHz, CDCI₃) δ 8.15-8.02 (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, 1 H), 7.00 (d, J = 4.6 Hz, 1 H), 5.95 - 5.80 (m, 1 H), 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).

¹⁹F NMR (376 MHz, CDCI3) δ -192.78 (ddd, J = 55.7, 24.6, 18.5 Hz).

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 x 3.00 mm

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1 % formic acid

Gradient: 0 min-1.4 min 2-100% ACN, 1.4 min-1.80 min 100% ACN, 1.8 min-1.85 min 100%-2% ACN, 1.85 min-2 min 2% ACN.

102

8d 8e

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.

To a solution of intermediate 8d (30 mgs, 0.053 mmol) in DMF (2 mL) was added 15Crown-5 (0.105 mL, 0.531 mmol) and sodium benzoate (77 mg, 0.531 mmol) at RT. The reaction was then heated to 105 °C. After 30 h, the reaction mixture was allowed to RT and was partitioned between 5% LiCI(_aq) and EtOAc. The phases were split and the aqueous phase was washed with EtOAc (2 x). The combined organic extracts were dried over anhydrous sodium sulfate, and were concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (0 - 60% EtOAc/Hex) to afford intermediate 8e.

¹H NMR (400 MHz, CDCI₃) δ 8.25 - 7.97 (m, 4H), 7.69 - 7.40 (m, 6H), 7.36 (d, J = 4.7 Hz, 1 H), 6.95 - 6.80 (m, 1 H), 5.90 (d, J = 25.0 Hz, 1 H), 5.65 (d, J = 1.9 Hz, 1 H), 5.62 - 5.48 (m, 1H), 4.69 (dd, J = 79.3, 12.0 Hz, 2H), 2.20 (s, 3H).

¹⁹F NMR (376 MHz, CDCI₃) δ -192.57 (ddd, J= 53.9, 25.1, 22.0 Hz).

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 x 3.00 mm

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid

Gradient: 0 min-1.4 min 2-100% ACN, 1.4 min-1.80 min 100% ACN, 1.8 min-1.85 min 100%-2% ACN, 1.85 min-2 min 2% ACN.

nh₃, ch₃oh

103

Example 19 - (2R,3R,4R,5S)-5-(4-aminopyrrolo[2_J1-f][1,2,4]triazin-7-yl)-2-azido-4-fluoro2-(hydroxymethyl)tetrahydrofuran-3-ol.

To intermediate 8e (24 mg, 0.043 mmol) was added 7N NH3 in CH3OH (2 mL) at RT. After 16 h, the resulting mixture was concentrated under reduced pressure. The crude residue was purified by reverse phase HPLC without acid modifier to afford example 19.

¹H NMR (400 MHz, Methanol-d₄) δ 7.81 (s, 1 H), 6.85 (d, J = 4.5 Hz, 1 H), 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).

¹⁹F NMR (376 MHz, Methanol-d₄) δ -195.30 (ddd, J = 55.5, 24.6, 22.9 Hz).

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 x 3.00 mm

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid

Gradient: 0 min-1.4 min 2-100% ACN, 1.4 min-1.80 min 100% ACN, 1.8 min-1.85 min 100%-2% ACN, 1.85 min-2 min 2% ACN.

NaH

BnBr

THF

9b

Intermediate 9b - ((3aR,5R,6S,6aR)-6-(benzyloxy)-5-((benzyloxy)methyl)-2,2dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)methanol ((3aR,6S,6aR)-6-(benzyloxy)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole-5,5diyl)dimethanol (9a, Purchased from Carbosynth, 10.0 g, 32.2 mmol) was added to a solution of sodium hydride (60% by wt, 1.55 g, 38.7 mmol) in THF (100 mL) at 0 °C under an argon atmosphère. After 10 min, benzyl bromide (4.54 mL, 38.6 mmol) was added and the reaction mixture was allowed to warm to RT. 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), was dried over anhydrous sodium sulfate, and was concentrated under reduced

104 pressure to afford a colorless oil. The crude residue was purified via S1O2 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.

¹H NMR (400 MHz, DMSO-d₆) δ 7.38 - 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 (brs, 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 x 4.6 mm; Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min-2.0 min 2100% ACN, 2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN, 3.2 min-3.5 min 2% ACN at 2pl/min.

HPLC: t_R = 3.79 min; HPLC System: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at2 mL/min.

TLC: eluent: 40% ethyl acetate in hexanes, Rf= 0.4 (UV)

Dess-Martin

Periodinane

CH₂CI₂

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

Dess-Martin Periodinane (3.1 g, 7.3 mmol) was added to a solution of intermediate 9b (1.95 g, 4.87 mmol) in dichloromethane (24.5 mL) at RT. After 1.5 h, the reaction mixture was purified via SiO2 column chromatography (80 g S1O2 Combiflash HP Gold Column, ΟΙ 00% ethyl acetate/hexanes) to afford intermdediate 9c (1.94 g, 100%) as a colorless oil.

¹H NMR (400 MHz, CDCb) δ 9.91 (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

105

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).

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: Kinetex2.6p XB-C18 100A, 50 x4.6 mm; Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min-2.C min 2100% ACN, 2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN, 3.2 min-3.5 min 2% ACN at 2pl/min.

HPLC: t_R = 4.09 min; HPLC system: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at 2 mL/min.

TLC: eluent: 40% ethyl acetate in hexanes, Rf= 0.6 (UV)

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

2.5M n-butyllithium (6.02 mL) was added to a solution of methyltriphenylphosphonium bromide (5.38 g, 15.1 mmol) in tetrahydrofuran (20 mL) at -78 °C. The reaction was allowed to warm 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 RT, and was stirred for 4 h. The reaction mixture was then quenched with saturated aqueous ammonium chloride solution (10 mL) and was partitioned between water (200 mL) and ethyl acetate (200 mL). The layers were split and the organic layer was washed with brine (200 mL), dried over anhydrous sodium sulfate, and was concentrated under reduced pressure. The crude residue was purified via SiO₂ column chromatography (120 g SiO₂ Combiflash HP Gold Column, 0-50% ethyl acetate/hexanes) to afford intermediate 9d (1.01 g, 51%) as a colorless oil.

106 ¹H NMR (400 MHz, CDCI₃) δ 7.42-7.17 (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, 1 H), 4.62 - 4.55 (m, 2H), 4.52 (d, J = 12.1 Hz, 1 H), 4.41 (d, J = 12.1 Hz, 1 H), 4.25 (d, J = 4.9 Hz, 1 H), 3.32 (d, J = 1.5 Hz, 2H), 1.52 (s, 3H), 1.29 (s, 3H)

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: Kinetex2.6p XB-C18 100A, 50 x4.6 mm; Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min-2.0 min 2100% ACN, 2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN, 3.2 min-3.5 min 2% ACN at 2pl/min.

HPLC: t_R = 4.37 min; HPLC system: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at 2 mL/min.

TLC: eluent: 50% ethyl acetate in hexanes, Rf= 0.55 (UV)

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

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

LC/MS: major anomer t_R = 2.00 min, MS m/z = 393.22 [M+Na], minor anomer t_R = 1.98 min, MS m/z = 393.22 [M+Na]; LC System: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; Column: Kinetex2.6p XB-C18 100A, 50 x 4.6 mm; Solvents: Acetonitrile with

107

0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% ACN, 2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN, 3.2 min-3.5 min 2% ACN at 2pl/min.

HPLC: major anomer t_R = 4.01 min, minor anomer t_R = 3.955 min; HPLC System: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at 2 mL/min.

TLC: eluent: 25% ethyl acetate in hexanes, Major anomer R_f = 0.30 (UV), Minor anomer R_f= 0.25 (UV)

O _o BnBr ^BnO^VO _o

J—( THF J—(

BnÔ OH BnÔ ÔBn

9e 9f

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

NaH (60% wt, 130 mg, 3.2 mmol) was added as a solid to a solution of intermediate 9e (1.0 g, 2.7 mmol) in THF (13.5 mL) at RT under an argon atmosphère. After 15 min, benzyl bromide (0.38 mL, 3.2 mmol) was added and the reaction mixture was stirred for 4 h. The reaction mixture was quenched with saturated aqueous ammonium chloride solution (5 mL) and was partitioned between ethyl acetate (100 mL) and brine (100 mL). The phases were split and the organic layer was dried over anhydrous sodium sulfate, and was concentrated under reduced pressure to afford crude intermediate 9f (1.57 g, ~2:1 mixture of 1' anomers) as a colorless oil.

LC/MS: major anomer t_R = 1.88 min, MS m/z = 483.36 [M+Na], minor anomer 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 x 4.6 mm; Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min-1.5 min 2-100% ACN, 1.5 min-2.2 min 100% ACN, 2.2 min-2.4 min 100%-2% ACN, 2.4 min-2.5 min 2% ACN at 2pl/min.

108

HPLC: major anomer t_R = 4.83 min, minoranomert_R = 4.62 min; HPLC System: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at 2 mL/min.

Intermediate 9g - (3R,4S,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)-5vinyltetrahydrofuran-2-ol

A solution of TFA (16 mL) and water (1.6 mL) was added to intermediate 9f (1.5 g, 3.2 mmol) at 0 °C, and the reaction mixture was allowed to warm to RT. After 9 h, water (1 mL) was added and the reaction mixture was allowed to stir an additional 10 h. The reaction mixture was then concentrated under reduced pressure. The crude residue was dissolved into ethyl acetate (200 mL) and was washed with saturated aqueous sodium bicarbonate solution (2 x 150 mL) and brine (150 mL). The organic layer was dried over anhydrous sodium sulfate and was concentrated under reduced pressure. The residue was purified via S1O2 column chromatography (24 g SiO₂ Combiflash HP Gold Column, 0-100% ethyl acetate/hexanes). The fractions containing the desired product were combined to afford intermediate 9g (580 mg) as a colorless oil that was a mixture with other impurities. The mixture was used directly in the following step.

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 x 4.6 mm; Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min-2.0 min 2100% ACN, 2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN, 3.2 min-3.5 min 2% ACN at 2pl/min.

HPLC: t_R = 4.34 min; HPLC system: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at 2 mL/min.

109

BnO °χ^0Η

ΤΡΑΡ ΝΜΟ

BnO

BnÔ ÔBn

CH₂CI₂

BnÔ ÔBn

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

Tetrapropylammonium perruthenate (45.7 mg, 130 pmol) and 4-methylmorpholine Λ/oxide (457 mg, 3.89 mmol) were added to a solution of intermediate 9g (580 mg, 1.30 mmol) and 4Â MS (100 mg) in DCM (6.45 mL) at RT. After 1 h, silica gel (-500 mg) was added to the reaction mixture and the resulting slurry was filtered through a plug of silica gel (-1 g). The filtrate was concentrated under reduced pressure. The crude residue was purified via S1O2 column chromatography (12 g S1O2 Combiflash HP Gold Column, 0-100% ethyl acetate/hexanes) to afford intermediate 9h (254 mg, 18% over two steps) as a colorless oil.

Ή NMR (400 MHz, CDCI3) δ 7.38 - 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)

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 x 4.6 mm; Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min-2.0 min 2100% ACN, 2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN, 3.2 min-3.5 min 2% ACN at 2μl/min.

HPLC: t_R = 4.53 min; HPLC system: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at2 mL/min.

TLC: eluent: 25% ethyl acetate in hexanes, Rf= 0.45 (UV)

110

9i

Intermediate 9i - (3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4bis(benzyloxy)-5-((benzyloxy)methyl)-5-vinyltetrahydrofuran-2-ol n-Butyllithium (2.5M in hexanes, 1.0 mL, 2.5 mmol) was added rapidly 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) at -78 °C under an argon atmosphère. 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 atmosphère. After 20 min, the reaction mixture was allowed to warm to 0 °C and was 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 was washed with saturated aqueous sodium bicarbonate solution (100 mL) and brine (100 mL). The organic layer was dried over anhydrous sodium sulfate, and was concentrated under reduced pressure. The crude residue was purified via SiO₂ column chromatography (12 g SiO₂ Combiflash HP Gold Column, 0-100% ethyl acetate/hexanes) to afford intermediate 9i (11.1 mg, 5%, mixture of isomers) as a colorless oil.

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 x 4.6 mm; Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min-2.0 min 2100% ACN, 2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN, 3.2 min-3.5 min 2% ACN 3ί2μΙ/ωΐη.

HPLC: t_R = 3.37 min; HPLC system: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at 2 mL/min.

TLC: eluent: ethyl acetate, Rf= 0.3 (UV)

111

9i 9j

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

To a solution of intermediate 9i (11.0 mg, 19.0 pmol) 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 atmosphère. After 1 h, the reaction mixture was slowly diluted with saturatecf aqueous sodium bicarbonate solution (10 mL), and the resulting mixture was extracted with ethyl acetate (2x10 mL), was dried over anhydrous sodium sulfate, and was concentrated under reduced pressure. The crude residue was purified via SiO2 column chromatography (4 g SiO₂ Combiflash HP Gold Column, 0-100% ethyl acetate/hexanes) to afford intermediate 9j (7.7 mg, 72%) as a colorless film.

¹H NMR (400 MHz, CDCI₃) δ 7.87 (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).

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: Kinetex2.6p XB-C18 100A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min-2.0 min 2100% ACN, 2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN, 3.2 min-3.5 min 2% ACN at 2μl/min.

HPLC: t_R = 3.51 min; HPLC system: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at 2 mL/min.

TLC: eluent: ethyl acetate, Rf= 0.40 (UV)

112

9j 20

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

Boron tribromide (1M, 0.06 mL, 60 pmol) was added dropwise to a solution of intermediate 9j (7.7 mg, 13.7 pmol) in dichloromethane (1 mL) at -78 °C under an argon atmosphère. After 1 h, the reaction mixture was allowed to warm to 0 °C, and was stirred an additional 1.5 h. The reaction was cooled to -78 °C and was quenched with a 2:1 methanol/pyridine solution (1.5 mL). The resulting mixture was allowed to warm to RT, and was concentrated under reduced pressure. The crude residue was purified by preparatory HPLC (Phenominex Synergi 4u Hydro-RR 80Â 150 x 30 mm column, 0-100% acetonitrile/water gradient) to afford example 20 (0.5 mg, 13%) as a white solid.

¹H NMR (400 MHz, CD₃OD) δ 7.78 (s, 1 H), 6.88 (d, J = 4.5 Hz, 1 H), 6.76 (d, J = 4.5 Hz, 1 H), 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)

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: Kinetex2.6p XB-C18 100A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min-1.5 min 2100% ACN, 1.5 min-2.2 min 100% ACN, 2.2 min-2.4 min 100%-2% ACN, 2.4 min-2.5 min 2% ACN at 2pl/min

HPLC: t_R = 2.181 min; HPLC System: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at 2 mL/min.

113

selectfluor

MeCN

Example 21 - (2R,3R,4R,5S)-5-(4-amino-5-fluoropyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-azido4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol

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 température for 30 min, and was then neutralized with sodium bicarbonate solution and filtered to remove solids. Upon concentration in vacuo, the residue was purified by préparative HPLC (acetonitrile 0 to 30% in water) to give example 21 (27 mg, 11%) as an off-white solid.

¹H NMR (400 MHz, CD₃OD) δ 7.73 (s, 1H), 6.63 (s, 1 H), 5.80 (dd, J = 23.9, 1.7 Hz, 1 H), 5.16 (ddd, J = 55.3, 5.0, 1.7 Hz, 1 H), 4.56 (dd, J = 23.5, 5.0 Hz, 1 H), 3.94 - 3.60 (m, 2H) ¹⁹F 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

fBuNO₂

HF/Pyr

11b

Intermediate 11 b - 2-fluoropyrrolo[2,1 -f][1,2,4]triazin-4-amine.

Intermediate 11a (2.0 g, 13.4 mmol) was charged to a polyTube vessel. The reaction vessel was then placed in an ice bath and both 70% HF/Pyr (18 mL) and pyridine (9 mL) were added sequentially. Immediately after the addition of pyridine, fBuNO₂(2.07 mL, 17.43 mmol) was then added slowly over 20 min. The solution goes from tan to black with an exotherm and outgassing. The reaction was then stirred for an additional 20 min, and the reaction mixture was then diluted with water, and was concentrated under reduced pressure. The crude residue was partitioned between ethyl acetate and water. The organics were

114 separated and the aqueous was washed with ethyl acetate three times. The organics were combined and washed with brine. The crude was dried over Na₂SO4, filtered, and the solvent was 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.

¹H NMR (400 MHz, DMSO-d₆) δ 8.49 - 8.09 (m, 2H), 7.58 (t, J = 2.0 Hz, 1 H), 6.95 (d, J = 4.5, 1 H), 6.59 (d, J =4.5, 1H).

¹⁹F NMR (376 MHz, DMSO-d₆) δ -73.42 (s).

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 x 3.00 mm;

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min1.4 min 2-100% ACN, 1.4 min-1.80 min 100% ACN, 1.8 min-1.85 min 100%-2% ACN, 1.85 min-2 min 2% ACN at 1.8mL/min.

dibromohydantoin

DMF

Intermediate 11c - 7-bromo-2-fluoropyrrolo[2,1-f][1,2,4]triazin-4-amîne.

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 by addition funnel over 40 min. After 1 h, the reaction was quenched with sat. Na2S₂O3(aq) and the crude was partitioned between EtOAc and 5% LiCI(_aq). The organics were extracted with 5% LiCI(_aq) (4 x) followed by brine. The organics were dried over Na₂SO4, the solids were removed by filtration, and the filtrate was concentrated under reduced pressure. The crude residue was sonicated with CH₂CI₂, and the solids were collected by filtration, and were dried under high vacuum to afford intermediate 11c (3.93 g, 77%) as a yellow solid.

115 ¹H NMR (400 MHz, DMSO-d₆) δ 8.49-8.44 (m, 2H), 7.08 (d, J= 4.6 Hz, 1H), 6.76 (d, J = 4.6 Hz, 1H).

¹⁹F NMR (376 MHz, DMSO-cfe) δ -71.45 (s).

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 x 3.00 mm;

Solvents: Acetonitrile with 0.1 % formic acid, Water with 0.1 % formic acid; Gradient: 0 min1.4 min 2-100% ACN, 1.4 min-1.80 min 100% ACN, 1.8 min-1.85 min 100%-2% ACN, 1.85 min-2 min 2% ACN at 1.8mL/min.

11c 11d

STABASE, nBuLi

THF, -78 °C

11e

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.

To a solution of 11c (2.09 g, 9.08 mmol) in THF (30 mL) was added 1,2bis(chlorodimethylsilyl)ethane (STABASE, 1.96 g, 9.08 mmol) in one portion, and the resulting mixture was allowed to stir at ambient température for 1 h. The reaction was then cooled to -78 °C using a dry ice bath with methanol. nBuLi (2.5M in hexanes, 10.9 mL, 27.2 mmol) was added in a manner to maintain an internai température of -65 °C. A solution of intermediate 11 d (Prepared according to WO2012012776, 2.5 g, 7.5 mmol) in THF (25 mL) was then added to the reaction mixture over 1 min. After 5 min, the reaction mixture was quenched with acetic acid and was allowed to corne to ambient température. The solvents were removed under reduced pressure and the residue was taken up into ethyl acetate. The organics were washed with water followed by brine. The layers were separated and the organics were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to afford crude 11e as a mixture of isomers, which was used as is for the next step.

LC/MS: t_R = 1.32 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 x 3.00 mm; Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0

116 min-1.4 min 2-100% ACN, 1.4min-1.80 min 100% ACN, 1.8min-1.85 min 100%-2%ACN, 1.85 min-2 min 2% ACN at 1.8mL/min.

11e

TES, BF₃.Et₂O

CH₂CI₂

11f

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

Intermediate 11e (1.99 g, 3.72 mmol) was dissolved into CH₂Cl2 (80 mL) and TES (4.75 mL, 29.7 mmol) was added to the mixture. The reaction mixture was cooled to 0 °C and BF₃ Et₂O (1.07 mL, 4.09 mmol) was slowly added. After 15 min, the reaction mixture was quenched with sat. NaHCO_3(aq) and the layers were separated. The aqueous layer was washed with CH₂CI₂. The organic layers were combined and were washed with sat. NaHCO₃₍aq). The organics were dried over Na₂SO₄, filtered, and were concentrated under reduced pressure. The crude was purified by silica gel chromatography (0-60% EtOAc/Hex) to afford intermediate 11f (1.12 g, 64%, 2:1 mixture of T anomers).

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 x 3.00 mm;

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min1.4 min 2-100% ACN, 1.4min-1.80 min 100% ACN, 1.8 min-1.85 min 100%-2% ACN, 1.85 min-2 min 2% ACN at 1.8mUmin.

h₂n

BnO F

11f

10% Pd/C, AcOH

H₂N

HO F

11g

117

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

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 backfilled with H₂(g) (3 x). After 1 h, the reaction vessel was purged with nitrogen. The resulting mixture was filtered through a pad of celite and the filter cake was washed with CH₃OH. The filtrate was concentrated under reduced pressure and then coevaporated with ethyl acetate followed by hexanes to afford intermediate 11g (503 mg, 98%, 2:1 mixture of T anomers).

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 x 3.00 mm; Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min1.4 min 2-100% ACN, 1.4 min-1.80 min 100% ACN, 1.8 min-1.85 min 100%-2%ACN, 1.85 min-2 min 2% ACN at 1.8ml_/min.

CH₃(OPh)₃Pl, DMF

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

To an argon purged flask was added a solution of 11g (283 mg, 0.989 mmol) in DMF (10 mL) followed by a solution of methyl triphenoxyphosphonium iodide (0.536 g, 1.19 mmol) in 4mL DMF. The reaction mixture was allowed to stir at 0 °C for 10 min and was then warmed to ambient température. After 30 min, the reaction was quenched with sat. Na₂S₂O₃(aq). The crude material was partitioned between EtOAc and 5% LiCI(aq). The organics were separated and washed with brine. The organics were dried over Na₂SO4, filtered, and concentrated under reduced pressure. The crude residue was taken up in ACN and purified by HPLC without acid modifier to afford intermediate 11h (201 mg, 52%, 2:1

118 mixture of T anomers) as a white solid.

LC/MS: îr = 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, 50x3.00 mm;

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1 % formic acid; Gradient 0 min1.4 min 2-100% ACN, 1.4 min-1.80 min 100% ACN, 1.8 min-1.85 min 100%-2% ACN, 1.85 min-2 min 2% ACN at1.8mL/min.

DBU, THF, 60 °C

11i

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

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 11 i (201 mg, 83%, 2:1 mixture of T anomers) as a white solid.

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 x 3.00 mm;

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min1.4 min 2-100% ACN, 1.4 min-1.80 min 100% ACN, 1.8 min-1.85 min 100%-2% ACN, 1.85 min-2 min 2% ACN at 1.8mUmin.

119

Bn(CH₃)₃NCI, NaN₃ l₂, NMM, ACN, THF

11j

Intermediate 11 j - (2S,3R,4R_J5S)-5-(4-amino-2-fluoropyrrolo[2,1^[1,2,4]triazin-7-yl)-2azido-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 température for 4 h. The mixture was filtered and the filtrate was added to a solution of 11 i (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 (4mL). After 15 min, N-acetyl cysteine was added portion wise until no outgassing was observed. Sat. Na₂S₂O₃(aq) was added until the solution was a light yellow. 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 11 j (183 mg, 56%) as a single isomer.

¹H NMR (400 MHz, DMSO-d₆) δ 8.44 (d, J = 28.6 Hz, 2H), 6.98 (d, J = 4.6 Hz, 1H), 6.79 (d, J = 4.6 Hz, 1 H), 6.32 (d, J = 6.9 Hz, 1 H), 5.60 (dd, J = 23.8, 2.5 Hz, 1 H), 5.36 (ddd, J = 54.9, 5.0, 2.6 Hz, 1 H), 4.60 (ddd, J = 21.5, 6.9, 5.0 Hz, 1 H), 3.63 (ABq, Δδ = 0.09ppm, J = 8Hz, 2H).

¹⁹F NMR (376 MHz, DMSO-d₆) δ -71.74 (s), -194.57 (ddd, J = 54.9, 24.0, 21.7 Hz)

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 x 3.00 mm; Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min2.4 min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8mL/min.

120

Isobutyric anhydride EfeN, DMAP, THF

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

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 allowed to stir at ambient température for 15 min, and the reaction was quenched with CH₃OH. 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.

¹H NMR (400 MHz, DMSO-d₆) δ 8.48 (d, J = 30.7 Hz, 2H), 6.99 (d, J= 4.6 Hz, 1H), 6.84 (d, J = 4.6 Hz, 1 H), 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).

¹⁹F NMR (376 MHz, DMSO-cfe) δ -71.58 (s), -194.89 (ddd, J = 55.0, 24.3, 16.8 Hz).

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 x 3.00 mm; Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min2.4 min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8mL/min.

h₂n

(NBu₄)HSO₄

Na₂HPO₄

3-chlorobenzoic acid

3-chloroperoxybenzoic acid

CH₂CI₂, H₂O

121

Intermediate 111 - ((2R,3R,4S,5S)-5-(4-amino-2-fluoropyrrolo[2,1-f][1,2,4]triazin-7-yl)-2azido-4-fluoro-3-(isobutyryloxy)tetrahydrofuran-2-yl)methyl 3-chlorobenzoate.

Intermediate 11k (0.153 g, 0.302 mmol) was dissolved in CH2CI2 (10 mL) and H₂O (6 mL). Potassium phosphate dibasic (0.138 g, 0.603 mmol), tetrabutylammonium bisulfate (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 allowed to warm to ambient température and was stirred for 16 h. The reaction mixture was then quenched with sat Na2S2O3(aq), and was concentrated under reduced pressure. The crude aqueous residue was diluted with ACN and purified by préparative HPLC without acid modifier to afford intermediate 111 (20 mg, 13%) as a white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 8.46 (d, J = 26.4 Hz, 2H), 7.96 - 7.81 (m, 2H), 7.81 - 7.66 (m, 1 H), 7.62 - 7.46 (m, 1 H), 6.94 (d, J = 4.5 Hz, 1 H), 6.80 (d, J = 4.5 Hz, 1 H), 5.73 (s, 3H), 4.60 (ABq, Δδ = 0.08ppm, J = 12 Hz, 2H), 2.66 (p, J = 7.0 Hz, 1 H), 1.20 - 1.01 (m, 6H).

¹⁹F NMR (376 MHz, DMSO-d₆) δ = -71.45 (s), -193.41 (ddd, J = 54.4, 25.4, 21.2 Hz,).

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 x 3.00 mm; Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min2.4 min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8ml_/min.

CH3OH, NH4OH

Example 22 - (2R,3R,4R,5S)-5-(4-amino-2-fluoropyrrolo[2,1-f][1,2,4]triazin-7-yl)-2-azido4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol

To a solution of intermediate 111 (22 mg, 0.041 mmol) in CH3OH (1 mL) was added

122 conc. NH4OH (1 mL) at RT. After 30 min, the reaction mixture was concentrated under reduced pressure. The crude residue was diluted with minimal H₂O and was purified by préparative HPLC without modifier to afford example 22 (10 mg, 77%) as a white solid.

¹H NMR (400 MHz, DMSO-cfe) δ 8.40 (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).

¹⁹F NMR (376 MHz, DMSO-cfe) δ -71.18 (s), -193.48 (dt, J= 55.3, 23.8 Hz).

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 x 3.00 mm; Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient 0 min2.4 min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8mL/min.

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)-4fluorotetrahydrofuran-2-carbonitrile

To a solution of intermediate 3d (57 mg, 0.109 mmol) in ACN (3 mL), was added

Selectfluor II in one portion (52 mg, 0.164 mmol). After 1.5 h, the reaction was quenched by the addition of saturated NaHCO₃(_aq). Ethyl acetate (4mL) was added and the biphasic mixture was stirred vigorously for 5 min. The reaction was further diluted with EtOAc and saturated NaHCO_3(aq). 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 ramp: 0% EtOAc in hexanes

123 ramping to 70% EtOAc in hexanes, quickly ramping to 100% EtOAc once the starting material elutes off the column.

¹H NMR (400 MHz, CD₃OD) δ 7.72 (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_ΑΒ = 0.141pprn, J= 11

Hz, 2H), 0.99 (s, 9H), 0.86 (s, 9H), 0.21 (s, 6H), 0.07 (s, 3H), -0.02 (s, 3H).

¹⁹F NMR (376 MHz, CD₃OD) δ -161.795 (s), -194.806 (ddd, J = 54.5, 19.2, 18.8 Hz).

LC/MS: Rt = 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 x 3.00 mm; Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min-2.4 min 2100% ACN, 2.4 min-2.8 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3 min 2% ACN.

12a

Example 23 - (2R,3R,4R,5S)-5-(4-amino-5-fluoropyrrolo[2,1-f][1,2,4]triazin-7-yl)-420 fluoro-3-hydroxy-2-(hydroxymethyl)tetrahydrofuran-2-carbonitrile

To a solution of intermediate 12a (29 mg, 0.054 mmol) in THF (2 mL) in a polypropylene tube was added 70% HF Pyridine in pyridine (51 qL, 1.97 mmol) at 0 °C, under an N2 atmosphère. After 1.5 h, the reaction was removed from the ice bath. Additional 70% HF Pyridine in pyridine was added after 3 h (150 qL), 5 h 45 min (200 qL), and 21 h 15 25 min (0.7 mL). The reaction mixture was then stirred for another 24 h at which point the reaction mixture was cooled in an ice bath and then quenched with water and saturated NaHCO₃(aq). The mixture was concentrated under reduced pressure and the residue was taken up in DMF. The resulting solution/suspension was filtered through a syringe filter (Whatman 0.45qm PTFE w/GMF). The filtrate was injected on an HPLC and the semi30 purified product was further purified by silica gel column chromatography using the following

124 solvent ramp: 0% MeOH in DCM ramping to 20% MeOH in DCM. Product containing fractions were concentrated and the residue was lyophilized to afford example 23 (5 mg, 30%) as a white powder.

¹H NMR (400 MHz, DMF-d₇) δ 7.74 (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, 1 H), 4.64 (dd, J = 22, 4.4 Hz, 1 H), 3.90 (ABq, Δ5αβ = 0.151ppm, 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).

.0

LC/MS: Rt = 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 x 3.00 mm; Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min-2.4 min 2-100% ACN, 2.4 min-2.8 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3 min 2% ACN.

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

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 température. Upon complété dissolution of iodine, a solution of compound 2g (650 mg, 0.93 mmol) in THF (5 mL) was added dropwise slowly. The resulting mixture was stirred at 80 °C for 3 days and was 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.

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

125

13a

13b

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

Intermediate 13a (200 mg, 0.243 mmol) was dissolved in methanol (10 mL) and under nitrogen atmosphère, 10% Pd/C (100 mg, 0.094 mmol) and TEA (0.035 mL, 0.243 mmol) were added. The resulting mixture was then stirred under H₂ atmosphère (balloon) at room température for 40 min. The resulting mixture was filtered, and concentrated in vacuo and the residue was purified by silica gel column chromatography (0 to 40% EtOAc in hexanes) to give intermediate 13b (145 mg, 72%) as a white solid with 75% purity.

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

13b

TBAF, THF;

NH₃, MeOH

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.

Intermediate 13b (145 mg, 75% purity, 0.177 mmol) was dissolved in THF (10 mL) and TBAF (1M in THF, 0.53 mL, 0.531 mmol) was added. The resulting mixture was stirred at room température for 2 h and then methanolic ammonia (7N, 10 mL) was added. The resulting mixture was stirred for 24 h and was concentrated under reduced pressure. The

126 crude residue was purified by préparative HPLC (0 to 35% acetonitrile in water in 20 min) to afford example 24 (30 mg, 60%) as a white solid.

¹H NMR (400 MHz, CD₃OD) δ 7.78 (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, 1 H), 3.65 - 3.43 (m, 2H), 1.27 (s, 3H) ¹⁹F 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

H₂N

BnÔ ÔBn

1e

10%Pd/C, AcOH

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

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 backfilled with H₂(g) three times. The reaction mixture was stirred under an atmosphère of H_2(g). After 1 h, the flask was purged with nitrogen and the reaction mixture was filtered through a celite pad with CH3OH washings. The filtrate was concentrated under reduced pressure and then coevaporated with EtOAc followed by hexanes. The residue was placed under high vacuum to afford intermediate 14a (1.31 g, 99%) as a white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 7.80 (s, 1 H), 7.66 (s, 2H), 6.82 (d, J = 4.4 Hz, 1 H), 6.66 (d,

J = 4.4 Hz, 1 H), 5.09 (d, J = 6.5 Hz, 1 H), 5.06-4.56 (m, 3H), 4.21 (t, J = 5.9 Hz, 1 H), 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).

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 x 3.00 mm;

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient 0 min127

2.4 min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8mL/min.

Intermediate 14b - ((3aR,4R,6S,6aS)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2,2dimethyltetrahydrofuro[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. Triethylorthoformate (6.0 mL, 36.1 mmol) was slowly added over 10 min. The resulting mixture was allowed to stir at ambient température overnight. Saturated aqueous sodium carbonate solution was added until the react on mixture was pH=8. The solids were removed by filtration and the filtrate was concentrated under reduced pressure. The crude residue was partitioned between EtOAc and brine. The phases were split and the organics were dried over Na2SO4, were filtered, and were concentrated under reduced pressure. The crude was purified by silica gel chromatography (60-100% EtOAc/Hex - 20% MeOH/EtOAc) to afford intermediate 14b (2.55 g, 71%) as a white solid.

¹H NMR (400 MHz, DMSO-de) δ 7.83 (s, 1 H), 7.71 (s, 2H), 6.83 (d, J = 4.4 Hz, 1 H), 6.73 (d, J = 4.5 Hz, 1 H), 5.21 (d, J = 4.9 Hz, 1 H), 5.01 (dd, J = 6.6, 4.9 Hz, 1 H), 4.84 (t, J = 5.7 Hz, 1 H), 4.71 (dd, J = 6.7, 3.7 Hz, 1 H), 3.99 - 3.85 (m, 1 H), 3.46 (t, J = 5.5 Hz, 2H), 1.48 (s, 3H), 1.29 (s, 3H).

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 x 3.00 mm;

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min1.4 min 2-100% ACN, 1.4 min-1.80 min 100% ACN, 1.8 min-1.85 min 100%-2%ACN, 1.85 min-2 min 2% ACN at 1.8ml_/min.

128 h₂n h₂n

14b 14c

Intermediate 14c - 7-((3aS,4S,6R,6aR)-6-(((tert-butyldîmethylsilyl)oxy)methyl)-2,2dimethyltetrahydrofuro[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 TBSCI (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 Na2SO4, were filtered, and were 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.

¹H NMR (400 MHz, DMSO-d₆) δ 7.83 (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).

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: Kinetex2.6p XB-C18 100A, 50 x 3.00 mm; Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient 0 min2.4 min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8mL/min.

129

BOC2O, TEA

DMAP, THF

TBSO

14d

Intermediate 14d -tert-butyl (7-((3aS,4S,6R,6aR)-6-(((tertbutyldimethylsilyl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4yl)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. Boc₂O (2.69 g, 12.3 mmol) and DMAP (0.3 g, 2.46 mmol) were then added. TEA (2.56 mL, 18.3 mmol) was slowly added and the reaction mixture was allowed to warm to RT. After 3 h, the reaction mixture was cooled to 0 °C and MeOH (10 mL) was added followed conc. NH₄OH_(aq) (50 mL). The resulting mixture was allowed to warm to RT and was 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, was filtered, and was 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.

¹H NMR (400 MHz, DMSO-cfe) δ 10.46 (s, 1H), 8.20 (s, 1H), 7.19 (d, J= 4.6 Hz, 1H), 6.90 (d, J = 4.6 Hz, 1 H), 5.33 (d, J = 4.2 Hz, 1 H), 5.02 (dd, J = 6.5, 4.3 Hz, 1 H), 4.72 (dd, J = 6.5, 3.6 Hz, 1 H), 4.01 (q, J = 5.0 Hz, 1 H), 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).

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 x 3.00 mm; Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient 0 min2.4 min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8mL/min.

130

TBAF, THF

14e

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

Intermediate 14d (2.8 g, 5.4 mmol) was dissolved in THF (50 mL), and TBAF (1.0M in THF, 5.92 mL, 5.92 mmol) was added. After 30 min, additional TBAF (1.0M in THF, 5.92 mL, 5.92 mmol) was added. After another 30 min, the reaction mixture was quenched with water and the resulting mixture was extracted with EtOAc (2 x). The combine the organic layers were washed with brine, were dried over Na₂SO4, were filtered, and were 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.

¹H NMR (400 MHz, DMSO-d₆) δ 10.46 (s, 1H), 8.22 (s, 1H), 7.20 (s, 1H), 6.95 (s, 1H), 5.29 (d, J = 4.6 Hz, 1 H), 5.03 (dd, J = 6.6, 4.7 Hz, 1 H), 4.85 (t, J = 5.7 Hz, 1 H), 4.72 (dd, J = 6.6, 3.6 Hz, 1 H), 4.05 - 3.90 (m, 1 H), 3.46 (t, J = 5.6 Hz, 2H), 1.50 (s, 12H), 1.29 (s, 3H).

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 x 3.00 mm; Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient 0 min2.4 min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8mL/min.

131

BocHN

1) EDCI, Pyr, TFA, DMSO

2) H₂CO, NaOH, Dioxane HO^

3) NaBH₄, EtOH Y° 7

HO— 'Λ_/

14f

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-4yl)carbamate.

Intermediate 14e (1.78 g, 4.38 mmol) was dissolved in DMSO (20 mL) and toluene (15mL). 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 CH2CI2. The aqueous was back extracted with CH2CI2. The organic layers were combined and were washed with brine, were dried over Na₂SO4, were filtered, and were concentrated under reduced pressure. The crude was put under high vacuum for 15 min then was used as is for the next step.

The crude residue was dissolved in dioxane (15 mL) and formaldéhyde (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 partitioned between sat. NaHCO₃(_aq) and CH₂CI₂. The aqueous layer was back extracted with CH₂CI₂. The organic layers were combined and were wash with brine, were dried over Na₂SO₄, were filtered, and were concentrated under reduced pressure. The crude was placed under high vacuum for 15 min then were taken directly into the next reaction.

The crude residue was dissolved in EtOH (50 mL), and NaBH₄ (0.324 g, 8.76 mmol) was added in small portions. After 20 min, the reaction mixture was quenched with AcOH and was concentrated under reduced pressure. The crude residue was partitioned between with EtOAc and sat. NaHCO_3(aq). The organic layer was split, was dried over Na₂SO₄, was filtered, and was concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (50-100% EtOAc/Hex) to afford intermediate 14f (1.91 g, 68%) as a white solid.

132 ¹H NMR (400 MHz, DMSO-d₆) δ 10.45 (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, 1 H), 3.73 - 3.46 (m, 3H), 3.40 - 3.30 (m, 1 H), 1.50 (s, 12H), 1.27 (s, 3H).

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 x 3.00 mm; Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1 % formic acid; Gradient: 0 min2.4 min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8mL/min.

DMTrCI, TEA, CH₂CI₂

Intermediate 14g -tert-butyl (7-((3aS,4S,6S,6aS)-6-((bis(4methoxyphenyl)(phenyl)methoxy)methyl)-6-(hydroxymethyl)-2,2dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyrrolo[2,1 -f][1,2,4]triazin-4yl)carbamate.

Intermediate 14f (1.15 g, 2.63 mmol) was dissolved in CH2CI2 (50 mL) and TEA (0.73 mL, 5.27 mmol) was added. The resulting solution was cooled to 0 °C and DMTrCI (1.35 g, 3.95 mmol) was added. After 10 min, the reaction mixture was quenched with CH₃OH as was then diluted with CH2CI2. The resulting mixture was washed with sat NaHCO₃(aq) and brine. The organic layer was dried over Na2SO4, was filtered, and was 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.

¹H NMR (400 MHz, DMSO-d₆) δ10.46 (s, 1H), 8.23 (s, 1H), 7.56-7.07 (m, 10H), 7.07133

6.70 (m, 5H), 5.24 (d, J = 5.2 Hz, 1 H), 5.04 (t, J = 5.9 Hz, 1 H), 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).

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: Kinetex2.6p XB-C18 100A, 50 x 3.00 mm; Solvents: Acetonitrile with 0.1 % formic acid, Water with 0.1 % formic acid; Gradient: 0 min2.4 min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8ml_/min.

TBSCI, Imid, DMF

Intermediate 14h -tert-butyl (7-((3aS,4S,6R,6aS)-6-((bis(4methoxyphenyl)(phenyl)methoxy)methyl)-6-(((tert-butyldimethylsilyl)oxy)methyl)-2,2dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyrrolo[2,1-f][1,2,4]triazin-415 yl)carbamate.

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 TBSCI (0.47 g, 3.11 mmol). After 1 h, the reaction was quenched with methanol and partitioned between EtOAc and 5% LiCI(_aq>- The phases were split and the organic layer was washed with brine, was dried over Na₂SO4, was 20 filtered, and was 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.

¹H NMR (400 MHz, DMSO-cfe) δ 10.47 (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,

H), 3.72 (s, 6H), 3.53 (d, J = 9.7 Hz, 1 H), 3.31 (m, 1 H), 3.08 (d, J = 9.8 Hz, 1 H), 1.50 (s,

134

9H), 1.25 (s, 3H), 1.22 (s, 3H), 0.75 (s, 9H), -0.04 (s, 3H), -0.08 (s, 3H).

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 x 3.00 mm;

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min1.0 min 2-100% ACN,1.0 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8mL/min.

TsOH, CHCI₃

MeOH

Intermediate 14i -tert-butyl (7-((3aS,4S,6R,6aS)-6-(((tertbutyldimethylsilyl)oxy)methyl)-6-(hydroxymethyi)-2,2-dimethyltetrahydrofuro[3,4d][1,3]dioxol-4-yl)pyrrolo[2,1-f][1,2,4]triazin-4-yl)carbamate.

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 CH3OH (16 mL) was then add slowly. After 30 min, the reaction was quenched with sat. NaHCO₃(aq), and the resulting mixture was partitioned between EtOAc and brine. The layers were split and the organic layer was dried over Na2SO₄, was filtered, and was 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.

¹H NMR (400 MHz, DMSO-cfe) δ 10.42 (s, 1H), 8.20 (s, 1H), 7.19 (s, 1H), 6.89 (s, 1H), 5.37 (d, J = 4.8 Hz, 1 H), 5.05 (dd, J = 6.2, 4.8 Hz, 1 H), 4.71 (d, J = 6.2 Hz, 1 H), 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, 1 H), 1.49 (s, 12H), 1.28 (s, 3H), 0.82 (s, 9H), -0.01 (s, 3H), -0.02 (s, 3H).

LC/MS: t_R = 1.88 min, MS m/z = 551.25 [M+H]; LC system: Thermo Accela 1250 UHPLC

135

MS System: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 x 3.00 mm;

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min1.0 min 2-100% ACN,1.0 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8mL/min.

1) EDCI, Pyr, TFA, DMSO, Toi

2) NH2OH*HCI, Pyr

3) CDI, ACN

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

Intermediate 141 (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 quenched with % sat. NaHCO₃(aq) and was partitioned between EtOAc and % sat. NaHCO_3(aq). The layers were separated and the organic layer was dried over Na₂SO4, was filtered, and was concentrated under reduced pressure. The residue was dissolved in CH2CI2 was concentrated under high vacuum for 1 h to afford a residue that was used directly in the next step.

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 was partitioned between EtOAc and water. The organic layer was dried over Na₂SO₄, was filtered, and was concentrated under reduced pressure. The crude residue was placed under high vacuum for 30 min and used as is for the third step.

136

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 quenched with ¹/₂ sat. NaHCO₃(_aq). The crude was partitioned between EtOAc and % sat. NaHCO_3(aq). The layers were separated and the organic layer was dried 5 over Na₂SO4, was filtered, and was 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.

¹H NMR (400 MHz, DMSO-d₆) δ 10.53 (s, 1H), 8.25 (s, 1H), 7.21 (s, 1H), 7.00 (d, J = 4.6 10 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).

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 x 3.00 mm;

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min2.4 min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8mL/min.

14k

Intermediate 14k - (3aS,4R,6S,6aS)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-420 (((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4d][1,3]dioxole-4-carbonitrile.

Intermediate 14j (0.688 g, 1.26 mmol) was dissolved in CH2CI2 (15 mL). Zinc bromide (0.567 g, 2.52 mmol) was added in one portion and the reaction mixture was stirred at ambient température. After 3 h, the reaction mixture was added to a silica load cartridge and was purified by silica gel chromatography (40100% EtOAc/Hex) to afford intermediate 14k (0.56 g, 99%) as a white solid.

137 ¹H NMR (400 MHz, DMSO-cfe) δ 7.86 (s, 1 H), 7.80 (s, 2H), 6.85 (d, J = 4.5 Hz, 1 H), 6.79 (d, J = 4.5 Hz, 1 H), 5.55 (d, J = 3.7 Hz, 1 H), 5.25 (dd, J = 6.6, 3.8 Hz, 1 H), 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).

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 x 3.00 mm; Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min-2.4 min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8mL/min.

AC2O, Pyridine

Intermediate 141 - N-(7-((3aS,4S,6R,6aS)-6-(((tert-butyldimethylsilyl)oxy)methyl)-6cyano-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)pyrrolo[2,1-f][1,2,4]triazin-4yl)acetamide.

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 température. After 30 min, the reaction mixture was quenched with methanol and was concentrated under reduced pressure. The crude residue was purified directly by silica gel chromatography (0-100% EtOAc/Hex) to afford intermediate 141 (0.185 g, 85%) as a white solid.

¹H NMR (400 MHz, DMSO-cfe) δ 10.87 (s, 1H), 8.31 (s, 1H), 7.24 (d, J = 4.7 Hz, 1H), 7.05 (d, j = 4.7 Hz, 1 H), 5.65 (d, J = 3.6 Hz, 1 H), 5.29 (dd, J = 6.6, 3.6 Hz, 1 H), 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).

138

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 x 3.00 mm;

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min1.4 min 2-100% ACN, 1.4 min-1.80 min 100% ACN, 1.8 min-1.85 min 100%-2% ACN, 1.85 min-2 min 2% ACN at 1.8ml_/min.

NBS, DMF

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

Intermediate 141 (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 afford intermediate 14m (50 mg, 54%) as an off-white solid.

¹H NMR (400 MHz, DMSO-cfe) δ 10.13 (s, 1H), 8.42 (s, 1H), 7.29 (s, 1H), 5.65 (d, J= 3.2 Hz, 1 H), 5.29 (dd, J = 6.6, 3.2 Hz, 1 H), 4.91 (d, J = 6.5 Hz, 1 H), 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).

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 x 3.00 mm ; Solvents: Acetonitrile with 0.1 % formic acid, Water with 0.1 % formic acid; Gradient 0 min1.4 min 2-100% ACN, 1.4 min-1.80 min 100% ACN, 1.8 min-1.85 min 100%-2% ACN, 1.85 min-2 min 2% ACN at 1.8mL/min.

139

nBuLi, NFSI, THF

Intermediate 14n - N-(7-((3aS,4S,6R,6aS)-6-(((tert-butyldimethylsilyl)oxy)methyl)-6cyano-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-5-fluoropyrrolo[2,1f][1,2,4]triazin-4-yl)acetamide.

Intermediate 14m (50 mg, 0.088 mmol) was dissolved in THF (2 mL) and th€ï solution was cooled to -78 °C. nBuLi (2.5M in hexanes, 0.071 mL, 0.18 mmol) was added. After 5 min, A/-fluorobenzenesulfonimide (NSFI, 33.4 mg, 0.106 mmol) was added and the réaction mixture was stirred for 5 min. The reaction was then quenched with AcOH. The solvents were removed under reduced pressure. The crude residue was purified by reverse phase HPLC to afford intermediate 14n (10 mg, 22%) as a white solid.

¹H 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).

¹⁹F NMR (376 MHz, Methanol-cL) δ -156.43 (s).

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 x 3.00 mm;

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min2.4 min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8mL/min.

140

1) TFA, H₂O

2) NH4OH, CH₃OH

HÔ OH

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

Intermediate 14n (11 mg, 0.022 mmol) was taken up into a solution of 50% TFA in water at ambient température. After 2 h, the reaction mixture was quenched with solid Na₂CO₃ to achieve a pH = 8. The solvent was removed under reduced pressure and the crude residue was purified by reverse phase HPLC. The fractions containing example 25 were combined and set aside and the fractions containing the N6-Acyl were combined and concentrated under reduced pressure. The N6-Acyl intermediate residue was taken up in conc. NH₄OH₍aq) (1 mL) and the mixture was allowed to stir at ambient température. After 30 min, 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 afford example 25 (4 mg , 53%) as a white solid.

¹H 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).

¹⁹F NMR (376 MHz, Methanol-d₄) δ -161.81 (s).

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 x 3.00 mm;

Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min1.4 min 2-100% ACN, 1.4 min-1.80 min 100% ACN, 1.8 min-1.85 min 100%-2% ACN, 1.85 min-2 min 2% ACN at 1.8mL/min.

141

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

Intermediate 14i (100 mg, 0.18 mmol) was dissolved in anhydrous pyridine (5 mL). Trifluoromethanesulfonyl chloride (23 pL, 0.22 mmol) was added in one portion and the reaction mixture was stirred for 45 min at RT. Additional trifluoromethanesulfonyl chloride (100 pL) was then added. After 30 min, additional trifluoromethanesulfonyl chloride (100 pL) was added. After an additional 30 min, more trifluoromethanesulfonyl chloride (100 pL) was added and the reaction was stirred for 30 min, at which point the reaction mixture was concentrated under reduced pressure. The crude residue was dissolved in anhydrous DMF (5 mL) and lithium chloride (153 mg, 3.6 mmol) was then added in one portion. The resulting mixture was stirred at RT for 16 h. The reaction mixture was diluted with ethyl acetate (50 mL) and was washed with saturated aqueous sodium chloride solution (3 x 20 mL). The organic layer was dried over anhydrous sodium sulfate and was concentrated under reduced pressure. The crude residue was purified with silica gel chromatography (0-20% ethyl acetate in hexanes) to afford intermediate 15a.

MS m/z = 569.0 [M+H]. MS system: Thermo LCQ Advantage

15a

142

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.

Intermediate 15a was dissolved in a solution of TFA and water (1:1,5 mL) and the resulting mixture was stirred for 16 h. The reaction mixture was then concentrated under reduced pressure. The crude residue was dissolved in aqueous sodium bicarbonate solution and acetonitrile and was purified with prep HPLC to afford example 26 (19 mg, 34%) as white powder.

¹H NMR (400 MHz, D₂O) δ 7.61 (s, 1H), 6.72-6.64 (m, 2H), 5.19 (d, 9=9.1 Hz, 1H), 4.734.66 (m, 1 H), 4.28 (d, J = 5.2 Hz, 1 H), 3.78 (s, 2H), 3.72 - 3.57 (m, 2H).

MS m/z = 315.3 [M+H], MS system: Thermo LCQ Advantage

(TP7)

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

Example 27 was prepared as the tetra-sodium sait in a manner similar to that descr bed for example TP4 starting with example 19.

Ή NMR (400 MHz, D₂O) δ 7.76 (s, 1H), 6.83 (d, 9= 4.4 Hz, 1H), 6.80 (d, 9= 4.8 Hz, 1H), 5.94 (d, J = 25.2 Hz, 1 H), 5.24 (dd, J = 55.2, 5.2 Hz, 1 H), 4.78 (dd, J = 26.8, 5.2 Hz, 1 H), 4.08-4.18 (m, 2H).

¹⁹F NMR (376 MHz, D₂O) δ -193.74 - -194.02 (m).

143 ³¹P 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).

HÔ F p(Q)ci₃;

[nBu₃NH]₂P₂O₇H₂

Example 28 - ((2R,3R,4R,5S)-5-(4-amino-5-fluoropyrrolo[2,1-f][1,2,4]triazin-7-yl]i-25 azido-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyi tetrahydrogen triphosphate.

Example 28 was prepared as the tetra-sodium sait in a manner similar to that described for example TP4 starting with example 21.

¹H NMR (400 MHz, D₂O) δ 7.64 (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).

¹⁹F NMR (376 MHz, D₂O) δ -161.00 (s), -196.39 - -196.69 (m).

³¹P 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

p(Q)ci₃;

[nBu₃NH]₂P₂O₇H₂

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

144

Example 29 was prepared as the tetra-sodium sait in a manner similar to that described for example TP4 starting with example 22.

¹H NMR (400 MHz, D₂O) δ 6.81 (d, J = 4.4 Hz, 1 H), 6.75 (d, J = 4.8 Hz, 1 H), 5.81 (cl, 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).

¹⁹F NMR (376 MHz, D₂O) δ -75.95 (s), -196.51 --196.80 (m).

³¹P 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

30

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

Example 30 was prepared as the tetra-sodium sait in a manner similar to that described for example TP4 starting with example 23.

¹H NMR (400 MHz, D₂O) δ 7.64 (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).

¹⁹F NMR (376 MHz, D₂O) δ -161.05 (s), -194.92--195.19 (m).

³¹P 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

145

TP11

Example 31 (also TP11) - ((2R,3R,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-4fluoro-3-hydroxy-2-methyltetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate.

Example 31 was prepared as the tetra-sodium sait in a manner similar to that described for example TP4 starting with example 24 ¹H NMR (400 MHz, D₂O) δ 7.66 (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).

¹⁹F NMR (376 MHz, D₂O) δ -195.74 - -196.01 (m).

³¹P 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).

Example 32 (also TP12) - ((2R_J3S,4R,5S)-5-(4-amino-5-fluoropyrrolo[2,1-f][1,2,4]triazin7-y|)-2-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate.

Example 32 was prepared as the tetra-sodium sait in a manner similar to that described for example TP4 starting with example 25.

146 ¹H NMR (400 MHz, D₂O) δ 7.59 (s, 1 H), 6.57 (s, 1H), 5.44 (d, J=6.0Hz, 1H), 4.56 (d, J = 5.2 Hz, 1 H), 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).

¹⁹F NMR (376 MHz, D₂O) δ -161.25 (s).

³¹P 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

Example 33 (also TP13) - ((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2_J4]triazin-7-yl)-210 (chloromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate.

Example 33 was prepared as the tetra-triethylamine sait in a manner similar to that described for example TP3 starting with example 26.

¹H NMR (400 MHz, D₂O) δ 7.76 (s, 1 H), 6.92 (br s, 1 H), 6.85 (br s, 1 H), 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), 15 3.83 - 3.95 (m, 3H), 3.07 (q, J = 7.6 Hz, 24 H), 1.16 (t, J = 7.6 Hz, 36 H).

³¹P 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

147

PD3c tBuMgCI

DMF, THF

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

Example 1 (3.8 mg, 0.013 mmol) was dissolved in anhydrous /V-methyl-2-pyrrolidone (0.2 mL) and THF (0.1 mL) was added under an argon atmosphère. tert-Butyl magnésium chloride (1M in THF, 20 pL, 0.024 mmol) was then added at RT, and white solids precipitated. After 5 min, a solution of p-nitrophenylphosphoramidate PD3c (12 mg, 0.026 mmol) in THF (0.1 mL) was added to the reaction mixture in one portion, and the resulting mixture was heated to 50 °C. After 20 h, the reaction mixture was allowed to cool to RT and was then purified directly by preparatory HPLC (Phenominex Synergi 4u Hydro-RR 80Â 150 x 30 mm column, 40-100% acetonitrile/water gradient). The fractions containing the desired product were combined and were lyophilized to afford example 34 (2.9 mg, 37%, 3:2 diastereorneric mixture) as a white solid.

¹H NMR (400 MHz, CD₃OD) δ 7.80 (s, 0.3H), 7.78 (s, 0.6H), 7.38 - 7.10 (m, 5H), 6.85 (br dd, J = 4.7, 2.2 Hz, 1 H), 6.75 - 6.71 (m, 1 H), 5.54 - 5.46 (m, 1 H), 4.65 - 4.58 (m, 1 H), 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 ³¹P NMR (162 MHz, CD3OD) δ 3.25 (br s).

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 2.6p XB-C18 100A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% acetic acid, Water with 0.1% acetic acid; Gradient: 0 min-2.0 min 2100% ACN, 2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN, 3.2 min-3.5 min 2% ACN at2pl/min.

148

HPLC: t_R = 2.98 min; HPLC system: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at 2 mL/min.

PD7a (BuMgCI

DMF, THF

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

Example 1 (19 mg, 65.3 pmol) was dissolved in NMP (0.2 mL). THF (0.1 ml.) was added followed by tert-butyl magnésium chloride (1.0M solution in tetrahydrofuran, 0.098 mL) at RT under an argon atmosphère. After 5 min, a solution of intermediate PD7a (Prepared according to US20120009147A1, 51.4 mg, 130 pmol) in THF (0.1 mL) was added, and the resulting mixture was warmed to 50 °C. After 1 h, the reaction mixture was allowed to cool to RT and was purified directly by preparatory HPLC (Phenominex Synergi 4u Hydro-RR 80Â 150 x 30 mm column, 5-100% acetonitrile/water gradient). The fractions containing product were combined, and concentrated and the resulting residue was repurified by preparatory HPLC (Phenominex Luna 5u C18 100 x 30 mm column, 5-100% acetonitrile/water gradient) to afford example 35 (12 mg, 34%, 3:2 mixture of diastereomers) as a white solid.

¹H NMR (400 MHz, CD₃OD) δ 7.80 (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).

³¹P NMR (162 MHz, CD3OD) δ 3.27 (br s).

LC/MS: major diastereomer t_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 2.6p XB-C18 100A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% aceticacid, Water with 0.1% acetic acid; Gradient: 0 min-2.0 min 2-100% ACN,

149

2.0 min-3.05 min 100% ACN, 3.05 min-3.2 min 100%-2% ACN, 3.2 min-3.5 min 2% ACN at 2pl/min.

HPLC: major diastereomer t_R = 2.44 min, minor diastereomer t_R = 2.46 min; HPLC System: Agilent 1100 sériés.; Column: Gemini 5μ C18 110A, 50 x 4.6 mm; Solvents: Acetonitrile with 0.1% TFA, Water with 0.1% TFA; Gradient: 0 min-5.0 min 2-98% ACN, 5.0 min-6.0 min 98% ACN at 2 mL/min.

Also provided are separate embodiments comprising, respectively, compounds of Formula (A), Formula (B), Formula (C), Formula (D), and Formula (E):

wherein, in each instance, X¹ represents an oxygen protecting group and X² represents an amine protecting group.

Useful oxygen protecting groups include a silyl ether protecting group or a benzyltype protecting group, including methoxybenzyl groups.

Useful silyl ether protecting groups include Trimethylsilyl (TMS), Triethylsilyl (TES), Dimethylisopropylsilyl (IPDMS), Diethylisopropylsilyl (DEIPS), Dimethylthexylsilyl (TDS), tButyldimethylsilyl (TBS orTBDMS), t-Butyldiphenylsilyl (TBDPS), Tribenzylsilyl, Tri-pxylylxilyl, Triisopropylsilyl (TIPS), Diphenylmethylsilyl (DPMS), Di-f-butylmethylsilyl (DTBMS), Triphenylsilyl (TPS),Methyldiphenylsilyl (MDPS), t-butylmethoxyphenylsilyl, Tris(trimethylsilyl)silyl (sisyl), (2-Hydroxystyryl)dimethylsilyl (HSDMS), (2Hydroxystyryl)diisopropylsilyl (HSDIS). f-Butylmethoxyphenylsilyl (TBMPS), and tButoxydiphenylsilyl (DPTBOS) protecting groups.

150

Useful benzyl-type protecting groups include benzyl, halogenated benzyl, pmethoxybenzyl, benzyloxymethyl, 2,4-dimethoxybenzyl, 3,4-dimethoxybenzyl, 2,6dimethoxybenzyl, p-CFa-benzyl, p-methylbenzyl, p-methoxylbenzyl, 3,5-dimethylbenzyl, ptert-butylbenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, including p-Br-benzyl, 2,6dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2,6-difluorobenzyl, p-acylaminobenzyl (PAB), p-azidobenzyl (Azb), 4-azido-3-chlorobenzyl, 2-trifluoromethylbenzyl, p(methylsulfinyl)benzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido, 2-quinolinylmethyl, diphenylmethyl (DPM), ρ,ρ-dinitrobenzhydryl, triphenylmethyl, alphanaphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(pmethoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4,4’,4”tris(benzoyloxyphenyl)methyl, and 2-naphthylmethyl protecting groups.

Useful amine protecting groups include p-methoxybenzyl carbonyl (Moz or MeOZ), acetyl (Ac), benzoyl (Bz), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), pmethoxyphenyl (PMP), tosyl (Ts orTos), 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 9fluorenylmethyloxycarbonyl (FMOC), 9-(2-sulfo)fluorenylmethyl, 9-(2,7dibromo)fluorenylmethyl, 17-tetrabenzo[a,c,g,/]fluorenylmethyl (Tbfmoc), 2-chloro-3indenylmethyl (Climoc), benz[f]inden-3-ylmethyl (Bimoc), 2,7-di-t-butyl[9-(10,10-dioxo10,10,10,10-tetrahydrothioxanyl)]methyl (DBD-Tmoc), [2-(1,3-dithianyl)methyl (Dmoc), and 1,1-dioxobenzo[b]thiophene-2-ylmethyl (Bsmoc) carbamates.

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), 1l,1-dimethyl-2-bromoethyl, 1,1-dimethyl-2-chloroethyl, 1,1-dimethyl-2,2dibromoethyl (DB-LBOC), 1,1-dimethyl-2,2,2-trichloroethyl (TCBOC), 1-methyl-1-(4biphenylyl)ethyl (Bpoc), 1-(3,5-di-f-butylphenyl)-1-methylethyl (LBumeoc), 2-(2’pyridyl)ethyl, 2-(4’pyridyl)ethyl, 2,2-bis(4’-nitrophenyl)ethyl (Bnpeoc), A/-(2-pivaloylamino)1,1,dimethylethyl, 2-[(2-nitrophenyl)dithio]-1-phenylethyl (NpSSPeoc), 2-(N,Ndicyclohexylcarboxamido)ethyl, t-butyl (Boc or BOC), 1-adamantyl (1-Adoc), 2-adarnantyl (2Adoc), vinyl (Voc), allyl (Aloc or alloc), 1 -isopropylallyl (Ipaoc), cinnamyl (Coc), 4nitrocinnamyl (Noc), 3-(3’-pyridyl)prop-2-enyl (Paloc), 8-quinolyl, and /V-hydroxypiperidinyl,

151 carbamates, as well as alkyldithio carbamates, including methyldithio, ethyldithio, isopropyldithio, t-butyldithio, and phenyldithio carbamates.

Also useful are aryl-containing and substituted aryl-containing carbamates such as 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-phenylacetoxybenzyl (PhAcOZ), 4-azidobenzyl (ACBZ), 4azidomethoxybenzyl, 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 carbamates.

Useful amide protecting groups for amines include /V-formyl, /V-acetyl, /V-chloroacetyl, /V-trichloroacetyl, /V-trifluoroacetyl (TFA), /V-phenylacetyl, A/-3-phenylpropionyl, Λ/-4pentenoyl, /V-picolinoyl, A/-3-pyridylcarboxamido, A/-benzoylphenylalanyl, /V-benzoyl, and Np-phenylbenzoyl amides.

Antiviral Activity

Another embodiment relates to methods of inhibiting viral infections, comprising the step of treating a sample or subject suspected of needing such inhibition with a composition herein.

Considered herein are samples suspected of containing a virus include natural or man-made materials such as living organisms; tissue or cell cultures; biological samples such as biological material samples (blood, sérum, urine, cerebrospinal fluid, tears, sputum, saliva, tissue samples, and the like); laboratory samples; food, water, or air samples; bioproduct samples such as extracts of cells, particularly recombinant cells synthesizing a desired glycoprotein; and the like. Typically the sample will be suspected of containing an organism which induces a viral infection, frequently 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 manmade materials such as cell cultures.

If desired, the anti-virus activity of a compound after application of the composition

152 can be observed by any method including direct and indirect methods of detecting such activity. Quantitative, qualitative, and semiquantitative methods of determining such activity are ail contemplated. Typically one of the screening methods described above are applied, however, any other method such as observation of the physiological properties of a living organism are also applicable.

The antiviral activity of a compound can be measured using standard screening protocols that are known. For example, the antiviral activity of a compound can be measured using the following general protocols.

Respiratory syncytial virus (RSV) antiviral activity and cytotoxicity assays

Anti-RSV activity

Antiviral activity against RSV is determined using an infectious cytopathic cell protection assay in HEp-2 cells. In this assay, compounds inhibiting viral infection and/or réplication produce a cytoprotective effect against the virus-induced cell killing that can be quantified using a cell viability reagent. The techniques used here are novel adaptations of methods described in published literature (Chapman et al., Antimicrob Agents Chemother. 2007, 57(9):3346-53.)

HEp-2 cells are obtained from ATCC (Manassas, VI) and maintained in MEM media supplemented with 10% fêtai bovine sérum and penicillin/streptomycin. Cells are passaged twice a week and kept at subconfluent stage. Commercial stock of RSV strain A2 (Advanced Biotechnologies, Columbia, MD) is titered before compound testing to détermine the appropriate dilution of the virus stock that generates désirable cytopathic effect in HEp-2 cells.

For antiviral tests, HEp-2 cells are grown in large cell culture flasks to near confluency but not fully so. The compounds to be tested are prediluted in DMSO in 384-well compound dilution plates, either in an 8 or 40 sample per plate standardized dose response format. 3-fold serial dilution incréments of each test compound are prepared in the plates and test samples are transferred via acoustic transfer apparatus (Echo, Labcyte) at 100 ni per well into cell culture assay 384-well plates. Each compound dilution is transferred in single or quadruplicate samples into dry assay plates, which are stored until assay is ready to go. The positive and négative Controls are laid out in opposite on ends of the plate in vertical blocks (1 column).

153

Subsequently, an infectious mixture is prepared using an appropriate dilution of virus stock previously determined by titration with cells at a density of 50,000/ml and 20 uL/well is added to test plates w/compounds via automation (uFlow, Biotek). Each plate includes négative and positive Controls (16 replicates each) to create 0% and 100% virus inhibition standards, respectively. Following the infection with RSV, testing plates are incubated for 4 days in a 37 °C cell culture incubator. After the incubation, a cell viability reagent, Cell TiterGIo (Promega, Madison, Wl) is added to the assay plates, which are incubated briefly, and a luminescent readout is measured (Envision, Perkin Elmer) in ail the assay plates. The RSV-induced cytopathic effect, percentage inhibition, is determined from the levels of remaining cell viability. These numbers are calculated for each tested concentration relative to the 0% and 100% inhibition Controls, and the ECso value for each compound is determined by non-linear régression as a concentration inhibiting the RSV-induced cytopathic effect by 50%. Various potent anti-RSV tool compounds are used as positive Controls for antiviral activity.

Cytotoxicity Assay in HEp-2 Cells

Cytotoxicity of tested compounds is determined in uninfected HEp-2 cells in parallel with the antiviral activity using the cell viability reagent in a similar fashion as described before for other cell types (Cihlar et al., Antimicrob Agents Chemother. 2008,52(2):655-65.). The same protocol as for the détermination of antiviral activity is used for the measurement of compound cytotoxicity except that the cells are not infected with RSV. Instead, an uninfected cell mixture at the same density is added at 20 ul/well to plates containing prediluted compounds, also at 100 nl/sample. Assay plates are then incubated for 4 days followed by a cell viability test using the same CelITiter Glo reagent addition and measurement of luminescent readouts. Untreated cell and cells treated at 2 uM puromycin (Sigma, St. Louis, MO) serve as 100% and 0% cell viability control, respectively. The percent of cell viability is calculated for each tested compound concentration relative to the 0% and 100% Controls and the CCso value is determined by non-linear régression as a compound concentration reducing the cell viability by 50%.

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, Cat# 9160) supplemented with 10% FBS, 100 units/mL penicillin, 100

154 units/mL streptomycin, and 2 mM L-Glutamine. The MT-4 cells were passaged twice per week to maintain cell densities below 0.6 x 10⁶cells/mL. Complété RPMI-1640 media containing 100x concentrations of 3-fold serially diluted compound, ranging from 26 nM to 530 μΜ, were stamped in quadruplicate into black 384-well plates. After compound addition, 5 2 x 10³ MT-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.

Following the incubation the cells were allowed to equilibrate to 25 °C and cell viability was determined by adding 25 pL of Cell-Titer Glo viability reagent. The mixture was incubated for 10 minutes at 25 °C, and the luminescence signal was quantified on a Victor

Luminescence plate reader. The CC50 value is defined as the concentration of compound that reduces cell viability by 50% as determined by the Cell-Titer Glo signal. The data were analyzed using Pipeline Pilot Plate Data Analytics Collection software (Version 7.0, Accelrys, San Diego, CA). CC50 values were calculated from a non-linear régression analysis using a 4-parameter sigmoidal dose-response équation: Y = Bottom + (Top15 Bottom)/(1+10^A[(LogCC50-X)*HillSlope]) where the Top and Bottom were fixed at 100% and 0% cell viability, respectively. CC50 values were calculated as the average ± standard déviation of 3 independent experiments.

Example	EC50 / pM	HEp-2 CC50 / μΜ	MT-4 CC50 / pM
1	7.3	>50	>53
2	9.6	>100	>106
3	2.0	>100	>106
4	30	>50	>53
5	2.1	>50	>53
10 (PD1)	1.0	39	7.3
11 (PD2)	1.2	42	19.7
12 (PD3)	3.0	>50	27
13	10.5	>100	>106

155

14	40	>100	>106
15 (PD4)	1.6	35	7.9
16 (PD5)	0.3	18	25
19	0.40	>44	16
20	>50	>50
21	50	36	15
22	0.57	>100	32
23	23.5	72	21
24	1.0	>100	11
25	9.2	>100	>93
26	>50	42	>57
34 (PD6)	0.21	>50	>50
35 (PD7)	0.34	>50	47

Another benefit relates to advantage that compounds bearing R⁴ substitution provides in comparison to compounds lacking R⁴ substitution (i.e. those in which R⁴= H) with regard to MT-4 cytotoxicity. For example, compound (2S,3R,4S,5R)-2-(4-aminopyrrolo[1,25 f][1,2,4]triazin-7-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (Patil, S. A.; Otter, R. A.; Klein, R. S. Tetrahedron Lett. 1994, 35, 5339-5342), having the structure:

156 exhibits a MT-4 CCso = 0.007 μΜ; whereas Examples 1, 4, 5, 20, and 26 ail exhibit a MT-4 CC50 > 53 μΜ. Furthermore, compound (2R,3R,4F?,5S)-5-(4-aminopyrrolo[1,2/][1,2,4]triazin-7-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol (WO201203703SA1), 5 having the structure:

exhibits a MT-4 CC50 = 30 μΜ; whereas Examples 2, 3, 13, and 14 ail exhibit a MT-4 CC50 > 106 μΜ.

Another benefit relates to advantage that R³ = F compounds bearing R⁴ substitution provides 10 in comparison to compounds lacking R⁴ substitution (i.e. those in which R⁴= H) with regard to HEp-2 anti-RSV activity. For example the compound (2R,3R,4R,5S)-5-(4aminopyrrolo[1,2-f|[1,2,4]triazin-7-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ot (WO2012037038A1) having the structure above has a HEp-2 EC50 = >100 μΜ; whereas examples 2, 3, 13, 14, 19, 21, 22, 23,and 24 ail exhibit EC50 = <100uM.

157

RSV RNP Préparation

RSV ribonucleoprotein (RNP) complexes were prepared from a method modified from Mason et al (1). HEp-2 cells were plated at a density of 7.1 x 10⁴ cells/cm² in MEM + 10% fêtai bovine sérum (FBS) and allowed to attach ovemight at 37°C (5% CO₂). Following attachment, the cells were infected with RSV A2 (MOI=5) in 35 mL MEM + 2% FBS. At 20 hours post-infection, the media was replaced with MEM + 2% FBS supplemented with 2 pg/mL actinomycin D and retumed to 37°C for one hour. The cells were then washed once with PBS and treated with 35 mL of PBS + 250 pg/mL lyso-lecithin for one minute, after which ail liquid was aspirated. The cells were harvested by scrapping them into 1.2 mL of buffer A [50 mM TRIS acetate (pH 8.0), 100 mM potassium acetate, 1 mM DTT and 2 pg/mL actinomycin D] and lysed by repeated passage through an 18 gauge needle (10 times). The cell lysate was placed in ice for 10 minutes and then centrifuged at 2400g for 10 minutes at 4°C. The supernatant (S1) was removed and the pellet (P1) was disrupted in 600 uL of Buffer B [10 mM TRIS acetate (pH 8.0), 10mM potassium acetate and 1.5 mM MgCI₂] supplemented with 1% Triton X-100 by repeated passage through an 18 gauge needle (10 times). The resuspended pellet was placed in ice for 10 minutes and then centrifuged at 2400g for 10 minutes at 4°C. The supernatant (S2) was removed and the pellet (P2) was disrupted in 600 uL of Buffer B supplemented with 0.5% deoxycholate and 0.1% Tween 40. The resuspended pellet was placed in ice for 10 minutes and then centrifuged at 2400g for 10 minutes at 4°C. The supernatant (S3) fraction, containing the enriched RSV RNP complexes, was collected and the protein concentration determined by UV absorbance at 280 nm. Aliquoted RSV RNP S3 fractions were stored at -80°C.

RSV RNP Assay

Transcription reactions contained 25pg of crude RSV RNP complexes in 30 pL of reaction buffer [50 mM TRIS-acetate (pH 8.0), 120 mM potassium acetate, 5% glycerol, 4.5 mM MgCI₂, 3 mM DTT, 2 mM ethyleneglycol-bis(2-aminoethylether)-tetraacetic acid (EGTA), 50 pg/mL BSA, 2.5 U RNasin (Promega), ATP, GTP, UTP, CTP and 1.5 uCi [α-³²Ρ] NTP (3000 Ci/mmol)]. The radiolabled nucléotide used in the transcription assay was selected to match the nucléotide analog being evaluated for inhibition of RSV RNP transcription. Cold, compétitive NTP was added at a final concentration of one-half its K_m (ATP= 20 pM, GTP= 12.5 pM, IJTP= 6 pM and CTP= 2 pM). The three remaining nucléotides were added at a final concentration of 100 pM.

158

To détermine whether nucléotide analogs inhibited RSV RNP transcription, compounds were added using a 6 step serial dilution in 5-fold incréments. Following a 90 minute incubation at 30°C, the RNP reactions were stopped with 350 pL of Qiagen RLT lysis buffer and the RNA was purified using a Qiagen RNeasy 96 kit. Purified RNA was denatured in RNA sample loading buffer (Sigma) at 65°C for 10 minutes and run on a 1.2% agarose/MOPS gel containing 2M formaldéhyde. The agarose gel was dried and exposed to a Storm phosphorimager screen and developed using a Storm phosphorimager (GE: Healthcare). The concentration of compound that reduced total radiolabled transcripts by 50% (ICso) was calculated by non-linear régression analysis of two replicates.

ReferenceMason, 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 Reseiarch, 32, 4758-4767.

Example	IC501 pM
6 (TP1)	0.086
7 (TP2)	1
8 (TP3)	0.025
9 (TP4)	0.12
17 (TP5)	0.56
18(TP6)	0.42
27 (TP7)	0.097
28 (TP8)	0.086
29 (TP9)	0.080
30 (TP10)
31 (TP11)
32 (TP12)	0.022
33 (TP13)

159

A further considération relates to advantage that compounds exemplified exhibit potent inhibition of the RSV RNP transcription in comparison to compounds with 2’CMe substitution. For example, ((2R,3R,4R,5S)-5-(4-aminopyrrolo[2,1-/][1,2,4]triazin-7-yl)-3,4dihydroxy-4-methyltetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate (WO2008089105A2, and WO2010002877A2), having the structure:

exhibits an IC50 = 8.5 μΜ; whereas Example TP3 exhibits an IC50 = 0.025 pM. Furthermore, compound ((2R,3/?,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-4-fluoro-3-hydroxy-4methyltetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate (WO 2011035231 A1), having the structure:

exhibits an IC₅₀ = >100 pM; whereas Examples TP1 exhibits an IC50 = 0.086 pM.and TP2 exhibits an IC50 = 1 pM.

Claims (40)

1. What is Claimed:

   1. A compound of Formula (I), or a pharmaceutically acceptable sait thereof:

   wherein:

   R¹ is H or F;

   R² is H or F;

   R³ is OH or F;

   R⁴ is CN, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C4 cycloalkyl, azido, halogen, or C1-C2 haloalkyl;

   R⁶ is OH; R⁵ is selected from the group of H and: ⁰ , n U- - u J HO , °l j , HQ _HO A O n A ï » 1 ^HO HO ^? ,

   161

   wherein:

   n’ is selected from 1, 2, 3, and 4;

   R⁸ is selected from Ci-Cs alkyl, -O-Ci-Cs alkyl, benzyl, -O-benzyl, -CH2-C3-C6 cycloalkyl, -O-CH2-C3-C6 cycloalkyl, and CF₃;

   R⁹ is selected from phenyl, 1-naphthyl, 2-naphthyl,

   R¹⁰ is selected from H and CH₃;

   R¹¹ is selected from H or Ci-Ce alkyl;

   R¹² is selected from H, Ci-Cs alkyl, benzyl, C3-C6 cycloalkyl, and -CH2-C3-C6 cycloalkyl.
2. 2. A compound according to Claim 1, or a pharmaceutically acceptable sait thereof, wherein R¹ is H.
3. 3. A compound according to Claim 1, or a pharmaceutically acceptable sait thereof, wherein R² is H.
4. 4. A compound according to any of Claims 1,2, or 3 wherein both R¹ and R² are H.

   162
5. 5. A compound according to any of Claims 1,2,3, or 4, or a pharmaceutically acceptable sait thereof, wherein R¹, R², and R⁵ are each H.
6. 6. A compound according to any of Claims 1,2,3, 4, or 5, or a pharmaceuticéilly acceptable sait thereof, wherein both R¹ and R² are H, and R³ is OH.
7. 7. A compound according to any of Claims 1, 2, 3, 4, or 5, or a pharmaceutically acceptable sait thereof, wherein both R¹ and R² are H, R³ is F.
8. 8. A compound according to any of Claims 1,2,3, or 4, or a pharmaceutically acceptable sait thereof, of Formula (II):

   wherein:

   R³ is OH or F;

   R⁴ is CN, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C4 cycloalkyl, azido, halogen, or C1-C2 haloalkyl;

   R⁵ is selected from the group of H and:

   II

   HO—P--;

   HO

   163

   wherein:

   ri is selected from 1, 2, 3, and 4;

   R⁸ is selected from Ci-Cs alkyl, -O-Ci-Ce alkyl, benzyl, -O-benzyl, -CH2-C3-C6 cycloalkyl, -O-CH2-C3-C6 cycloalkyl, and CF3;

   R⁹ is phenyl;

   R¹⁰ is selected from H and CH₃;

   R¹¹ is selected from H or Ci-Ce alkyl;

   R¹² is selected from H, Ci-Cs alkyl, benzyl, C3-C6 cycloalkyl, and -CH2-C3-C6 cycloalkyl.
9. 9. A compound according to any of Claims 1,2,3, 4, 5, or 8, or a pharmaceutically acceptable sait thereof, wherein R⁴ is CN, methyl, ethyl, ethenyl, ethynyl, azido, F, Cl, CH2CI, -CH2F, -CHF₂, or -CF₃.
10. 10. A compound according to any of Claims 1, 2, 3, 4, 5, 7, 8, or 9, or a pharmaceutically acceptable sait thereof, wherein R³ is F.
11. 11. A compound according to any of Claims 1,2,3, 4, 5, 6, 8, or 9, or a pharmaceutically acceptable sait thereof, wherein R³ is OH.

    164
12. 12. A compound according to any of Claims 1, 2, 3, 4, 5, 7, 8, 9, or 10, or a pharmaceutically acceptable sait thereof, wherein R³ is F and R⁴ is CN.
13. 13. A compound according to any of Claims 1,2,3, 4, 5, 7, 8, 9, or 11, or a pharmaceutically acceptable sait thereof, wherein R³ is OH and R⁴ is CN.
14. 14. A compound according to any of Claims 1,2, 3, 4, 5, 7, 8, 9, or 10, or a pharmaceutically acceptable sait thereof, wherein both R¹ and R² are H, R³ is F, and R⁴ is methyl, ethyl, vinyl, or ethynyl.
15. 15. A compound according to any of Claims 1,2,3, 4, 5, 7, 8, 9, or 11, or a pharmaceutically acceptable sait thereof, wherein R³ is OH, and R⁴ is methyl, ethyl, vinyl, or ethynyl.
16. 16. A compound according to any of Claims 1,2, 3, 4, 5, 7, 8, 9, or 10, or a pharmaceutically acceptable sait thereof, wherein R³ is F and R⁴ is halomethyl.
17. 17. A compound according to any of Claims 1,2,3, 4, 5, 7, 8, 9, or 11, or a pharmaceutically acceptable sait thereof, wherein R³ is OH and R⁴ is halomethyl.
18. 18. A compound according to any of Claims 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17, or a pharmaceutically acceptable sait thereof, wherein R⁵ is H.
19. 19. A compound according to any of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17, or a pharmaceutically acceptable sait thereof, wherein R⁵ is selected from the group of:

    165

    wherein:

    R⁸ is selected from Ci-C₈ alkyl, -O-Ci-C₈ alkyl, benzyl, and -CH2-C3-C6 cycloalkyl; and

    R¹² is selected from Ci-C₈ alkyl, benzyl, C3-C6 cycloalkyl, and -CH2-C3-C6 cycloalkyl.
20. 20. A compound according to any of Claims 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 19, or a pharmaceutically acceptable sait thereof, wherein R⁸ and R⁹ are each C1C₈ alkyl.
21. 21. A compound according to any of Claims 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 19, or a pharmaceutically acceptable sait thereof, wherein R⁸ and R⁹ are each selected from Ci-Ce alkyl.
22. 22. A compound according to any of Claims 1,2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, Ί 9, 20, or 21, or a pharmaceutically acceptable sait thereof, wherein R⁸ and R⁹ are each selected from C1-C5 alkyl.

    166
23. 23. A compound according to any of Claims 1,2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, or 22, or a pharmaceutically acceptable sait thereof, wherein R^& and R^s are each selected from Ci-C₄ alkyl.
24. 24. A compound according to any of Claims 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 sait thereof, further comprising the proviso that when R³ is F, R⁴ is not methyl.
25. 25. A compound according to any of Claims 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 sait thereof, wherein R⁵ is selected from the group of H and:

    O

    II

    HO—P--;

    (a) HC)

    O

    HO—P^ (b) HO
26. 26. A compound according to any of Claims 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 sait thereof, wherein R⁵ is:

    HO—P--HO
27. 27. A compound according to any of Claims 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 sait thereof, wherein R⁵ is:

    HO—P. p

    HO pA

    167
28. 28. A compound according to any of Claims 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 sait thereof, wherein R⁵ is:
29. 29. A compound according to any of Claims 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, or28, ora pharmaceutically acceptable sait thereof, selected from the group of:
30. 30. A compound according to any of Claims 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, or28, ora pharmaceutically acceptable sait thereof, selected from the group of:

    168
31. 31. A compound according to any of Claims 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 sait thereof, selected from the group of:

    169
32. 32. A compound according to any of Claims 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 sait thereof, selected from the group of:
33. 33. A compound according to any of Claims 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 sait thereof, selected from the group of:

    170
34. 34. A compound according to any of Claims 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, ora pharmaceutically acceptable sait thereof, selected from the group of:

    171
35. 35. A compound of any of Claims 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, or 34, or a pharmaceutically acceptable sait thereof for use in the treatment of a Pneumovirinae virus infection in a human.
36. 36. A compound of any of Claims 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, or 34, or a pharmaceutically acceptable sait thereof for use in the treatment of a respiratory syncytial virus infection in a human.
37. 37. A pharmaceutical formulation comprising a pharmaceutically effective amount of a compound of any of Claims 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, or 34, or a pharmaceutically acceptable s ait thereof, and a pharmaceutically acceptable carrier or excipient.
38. 38. Use of a compound of any of Claims 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, or 34, or a pharmaceutically

    172 acceptable sait thereof in the manufacture of a médicament for treating a Pneumovirinae virus infection or a respiratory syncytial virus infection in a human.
39. 39. A compound selected from Formula (A), Formula (B), Formula (C), Formula (D), and Formula (E):

    X¹-0 F (A) :

    wherein X¹ is an oxygen protecting group and X² is an amine protecting group.
40. 40. A compound of Claim 39 selected from: