EA045066B1 - PYRROLO[1.2-f][1.2.4]TRIAZINES USED IN THE TREATMENT OF RESPIRATORY SYNCYTIAL VIRUS INFECTIONS - Google Patents

Abstract

The invention provides compositions, methods of using and compounds of substituted tetrahydrofuranylpyrrolo[1.2-f][1.2.4]triazin-4-amines of formula (I) for the treatment of Pneumovirinaes virus infection, including respiratory syncytial virus infections, as well as methods and intermediates for the synthesis of tetrahydrofuranylpyrrolo[1.2-f][1.2.4]triazin-4-amine compounds.

Description

Field of invention

The present invention provides substituted tetrahydrofuranylpyrrolo[1.2^[1.2.4]triazin-4-amine compounds, methods and pharmaceutical compositions for the treatment of viral infection.

Pneumovirinaes, in particular including respiratory syncytial virus infections, and methods and intermediates used to prepare the compound.

Background of the invention

Pneumovirinae viruses are single-stranded, negative-sense RNA viruses that cause many common diseases in humans and animals. The Pneumovirinae subfamily of viruses is part of the Paramyxoviridae family and includes human respiratory syncytial virus (HRSV). Almost all children in the first two years of life are infected with HRSV. HRSV is a leading cause of lower respiratory tract infection in infancy and childhood, requiring hospitalization in 0.5 to 2% of all infections. Older adults and adults with chronic heart disease, lung disease, or those with weakened immune systems are also at high risk of developing severe disease from HRSV (http://www.cdc.gov/rsv/index.html). No vaccine is currently available to prevent HRSV infection. The monoclonal antibody palivizumab is available for immunoprophylaxis, but its use in infants is limited due to high risk, such as in premature infants or in children with congenital heart defects or lung disease. Its cost is often prohibitive for widespread use. Furthermore, the nucleoside analogue ribavirin has been approved as the only antiviral agent for the treatment of HRSV infection, but its efficacy is limited. Therefore, there is a need for anti-Pneumovirinae drugs.

Examples of pyrrolo[2,3-d]pyrimidine compounds used to treat 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 Al (Butler et al.), WO 2011/150288 Al (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 effective antiviral agents with acceptable toxicity profiles that can be used in the treatment of Paramyxoviridae viral infections, including Pneumovirinae viral infections such as HRSV infections.

The essence of the invention

Proposed are compounds, methods, and pharmaceutical compositions for the treatment of infections caused by the Pneumovirinae virus family, including the treatment of infections caused by respiratory syncytial virus in humans.

A compound of formula (I) or a pharmaceutically acceptable salt thereof is provided:

R ⁶ where

R ¹ is H;

R ² is H or F;

R ³ is OH or F;

R ⁴ is CN, C1-C4 alkyl, C2-C4 alkenyl, or C1-C2 haloalkyl, wherein when R ³ is OH, R ⁴ is not C2-C ₄ alkenyl;

R ⁶ is OH;

R ⁵ is selected from the group consisting of H and

- 1 045066

where n' is selected from 1, 2, 3 and 4;

R ⁸ is C ₁ -C ₈ alkyl;

R ⁹ is phenyl;

R ¹⁰ is selected from H and CH ₃ ;

R ¹¹ is selected from H or C1-C6 alkyl;

R ¹² is C1-C8 alkyl.

Detailed description of the invention

One embodiment of the present invention includes a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R ¹ is H and all other variables, including R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² and n', have the meanings defined above for formula (I).

Another embodiment of the present invention includes a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R ² is H, and all other variables, including R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² and n' are as defined above for formula (I).

A further embodiment of the present invention includes a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein both R ¹ and R ² are H, and all other variables, including R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² and n' have the meanings defined above for formula (I).

Another embodiment of the present invention includes a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein both R ¹ , R ² , and R ⁵ are H, and all other variables, including R ³ , R ⁴ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² and n' are as defined above for formula (I).

Another specific embodiment of the present invention includes a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein both R ¹ and R ² are H, R ³ is OH, and all other variables, including R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² and n', have the meanings defined above for formula (I).

Another specific embodiment of the present invention includes a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein both R ¹ and R ² are H, R ³ is F, and all other variables, including R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² and n', have the meanings defined above for formula (I).

Another embodiment of the present invention includes a compound of formula (II) or a pharmaceutically acceptable salt thereof:

Where

R ³ is OH or F;

R ⁴ is CN, C1-C4 alkyl, C2-C4 alkenyl, or C1-C2 haloalkyl, wherein when R ³ is OH, R ⁴ is not C2-C ₄ alkenyl;

R ⁵ is selected from the group consisting of H and

- 2 045066

where n' is selected from 1, 2, 3 and 4;

R ⁸ is C ₁ -C ₈ alkyl;

R ⁹ is phenyl;

R ¹⁰ is selected from H and CH ₃ ;

R ¹¹ is selected from H or C1-C6 alkyl;

R ¹² is C ₁ -C ₈ alkyl.

A further embodiment of the invention includes a compound of formula (II) or a pharmaceutically acceptable salt thereof, wherein:

R ³ is OH or F;

R ⁴ is CN, methyl, ethyl, -CH ₂ Cl, -CH ₂ F, -CHF ₂ or -CF ₃ ;

and R ⁵ and all other groups are as defined for formula (II).

Also provided is an embodiment of the invention comprising a compound of formula (II) or a pharmaceutically acceptable salt thereof, as described above, wherein R ³ is F.

Also provided is an embodiment of the invention comprising a compound of formula (II) or a pharmaceutically acceptable salt thereof, as described above, wherein R ³ is OH.

Also provided is an embodiment of the invention comprising a compound of formula (II) or a pharmaceutically acceptable salt thereof, as described above, wherein R ³ is F and R ⁴ is CN.

Also provided is an embodiment of the invention comprising a compound of formula (II) or a pharmaceutically acceptable salt thereof, as described above, wherein R ³ is OH and R ⁴ is CN.

Also provided is an embodiment of the invention comprising a compound of formula (II) or a pharmaceutically acceptable salt thereof as described above, wherein both R ¹ and R ² are H, R ³ is F, and R ⁴ is methyl, ethyl, vinyl.

Also provided is an embodiment of the invention comprising a compound of formula (II) or a pharmaceutically acceptable salt thereof, as described above, wherein R ³ is OH, and R ⁴ is methyl, ethyl, vinyl.

Also provided is an embodiment of the invention comprising a compound of formula (II) or a pharmaceutically acceptable salt thereof, as described above, wherein R ³ is F and R ⁴ is halomethyl.

Also provided is an embodiment of the invention comprising a compound of formula (II) or a pharmaceutically acceptable salt thereof, as described above, wherein R ³ is OH and R ⁴ is halomethyl.

Also provided is an embodiment of the invention comprising a compound of formula (II) or a pharmaceutically acceptable salt thereof, as described above, wherein R ⁵ is H.

Also provided is an embodiment of the invention comprising a compound of formula (II) or a pharmaceutically acceptable salt thereof as described above, wherein each R ⁵ is H, R ³ is OH, and R ⁴ is methyl, ethyl, vinyl.

Also provided is an embodiment of the invention comprising a compound of formula (II) or a pharmaceutically acceptable salt thereof as described above, wherein R ⁵ is H, R ³ is F and R ⁴ is halomethyl.

Also provided is an embodiment of the invention comprising a compound of formula (II) or a pharmaceutically acceptable salt thereof, as described above, wherein R ⁵ is H, R ³ is OH, and R ⁴ is halomethyl.

In each of the above embodiments of the invention, comprising a compound of formula (II) or a pharmaceutically acceptable salt thereof, wherein R ⁵ may be other than H, there is a further embodiment, wherein all other variables have the meanings defined above for that embodiment, and R ⁵ is selected from the group consisting of:

- 3 045066

Where

R ⁸ is C ₁ -C ₈ alkyl and

R ¹² is C1-C8 alkyl.

In each of the embodiments of the invention described herein comprising a compound of formula (I) or formula (II), there is a further embodiment wherein all variables are as defined for the particular embodiment and meeting the additional condition that when R ³ is F, R ⁴ is not methyl.

Definitions

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

The term "haloalkyl," as used herein, refers to an alkyl group, as defined herein, wherein one or more hydrogen atoms are each replaced with a halogen substituent. For example, ( _C1 - _C6 ) haloalkyl is (C1-C6) alkyl wherein one or more hydrogen atoms are replaced with a halogen substituent. This range includes from a single halogen substituent on the alkyl group to complete halogenation of the alkyl group.

The term (C _1-n )haloalkyl, as used herein, where n is an integer, either alone or in combination with another radical, is intended to mean an alkyl radical having from 1 to n carbon atoms as defined above, wherein one or more hydrogen atoms, each, are replaced by a halogen substituent. Examples of (C _1-n )haloalkyl, where n is 2, include, but are not limited to, chloromethyl, chloroethyl, dichloroethyl, bromomethyl, bromoethyl, dibromoethyl, fluoromethyl, difluoromethyl, trifluoromethyl, fluoroethyl, and difluoroethyl.

The term (C _1-n ) alkyl, as used herein, where n is an integer, either alone or in combination with another radical, is intended to denote acyclic straight- or branched-chain alkyl radicals containing from 1 to n carbon atoms. (C _1-4 ) alkyl includes, but is not limited to, methyl, ethyl, propyl (n-propyl), butyl (n-butyl), 1-methylethyl (isopropyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (isobutyl), and 1,1-dimethylethyl (tert-butyl or t-butyl). The abbreviation Me denotes a methyl group; Et denotes an ethyl group, Pr denotes a propyl group, iPr denotes a 1-methylethyl group, Bu denotes a butyl group, and tBu denotes a 1,1-dimethylethyl group.

The term alkyl refers to a hydrocarbon containing normal, secondary, or tertiary atoms. For example, an alkyl group may contain 1 to 4 carbon atoms (i.e., (C1- _C4 )alkyl), 1 to 3 carbon atoms (i.e., ( _C1 - _C3 )alkyl), or 1 or 2 carbon atoms (i.e., ( _C1 - _C2 )alkyl). Examples of suitable alkyl groups include, but are not limited to, methyl (Me, -CH ₃ ), ethyl (Et, -CH ₂ CH ₃ ), 1-propyl (n-Pr, n-propyl, -CH ₂ CH ₂ CH ₃ ), 2-propyl (i-Pr, isopropyl, -CH(CH ₃ ) ₂ ), 1-butyl (n-Bu, n-butyl, -CH ₂ CH ₂ CH ₂ CH ₃ ), 2-methyl-1-propyl (i-Bu, isobutyl, -CH ₂ CH(CH ₃ ) ₂ ), 2-butyl (sec-Bu, sec-butyl, -CH (CH ₃ )CH ₂ CH ₃ ) and 2-methyl-2-propyl (tert-Bu, tert-butyl, -C(CH ₃ ) ₃ ) and the like. The term alkyl also refers to a saturated hydrocarbon radical, either branched or straight chain, containing two monovalent centers within the radical formed by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkyl radicals include, but are not limited to, methylene (-CH ₂ -), 1,1-ethyl (-CH(CH ₃ )-), 1,2-ethyl (-CH ₂ CH ₂ -), 1,1-propyl (-CH(CH ₂ CH ₃ )-), 1,2-propyl (-CH ₂ CH(CH ₃ )-), 1,3-propyl (-CH ₂ CH ₂ CH ₂ -), 1,4-butyl (-CH2CH2CH2CH2-), and the like.

An alkenyl is a straight or branched chain hydrocarbon containing normal, secondary, or tertiary carbon atoms with at least one site of unsaturation, i.e., a carbon-carbon, sp ² double bond. As examples, an alkenyl group may contain from 2 to 4 carbon atoms (i.e., C 2-C 4 alkenyl) or from 2 to 3 carbon atoms (i.e., C 2-C 3 alkenyl). Examples of suitable alkenyl groups include, but are not limited to, ethylene or vinyl (-CH=CH ₂ ) and allyl (-CH ₂ CH=CH ₂ ).

The term (C _2-n ) alkenyl as used herein, where n is an integer, either alone or in combination with another radical, is intended to denote an unsaturated, straight- or branched-chain, acyclic radical containing from two to n carbon atoms, at least two of which are bonded to each other by a double bond. Examples of such radicals

- 4 045066 include, but are not limited to, ethenyl (vinyl), 1-propenyl, 2-propenyl, and 1-butenyl. Unless otherwise specified, the term (C _2-n )alkenyl is understood to encompass individual stereoisomers where possible, including, but not limited to, the (E) and (Z) isomers and mixtures thereof. When a (C ₂ . _n )alkenyl group is substituted, it is understood to be substituted at any carbon atom thereof that is otherwise bonded to a hydrogen atom, unless otherwise specified, and substituted in such a manner that the substitution results in a chemically stable compound, as understood by those skilled in the art.

Pharmaceutical compositions

This document also provides a pharmaceutical composition comprising a pharmaceutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or ester thereof and a pharmaceutically acceptable carrier and excipient. Also provided are separate pharmaceutical compositions, each comprising a pharmaceutically effective amount of a compound of formula (II) or one of the specific compounds according to the examples given herein, or a pharmaceutically acceptable salt, solvate and/or ester thereof and a pharmaceutically acceptable carrier and excipient.

During preparation of the dosage form, the compounds of the present invention are combined with conventional carriers and excipients, which are selected in accordance with standard practice. Tablets will contain excipients, lubricants, fillers, binders, and the like. Aqueous compositions are prepared sterile and, when intended for delivery other than oral administration, are typically isotonic.

All compositions optionally contain excipients, such as those reviewed in the Handbook of Pharmaceutical Excipients (1986). Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextran, hydroxyalkyl cellulose, hydroxyalkyl methylcellulose, stearic acid, and the like. The pH of the formulations ranges from about 3 to about 11, but is typically from about 7 to 10.

While it is possible for the active ingredients to be administered alone, it may be preferable to present them as pharmaceutical compositions. Compositions for both veterinary and human use comprise at least one active ingredient as defined above, together with one or more acceptable carriers and, optionally, other therapeutic ingredients, particularly those additional therapeutic ingredients discussed herein. The carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and physiologically harmless to the recipient.

The compositions include those suitable for the above-mentioned routes of administration. The compositions may be presented as unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. A description of the methods and formulations can be found primarily in Remingtons Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). Such methods include the step of bringing into association the active ingredient with a carrier, which comprises one or more accessory ingredients. Generally, the compositions are prepared by uniformly and intimately mixing the active ingredient with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Compositions suitable for oral administration may be presented as discrete units such as capsules, caffeine, or tablets, each containing a predetermined amount of the active ingredient; as a powder or granule; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be administered as a bolus, caffeine, or paste.

Tablets are prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be obtained by compressing in a suitable machine the active ingredient in a free-flowing form, such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active agent, or dispersing agent. Molded tablets can be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. Tablets can optionally be coated or scored and, optionally, designed to provide slow or controlled release of the active ingredient.

In case of infections of the eye or other external tissues, for example, the mouth and skin, the compositions are preferably applied in the form of a topical ointment or cream containing the active ingredient(s) in an amount of, for example, from 0.075 to 20% w/w (including the active ingredient(s) in the range between 0.1% and 20% in increments of 0.1% w/w, for example 0.6% w/w, 0.7% w/w, etc.), preferably from 0.2 to 15% w/w, and most preferably from 0.5 to 10% w/w. When formulated as an ointment, the active ingredients can be used with either a paraffin or a water-miscible ointment base. Alternatively, the active ingredients 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 containing two or more hydroxyl groups, such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerin, and polyethylene glycol (including PEG 400), and mixtures thereof. Topical formulations may, if desired, include a compound that enhances the absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethyl sulfoxide and related analogs.

The oil phase of emulsions can be composed of known ingredients in a known manner. Although the phase may contain only an emulsifier (otherwise known as an emulsifier), it desirably contains a mixture of at least one emulsifier with a fat or oil, or both. Preferably, a hydrophilic emulsifier is included with a lipophilic emulsifier, which acts as a stabilizer. It is also preferable to include both oil and fat. Together, the emulsifier(s), with or without the stabilizer(s), forms the so-called emulsifying wax, and the wax, together with the oil and fat, forms the so-called emulsifying ointment base, which forms the oily dispersed phase of cream formulations.

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

The selection of suitable oils or fats for the formulation is based on the goal of achieving the desired cosmetic properties. The cream should preferably be non-greasy, non-staining, and rinseable, with a suitable consistency to prevent leakage from tubes or other containers. Mono- or dibasic straight- or branched-chain alkyl esters can be used, such as di-isoadipate, isocetyl stearate, coconut oil propylene glycol diester, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate, or a mixture of branched-chain esters known as Crodamol CAP. The last three are preferred esters. They can be used individually or in combination, depending on the desired properties. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils are used.

The pharmaceutical compositions of the present invention comprise a combination with one or more pharmaceutically acceptable carriers or excipients and, optionally, other therapeutic agents. Pharmaceutical compositions containing the active ingredient can be in any form suitable for the intended route of administration. For oral use, tablets, lozenges, troches, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, solutions, syrups, or elixirs can be prepared, for example. Compositions intended for oral use can be prepared according to any method known in the art for the production of pharmaceutical compositions, and such compositions can contain one or more agents, including sweeteners, flavorings, colorings, and preservatives, in order to develop a preparation with an attractive odor and taste. Tablets containing the active ingredient in a mixture with a non-toxic pharmaceutically acceptable excipient, which are suitable for the 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 corn starch or alginic acid; binding agents such as starch, gelatin, or acacia; and lubricating agents such as magnesium stearate, stearic acid, or talc. The tablets may be uncoated, or they may be coated by known methods, including microencapsulation, to delay disintegration and adsorption in the gastrointestinal tract, thereby providing prolonged action over a longer period. For example, a retaining material such as glyceryl monostearate or glyceryl distearate, alone or in combination with a wax, may be used.

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

Aqueous suspensions contain the active ingredients in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and acacia, and dispersing or wetting agents such as natural phosphatides (e.g., lecithin), alkylene oxide condensation product

- 6 045066 fatty acid (e.g. polyoxyethylene stearate), 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 hexitol anhydride (e.g. polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions can be made by suspending the active ingredient in a vegetable oil, such as peanut oil, olive oil, sesame oil, or coconut oil, or in a mineral oil, such as liquid paraffin. Oily suspensions may contain a thickening agent, such as beeswax, solid paraffin, or cetyl alcohol. Sweetening agents, such as those mentioned above, and flavoring agents may be added to the oral preparation to impart flavor. These compositions can be stabilized by the addition of an antioxidant, such as ascorbic acid.

Dispersible powders and granules suitable for aqueous suspension by the addition of water contain the active ingredient mixed with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are those described above. Additional excipients, such as sweeteners, flavorings, and colorings, may also be present.

Pharmaceutical compositions can also be in the form of oil-in-water emulsions. The oil phase can be a vegetable oil, such as olive oil or peanut oil, a mineral oil, such as liquid paraffin, or mixtures thereof. Suitable emulsifiers include natural gums, such as acacia and tragacanth, natural phosphatides, such as soy lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion can also contain sweeteners and flavoring agents. Syrups and elixirs can be prepared using sweeteners, such as glycerol, sorbitol, or sucrose. Such preparations may also contain a demulcent, preservative, flavoring, or coloring agent.

The pharmaceutical compositions may be in the form of sterile injectable or intravenous preparations, such as a sterile injectable aqueous or oily suspension. This suspension may be prepared according to the known art using suitable dispersing or wetting agents and suspending agents, as mentioned above. The sterile injectable or intravenous preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent such as a solution in 1,3-butanediol, or prepared as a lyophilized powder. Among the acceptable carriers and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils can generally be used as a solvent or suspending medium. Any fixed oil, including synthetic mono- or diglycerides, can be used for this purpose. In addition, fatty acids such as oleic acid can also be used in the preparation of injectables.

The amount of active ingredient that can be combined with a carrier material to produce a unit dosage form will vary depending on the treatment being administered and the specific route of administration. For example, a sustained-release formulation intended for oral administration to humans may contain approximately 1 to 1000 mg of the active substance mixed with an appropriate and convenient amount of carrier material, which can vary from about 5% to about 95% of the total composition (weight:weight). The pharmaceutical composition can be formulated to provide readily measurable amounts for administration. For example, an aqueous solution intended for intravenous infusion may contain approximately 3 to 500 mcg of active ingredient per milliliter of solution to allow infusion of a suitable volume at a rate of approximately 30 mL/hour.

Compositions suitable for topical application to the eye also include eye drops, wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient. The active ingredient is preferably present in such formulations at a concentration of 0.5 to 20%, preferably 0.5 to 10%, and in particular about 1.5% w/w.

Compositions suitable for topical application to the oral cavity include lozenges containing the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth; pastilles containing the active ingredient in an inert base such as gelatin and glycerol, or sucrose and acacia; and mouthwashes containing the active ingredient in a suitable liquid carrier.

Compositions for rectal administration may be presented as suppositories in a suitable base containing, for example, cocoa butter or a salicylate.

Compositions suitable for intrapulmonary or nasal administration have particle sizes ranging, for example, from 0.1 to 500 μm, such as 0.5, 1, 30, 35, etc., and are administered by rapid inhalation through the nasal passage or by inhalation through the mouth so as to reach the alveolar sacs. Suitable compositions include aqueous or oily solutions of the active ingredient. Dosage forms suitable for administration by aerosol or dry powder can be prepared according to conventional methods and can be delivered together with other therapeutic agents, such as compounds previously used for the treatment or prevention of Pneumovirinae infections, as described below.

Other embodiments of the invention relate to a new, effective, safe, non-irritating and physiologically compatible inhalation composition comprising a compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof, useful for the treatment of Pneumovirinae infections and possibly associated bronchiolitis. Preferred pharmaceutically acceptable salts are salts of inorganic acids, including hydrochloride, hydrobromide, sulfate or phosphate salts, since they may cause less pulmonary irritation. Preferably, the inhalation preparation is delivered to the endobronchial space as an aerosol containing particles with a mass median aerodynamic diameter (MMAD) in the range of from about 1 to about 5 μm. Preferably, the compound of formula (I) or formula (II) is formulated for delivery by aerosol using a nebulizer, a compressor metered dose inhaler (pMDI) or a dry powder inhaler (DPI).

Non-limiting examples of nebulizers include spray, jet, ultrasonic, compressor, vibrating porous plate, or equivalent nebulizers, including those that use an adaptive aerosol delivery device (Denyer, J. Aerosol Medicine Pulmonary Drug Delivery 2010, 23 Supp 1, S1-S10). A jet nebulizer uses air pressure to break up a liquid solution into aerosol droplets. An ultrasonic nebulizer uses a piezoelectric crystal to break up the liquid into small aerosol droplets. A compressor nebulizer system forces the solution under pressure through small pores to form aerosol droplets. A vibrating porous plate device uses rapid vibration to break up the liquid stream into appropriate droplet sizes.

In a preferred embodiment of the invention, the inhalation composition is delivered to the endobronchial space as an aerosol containing particles with an MMAD, predominantly in the range of about 1 μm to about 5 μm, using a nebulizer capable of converting the formulation with a compound of formula (I) or formula (II) into an aerosol with particles of the required MMAD. In order to be optimally therapeutically effective and avoid upper respiratory tract and systemic side effects, the majority of the aerosol particles should not have an MMAD greater than about 5 μm. If the aerosol contains a large number of particles with an MMAD greater than 5 μm, the particles settle in the upper respiratory tract, which reduces the amount of drug delivered to the site of inflammation and bronchospasm in the lower respiratory tract. If the MMAD of the aerosol is less than about 1 μm, the particles tend to remain suspended in the inhaled air and are subsequently exhaled during exhalation.

When manufactured and delivered in accordance with the method described in the present invention, the aerosol composition for inhalation provides a therapeutically effective dose of a compound of formula (I) or formula (II) to the site of Pneumovirinae infection, sufficient to treat the Pneumovirinae infection. The amount of drug administered should be adjusted taking into account the efficiency of delivering a therapeutically effective dose of a compound of formula (I) or formula (II). In a preferred embodiment of the invention, the combination of an aqueous aerosol composition with a spray, jet, compressor, vibrating porous plate or ultrasonic nebulizer allows for the delivery to the respiratory tract, depending on the nebulizer, of at least about 20% to about 90%, typically about 70% of the administered dose of a compound of formula (I) or formula (II). 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 of the invention, a compound of formula (I) or formula (II), or a pharmaceutically acceptable salt thereof, is delivered as a dry powder for inhalation. The compounds are administered endobronchially as a dry powder formulation to effectively deliver fine particles of the compound to the endobronchial space using a dry powder or metered dose inhaler. For delivery via a DDI, the compound of formula (I) or formula (II) is processed into particles with a predominantly MMAD in the range of between about 1 μm and about 5 μm by spray-drying milling, critical fluid processing, or precipitation from solution. Devices and methods for media milling, jet milling, and spray drying that can achieve particle sizes with an MMAD of from about 1 μm to about 5 μm are well known in the art. In one embodiment of the invention, excipients are added to the compound of formula (I) or formula (II) prior to processing to the desired particle size. In another embodiment, excipients are mixed with the particles of the desired size to facilitate dispersion of the drug particles, for example, using lactose as an excipient.

Particle size determination is performed using devices well known in the art. For example, a multistage Anderson cascade separator or other suitable method is specifically listed in USP Chapter 601 as representative devices for aerosols in metered-dose inhalers and dry powder inhalers.

In another preferred embodiment of the invention, the compound of formula (I) or formula (II) is delivered as a dry powder using a device such as a dry powder inhaler or other dry powder dispensing devices. Non-limiting examples of dry powder inhalers and devices include those described in US 5,458,135; US 5,740,794; US 5,775,320; US 5,785,049; US 3,906,950; US 4,013,075; US 4,069,819; US 4,995,385; US 5,522,385; US 4,668,218; US 4,667,668; US 4,805,811; and US 5,388,572. There are two basic designs of dry powder inhalers. One design is a metered-dose device, in which a drug reservoir is housed within the device, and the patient adds the drug dose to the inhalation chamber. The second design is a pre-measured device, in which each individual dose is placed in a separate container. Both systems rely on a formulation of fine particles with an MMAD of 1 μm and 5 μm and often include the formulation of drugs along with larger particles of excipients, such as, but not limited to, lactose. The drug powder is placed in the inhalation chamber (either by a metered-dose device or by compartmentalizing a metered pre-measured dose), and the patient's inhalation accelerates the release of the powder from the device into the oral cavity. The non-laminar flow characteristics of the powder pathway cause the disintegration of the excipient-drug aggregates, and the mass of large excipient particles leads to their retention in the back of the throat, while smaller drug particles are deposited deep in the lungs. In preferred embodiments of the invention, the compound of formula (I) or formula (II), or a pharmaceutically acceptable salt thereof, is delivered as a dry powder using any type of dry powder inhaler as described herein, wherein the MMAD of the dry powder, excluding any excipients, is preferably in the range of from 1 μm to about 5 μm.

In another embodiment of the invention, 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 described in U.S. Pat. No. 5,261,538; U.S. Pat. No. 5,544,647; U.S. Pat. No. 5,622,163; U.S. Pat. No. 4,955,371; U.S. Pat. No. 3,565,070; U.S. Pat. No. 3,361,306; and U.S. Pat. No. 6,116,234. In preferred embodiments of the invention, a compound of formula (I) or formula (II), or a pharmaceutically acceptable salt thereof, is delivered as a dry powder using a metered dose inhaler wherein the MMAD of the dry powder, excluding any excipients, is predominantly in the range of about 1-5 μM.

Compositions suitable for vaginal administration may be presented as vaginal suppositories, tampons, creams, gels, pastes, foams or sprays containing, in addition to the active ingredient, such carriers as are known in the art to be suitable for these purposes.

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

The compositions are presented in single-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored freeze-dried (lyophilized), requiring only the addition of a sterile liquid carrier, such as water for injection, immediately before use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules, and tablets of the type previously described. Preferred unit dosage formulations are those containing a daily dose or a single fraction of a daily dose, as described above, or an appropriate fraction thereof, of the active ingredient.

It should be taken into account that, in addition to the ingredients particularly mentioned above, the compositions may include other agents commonly used in the art, taking into account the type of composition in question, for example, compositions suitable for oral administration may include flavoring agents.

Furthermore, veterinary compositions are provided comprising at least one active ingredient as defined above, together with a veterinarily acceptable carrier.

Veterinarily acceptable carriers are substances used to administer the composition and may be solid, liquid or gaseous materials which, on the other hand,

- 9 045066 are inert or veterinarily acceptable and compatible with the active ingredient. These veterinary compositions can be administered orally, parenterally, or by any other desired route.

The compounds mentioned in this document are used to provide controlled release of a medicinal product containing one or more compounds as an active ingredient (controlled release formulations), in which the release of the active ingredient is controlled and regulated, which allows for a reduction in the frequency of administration or an improvement in the pharmacokinetics or toxicity profile of the active ingredient.

The effective dose of the active ingredient depends on, at least, the nature of the disease being treated, the toxicity, whether the compound is used prophylactically (lower doses) or against an active viral infection, the delivery method, and the pharmaceutical formulation, and will be determined by the treating physician using routine dose escalation studies. It may 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 0.01 to about 5 mg/kg body weight per day; most typically, from about 0.05 to about 0.5 mg/kg body weight per day. For example, the suggested daily dose for an adult weighing about 70 kg is in the range of 1 to 1000 mg, preferably 5 to 500 mg, and may be in single or multiple doses.

Routes of administration

One or more compounds (referred to herein as active ingredients) are administered by any route appropriate to the condition being treated. Suitable routes include oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), vaginal, and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural), among others. It should be noted that the preferred route may vary depending, for example, on the condition of the recipient. An advantage of the compounds discussed herein is that they are orally bioavailable and can be administered orally.

In another embodiment of the invention, the present application provides a method for treating a Pneumovirinae virus infection in a human, wherein the method comprises administering to the human a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or ester thereof. Also provided are certain methods for treating a Pneumovirinae virus infection in a human, each of which comprises administering to the human a therapeutically effective pharmaceutically effective amount of a compound of formula (II) or one of the specific compounds exemplified herein, or a pharmaceutically acceptable salt, solvate and/or ester thereof and a pharmaceutically acceptable carrier and excipient.

In another embodiment of the invention, there is provided a method of treating 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 solvate of a compound of formula (I) or a pharmaceutically acceptable salt or ester thereof.

Furthermore, certain methods for treating a Pneumovirinae infection in a human in need thereof are provided, wherein each method comprises administering to the human a therapeutically effective amount of a racemate, enantiomer, diastereomer, tautomer, polymorph, pseudopolymorph, amorphous form, hydrate or solvate of a compound of formula (II) or one of the specific compounds of the examples given herein, or a pharmaceutically acceptable salt, solvate and/or ester thereof.

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

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

Additionally provided are certain methods for treating human respiratory syncytial virus infection in a human in need thereof, wherein each method comprises administering to the human a therapeutically effective amount of a compound of formula (II) or one of the specific compounds exemplified herein, or a pharmaceutically acceptable salt, solvate and/or ester thereof.

Also provided are certain methods for treating human respiratory syncytial virus infection in a human, in need thereof, wherein each method comprises administering to the human a therapeutically effective amount of a compound of formula (II) or one of the specific compounds of

- 10 045066 examples given in this document, or a pharmaceutically acceptable salt, solvate and/or ester thereof and at least one additional active therapeutic agent.

Also provided are certain methods for treating human respiratory syncytial virus infection in a human, in need thereof, wherein the human further suffers from bronchiolitis, wherein each method comprises administering to the human a therapeutically effective amount of a compound of formula (I), formula (II), or one of the specific compounds of the examples given herein, or a pharmaceutically acceptable salt, solvate, and/or ester thereof.

Also provided are certain methods for treating human respiratory syncytial virus infection in a human in need thereof, wherein the human further suffers from pneumonia, wherein each method comprises administering to the human a therapeutically effective amount of a compound of formula (I), formula (II), or one of the specific compounds of the examples given herein, or a pharmaceutically acceptable salt, solvate, and/or ester thereof.

Also provided are certain methods for improving the symptoms of respiratory diseases in a human suffering from a human respiratory syncytial virus infection, wherein each method comprises administering to the human a therapeutically effective amount of a compound of formula (I), formula (II), or one of the specific compounds according to the examples given herein, a pharmaceutically acceptable salt, solvate and/or ester thereof.

Respiratory symptoms in a person with respiratory syncytial virus infection may include a stuffy or runny nose, cough, difficulty breathing, sneezing, rapid or labored breathing, shortness of breath, bronchiolitis, and pneumonia.

An embodiment of the invention is also provided, comprising the use of a compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or ester thereof in the manufacture of a medicament for the treatment of Pneumovirinae virus infection or respiratory syncytial virus infection.

An embodiment of the invention is also provided comprising the use of a compound of formula (II) or one of the specific compounds according to the examples given herein, or a pharmaceutically acceptable salt, solvate and/or ester thereof, in the manufacture of a medicament for the treatment of Pneumovirinae virus infection or respiratory syncytial virus infection.

Also provided are certain embodiments of the invention comprising a compound of formula (I), formula (II), or one of the specific compounds exemplified herein, or a pharmaceutically acceptable salt, solvate, and/or ester thereof, for use in the treatment of Pneumovirinae virus infection or respiratory syncytial virus infection in humans.

Also provided are certain embodiments of the invention comprising a compound of formula (I), formula (II), or one of the specific compounds exemplified herein, or a pharmaceutically acceptable salt, solvate, and/or ester thereof, for use as a medicament.

Also provided is a compound of formula (I) or a pharmaceutically acceptable salt, solvate and/or ester thereof for the treatment of Pneumovirinae virus infection or respiratory syncytial virus infection in humans.

Also provided are some embodiments of the invention comprising a compound of formula (II) or one of the specific compounds exemplified herein, or a pharmaceutically acceptable salt, solvate and/or ester thereof for the treatment of Pneumovirinae virus infection or respiratory syncytial virus infection in humans.

Furthermore, a compound described in the present description is provided. A pharmaceutical composition described in the present description is also provided. A method for using a compound of formula (I) described in the present description is also provided. Furthermore, a method for producing a compound of formula (I) described in the present description is also provided.

Metabolites of compounds

Furthermore, the scope of this document includes in vivo metabolic products of the compounds described herein, to the extent that such products are novel and non-obvious relative to the prior art. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, esterification, etc., of the administered compound, primarily through enzymatic processes. Accordingly, novel and non-obvious compounds formed by a process involving contacting the compound with a mammalian organism for a period of time sufficient to form a metabolic product of that compound are encompassed. Such products are typically identified by obtaining a radiolabeled compound (e.g., ^14C or ^3H ), administering it parenterally at a detectable dose (e.g., greater than about 0.5 mg/kg) to an animal such as a rat, mouse, guinea pig, monkey, or human, allowing sufficient time for metabolism to occur (usually about 30 seconds to 30 hours), and isolating their metabolites from urine, blood, or other biological

- 11,045,066 samples. These products can be easily isolated because they are labeled (others are isolated using antibodies capable of binding epitopes retained in the metabolite). The structures of the metabolites are determined routinely, for example, by MS or NMR analysis. Metabolite analysis is typically performed in the same manner as conventional drug metabolism studies, well known to those skilled in the art. Conversion products, provided they are otherwise undetectable in vivo, can be used in diagnostic assays for therapeutic dosing of compounds, even if they themselves do not possess HSV antiviral activity.

Methods and means for determining the stability of compounds in gastrointestinal substitution secretions are known. Compounds are defined herein as stable in the gastrointestinal tract when less than about 50 mole percent of the protected groups are deprotected in gastric or intestinal substitution juice upon incubation for 1 hour at 37°C. Mere stability of compounds in the gastrointestinal tract does not mean that they cannot be hydrolyzed in vivo. Prodrugs are generally stable in the digestive system but can be extensively hydrolyzed to the parent drug in the alimentary canal, liver, lungs, or other metabolic organs, or, primarily, in cells. As used herein, a prodrug is a compound that is chemically designed to effectively release the parent drug after it overcomes biological barriers during oral delivery.

Abbreviations

Several abbreviations and acronyms are used in describing experimental details. While most are understandable to those skilled in the art, Table 1 provides a list of many of these abbreviations and acronyms.

Table 1. List of abbreviations and acronyms

Abbreviations Meaning Ace acetate ACN acetonitrile AIBN azobisisobutyronitrile VP benzyl Vi butyl Bz benzoyl BzCl benzoyl chloride GDI 1,1'-carbonyldiimidazole DAST diethylaminosulfur trifluoride DCE 1,2-dichloroethane DCM dichloromethane DMAP 4-dimethylaminopyridine DMDO Dimethyldioxirane DMSO dimethyl sulfoxide DMF dimethylformamide DMTrCl 4,4-dimethoxytrityl chloride DMTr 4,4'-dimethoxytrityl EDCI N-(3-methylaminopropyl)-N'-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 MC or ms Mass spectrum NIS M-iodosuccinimide NMP V-methyl-2-pyrrolidone Ph phenyl Ph ₃ P triphenylphosphine PMB Lara-methoxybenzyl

- 12 045066

RMVS1 para-methoxybenzyl chloride PhOC(S)Cl phenylchlorothionoformate (PhO) ₃ PMeI Methyltriphenoxyphosphonium iodide Pyr pyridine RT room temperature TBAF Tetrabutylammonium fluoride TBS tert-butyldimethylsilyl TBSCl tert-butyldimethylsilyl chloride TMSN ₃ trimethylsilyl azide TEA triethylamine TES triethylsilane TFU trifluoroacetic acid THF tetrahydrofuran TMS trimethylsilyl TMSC1 trimethylsilyl chloride Ts 4-toluensul phonyl TsOH p-toluenesulfonic acid δ parts per million, reported relative to the residual peak of the non-deuterated solvent

General schemes

Scheme 1 nh ₂ nh ₂

Ι/γ-Ά ^NIS .

V-N,^ ρΆ

Sia S1b

Scheme 1 shows the general pathway for the synthesis of intermediates starting with an iodination reaction (e.g., NIS) to produce the nucleobase S1b.

Scheme 2

NH ₂ nh ₂ N ^H 2 €Y _n HBF ₄ Α _ν NIS rLm ^Ν 'Ν ^Λ ΝΗ ₂ NaNO ₂ γΆ

S2a S2b S2c

Scheme 2 shows a general route to the synthesis of intermediates, starting with a fluorination reaction (e.g., HBF4, _NaNO2 ) in a manner similar to that described in WO2012037038A1 to yield intermediate S2b. Intermediate S2b can then be iodinated (e.g., with NIS) to yield nucleobase S2c.

Scheme 3

Scheme 3 shows the general synthetic route to the compounds, starting with lithium-halogen exchange (e.g., n-BuLi, [-CH ₂ SiMe ₂ Cl] ₂ ) with the appropriate nucleobase S3b, followed by addition to lactone S3a. Reduction of the pendant 1' hydroxyl group in Lewis acid medium (e.g., BF ₃ -Et ₂ O, Et ₃ SiH) gives intermediate S3c. Intermediate S3c can first be protected (e.g., BzCl, Pyr; NH ₄ OH) at the nitrogen functionality, and the benzyl groups can then be removed under reducing conditions (e.g., HCO ₂ H, Pd/C, BCl ₃ , BBr ₃ ) to give intermediate S3d. A sequence involving first selective protection of the 5' hydroxyl (e.g., DMTrCl), then protection of the 2' and 3' hydroxyl (e.g., TBSCl), then selective removal of the 5' hydroxyl protecting group under acidic conditions (e.g., TsOH) results in

- 13 045066 to give intermediate S3e. The 5' hydroxyl group can then be converted to the corresponding iodide (e.g., (PhO) _3PMeI ), which is then subjected to basic conditions (i.e., KOtBu) to effect an elimination reaction, giving intermediate S3f. Oxidation of the olefin S3f (e.g., DMDO), followed by treatment with an appropriate nucleophile (e.g., TMSCN) in a Lewis acid medium (e.g., _InBr3 ) and removal of the hydroxyl protecting groups (e.g., CsF) gives intermediate S3g. Removal of the nitrogen protecting group (e.g., _NH2Me ) gives the final compound of type S3h.

Scheme 4

Scheme 4 shows the general route to the compounds, starting with oxidation of the S3f olefin (e.g., DMDO) followed by treatment with an appropriate nucleophile (e.g., TMSN ₃ ) in a Lewis acid medium (e.g., InBr ₃ ) 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 yields the intermediate S4a. Removal of the nitrogen protecting group (e.g., NH ₂ Me) yields the final compound of type S4b.

Scheme 5

Scheme 5 shows the general synthetic route to the compounds, starting with the oxidation of the olefin S3f (e.g., DMDO) in the presence of the corresponding alcohol HORa, followed by removal of the hydroxyl protecting groups (e.g., CsF) to yield the intermediate compound S5a. Removal of the nitrogen protecting group (e.g., NH ₂ Me) yields the final compound of type S5b.

Scheme 6

Scheme 6 shows a general route to the compounds starting with oxidation of the S3f olefin (e.g., DMDO) followed by treatment with an appropriate nucleophile (e.g., (HC=C) _3Al ) in a manner similar to that described in Nucleosides, Nucleotides, and Nucleic Acids 2005, 24, 343–347. Removal of the hydroxyl protecting groups (e.g., CsF) further affords intermediate S6a. Removal of the nitrogen protecting group (e.g., _NH2Me ) affords final compounds of type S6b. Treatment of the final compound under hydrogenating conditions (e.g., _H2 , Pd/C, or Lindlar's catalyst) can selectively afford final compounds of type S6c and S6d, respectively. Transformation of final compound S6d via formation of a cyclopropane ring (e.g., _CH2N2 ) can afford final compounds _of type S6e.

- 14 045066

Scheme 7

Scheme 7 shows the general synthetic route to the compounds starting with a synthetic sequence of nitrogen protection (e.g., TMSCl, Pyr; BzCl, Pyr; NH4OH) at intermediate S7a, synthesized in the same manner as described in WO 2012037038A1. Selective protection of the 5' hydroxyl group (e.g., DMTrCl) then yields intermediate S7b. Protection of the 2' hydroxyl group (e.g., TBSCl), then removal of the 5' hydroxyl group under acidic conditions (e.g., TsOH) yields intermediate S7e. Conversion of the 5' hydroxyl group to an aldehyde under oxidizing conditions (e.g., EDCI-HCl, Pyr, TFA, DMSO), then condensation of the corresponding enolate with formaldehyde and reduction (e.g., NaBH4) yields intermediate S7f. Subsequent selective protection of the hydroxyl groups with orthogonal protecting groups (e.g., DMTrCl and TBSCl), then removal of most of the labile protecting groups under acidic conditions (e.g., TsOH) further affords intermediate S7g. Conversion of the hydroxyl group to an aldehyde under oxidizing conditions (e.g., EDCI-HCl, Pyr, TFA, DMSO) affords intermediate S7h. Conversion of the aldehyde S7h to the halogen olefin intermediate S7i can be accomplished under Wittig olefination conditions (e.g., [Ph ₃ PCH ₂ Br] ⁺ Br ^- , KOtBu). Elimination reaction under basic conditions (e.g., KOtBu) affords the alkyne, and removal of the hydroxyl protecting groups (e.g., TBAF) and the nitrogen protecting group (e.g., NH ₄ OH) affords the final compound of type S7j.

Scheme 8

NHBz NH ₂

VI 1)NH ₂ OH«HCI _cp Vn A .

TBSO-x ₀ . ΗΟ^Ο,/ Ή Ή

Ο—γ/ 2) GDI % 3) NH ₄ OH ;I

TBSO F 4) HF* Pyr HOF

S7hS8a

Scheme 8 shows the general route to the synthesis of compounds starting with the formation of an oxime (e.g., NH ₂ OH-HC1) followed by conversion of the oxime to a nitrile group (e.g., CDI). Removal of the nitrogen protecting group (e.g., NH ₄ OH) and hydroxyl protecting groups (e.g., HF-Pyr) then yields the final compound of type S8a.

Scheme 9

Scheme 9 shows a general route to the synthesis of compounds starting with the conversion of an aldehyde S7h to an olefin under Wittig olefination conditions (e.g., _{[ Ph3PCH3} ] ^Br- , KOtBu). Removal of the hydroxyl protecting groups (e.g., TBAF) and the nitrogen protecting group (e.g., _NH4OH ) yields final compounds of type S9a. A reduction reaction (e.g., _H2 , Pd/C) can then yield final compounds of type S9b, and a cyclopropane ring formation reaction (e.g., _CH2N2 ) can yield final compounds _of type S9c.

- 15 045066

Scheme 10

Scheme 10 shows a general synthetic route to compounds starting with an Appell reaction (e.g., _Ph3P , _CCl4 ) to convert the hydroxyl group to chloride. Removal of the hydroxyl protecting groups (e.g., TBAF) and the nitrogen protecting group (e.g., NH4OH) yields the final compound of type S10a.

Scheme 11

Scheme 11 shows the general synthetic route starting with protection of the free hydroxyl group of intermediate S7g with a labile protecting group (e.g., PMBCl, _K2CO3 ). Selective removal of the 2' and 5' silyloxy protecting groups (e.g., TBAF), followed by reprotection with stable protecting groups (e.g., BnBr, NaH) and removal of labile hydroxyl protecting groups under acidic conditions (e.g., AcOH) yields intermediate S11a. Conversion of the hydroxyl group to fluorine (e.g., DAST), followed by removal of the hydroxyl protecting groups (e.g., BBr3) and the nitrogen protecting group (e.g., NH4OH) yields the final compound of type S11b.

Scheme 12

Scheme 12 shows a general synthetic route to the compounds starting with conversion of the 5' hydroxyl group to the corresponding iodide (e.g., (PhO) _3PMeI ), which is then treated under basic conditions (i.e., KOtBu) to effect an elimination reaction, affording intermediate S12a. Oxidation of the S12a olefin (e.g., DMDO), followed by treatment with an appropriate nucleophile (e.g., TMSN3) in a Lewis acid medium (e.g., _SnCl4 ) 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 the nitrogen protecting group (e.g., NH4OH) affords the final compound of type S12b.

Scheme 13

Scheme 13 shows the general synthetic route to compounds starting with the oxidation of an S12a olefin (e.g., DMDO) in the presence of the corresponding alcohol HOR ^a and removal of hydroxyl protecting groups (e.g., CsF). Removal of the nitrogen protecting group (e.g., NH ₂ Me) yields the final compound of type S13a.

Scheme 14

NHBz NHBz NH ₂

1)PhOC(S)CI /-f'N tosge—_ Eh. DMAP trchge Eh 1) TBAF - Sx-m Eh.

TBSOy ⁴ NR· ¹ ----------- TBSO-Ч ₀

2) (TMS) ₃ SiH, 2) NH ₄ OHY

J 1 AIBN / i

TBSO F TBSO FHO F

S7g S14aS14b

Scheme 14 shows a general synthetic route starting with the formation of a xanthate (e.g., PhOC(S)Cl, DMAP) followed by a Barton-McCombie deoxygenation reaction (e.g., (TMS) _3SiH , AIBN) to yield intermediate S14a. Removal of the hydroxyl protecting groups (e.g., TBAF) and the nitrogen protecting group (e.g., NH4OH) yields the final compound of type S14b.

- 16 045066 but

NH1) n-BuLi [-CH ₂ SiMe ₂ CI]2

2) Et ₃ SiH BF3«Et ₂ O

3) H ₂ , Pd black

R ¹

S15c

Scheme 15 o o ^NH 2

ΒηΟ^>θ...... ^0Ac 1) K ₂ CO ₃ , MeOH ΒηΟ'^^^ /=γ^Ν

ВпО 0Ас 2) NIS, Bu ₄ NI ВпО бВп / ^N N^R ¹

3) BpBg, Ag ₂ OI

S15a S15b S3b

Scheme 15 shows the general synthetic route to the compounds starting from intermediate S15a, prepared in a manner similar to that described in Biosci. Biotech. Biochem. 1993, 57, 1433–1438. Removal of acetate protecting groups under hydrolytic conditions (e.g., _K2CO3 , _MeOH ), followed by chemoselective oxidation (e.g., NIS, Bu4NI) and protection of the 2' hydroxyl group (e.g., BnBr, Ag2O) gives intermediate S15b. Exchange of a lithium halogen (e.g., n-BuLi, [-CH ₂ SiMe ₂ Cl] ₂ ) with the corresponding nucleobase S3b and addition to the lactone S15b, followed by reduction of the 1' hydroxyl group in a Lewis acid medium (e.g., BF ₃ -Et ₂ O, Et ₃ SiH) and removal of protecting groups (e.g., H ₂ , Pd black) gives the final compound of type S15c.

Scheme 16

S16b S16c

Scheme 16 shows the general synthetic route to the compounds, starting with the conversion of the 5' hydroxyl group to the aldehyde under oxidizing conditions (e.g., EDCI-HCl, Pyr, TFA, DMSO), followed by condensation of the corresponding enolate with formaldehyde and reduction (e.g., NaBH4) to afford intermediate S16a. Subsequent selective protection of the hydroxyl groups with orthogonal protecting groups (e.g., DMTrCl and TBSCl), followed by removal of the more labile protecting group under acidic conditions (e.g., TsOH) further affords intermediate S16b. An Appell reaction (e.g., _Ph3P , _CCl4 ) can then convert the hydroxyl group to the chloride, and removal of the hydroxyl protecting groups (e.g., TBAF) and the nitrogen protecting group (e.g., NH4OH) affords the final compound of type S16c.

Scheme 17

Scheme 17 shows a general synthetic route starting with protection of the free hydroxyl group of intermediate S16b with a labile protecting group (e.g., PMBCl, K2CO3). Selective removal of the 2', 3', and 5' silyloxy protecting groups (e.g., TBAF), followed by reprotection with stable protecting groups (e.g., BnBr, NaH) and removal of labile hydroxyl protecting groups under acidic conditions (e.g., AcOH) yields intermediate S17a. Conversion of the hydroxyl group to fluorine (e.g., DAST), followed by removal of hydroxyl protecting groups (e.g., _BBr3 ) and the nitrogen protecting group (e.g., NH4OH) yields the final compound of type S17b.

Scheme 18

S18c

Scheme 18 shows the general route to the synthesis of compounds via the appropriate electro- 17 045066 halogenation reactions of intermediate S18a to yield target compounds of the type

S18b (e.g. NCS), S18c (e.g. NIS), and S18d (e.g. Selectfluor).

Scheme 19

Scheme 19 shows a general route to the synthesis of compounds starting with a cross-coupling reaction (e.g., Zn(CN) ₂ , Pd(PtBu) ₃ ) to yield target compounds of type S19a. Compound S19a can then be synthesized via a _nitrile hydrolysis reaction (e.g., _H2O2 , NH4OH, _H2O ) to yield compound of type S19b.

Scheme 20

Scheme 20 shows a general route to the synthesis of compounds starting from the Sonogashira reaction (e.g., CuI, PdCl ₂ (PPh ₃ ) ₂ ) to give target compounds of type S20a.

Scheme 21

Scheme 21 shows a general route to the synthesis of compounds starting from a cross-coupling reaction (e.g., Pd(dppf)Cl ₂ , Cs ₂ CO ₃ ) to yield target compounds of type S21a.

Scheme 22

synthesis

Scheme 22 shows the general path of S22b type analogs.

compounds, including the synthesis of phosphorylated

Scheme 23

a common pathway of compounds involving the synthesis of phosphorylated peptides

Scheme 23 shows an analog of type S23b.

Scheme 24

a common pathway of compounds involving the synthesis of phosphorylated peptides

Scheme 24 shows an analog of type S24b.

- 18 045066

Experimental part

Intermediate compound 1b.

N-iodosuccinimide (84 mg, 373 mmol) was added as a solid to a solution of intermediate 1a (50 mg, 373 mmol) in DMF (1 mL) at room temperature. After 1.5 h, the reaction mixture was diluted with 1 M NaOH (10 mL), and the resulting slurry was stirred at room temperature. After 1 h, the solids were collected by vacuum filtration and dried under reduced pressure to yield intermediate 1b.

¹ H 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, 1H), 3.30 (br-s, 1H).

GC/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 2.6 μ HV-S18 100A, 50 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 2 µL/min.

HPLC: t _r = 1.536 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 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.

(2S,3R,4R,5R)-2-(4-aminopyrrolo[1.2-f][1.2.4]triazin-7-yl)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)tetrahydrofuran-2-ol intermediate 1d.

To a suspension of 7-iodopyrrolo[1.2-f][1.2.4]triazin-4-amine 1b (6.84 g, 26.3 mmol) and 1,2-bis(chlorodimethylsilyl)ethane (5.66 g, 26.3 mmol) in THF (200 mL) at -78°C under argon was rapidly added n-butyllithium (2.5 M in hexane, 34.4 mL, 86.0 mmol). During the addition, the internal temperature of the reaction mixture rose to -40.5°C, and the reaction mixture became a clear brown solution. After 15 min, a solution of (3R,4R,5R)-3,4-bis(benzyloxy)-5-(benzyloxymethyl)dihydrofuran-2(3H)-one (1c, purchased from Carbosynth, 10 g, 23.9 mmol) in tetrahydrofuran (40 mL) pre-cooled to -78°C was added quickly via cannula. After 1 h, the reaction mixture was quenched with acetic acid (15 mL), and the resulting mixture was allowed to warm to room temperature. The resulting mixture was diluted with ethyl acetate (800 mL) and washed with saturated aqueous sodium bicarbonate (500 mL) and brine (500 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by SiO ₂ column chromatography (220 g SiO ₂ Combiflash HP Gold Column, 0-100% ethyl acetate/hexane) to afford intermediate 1d.

GC/MS: t _R = 1.50 min, MS m/z = 553.34 [M+1]; LC system: Thermo Accela 1250 UHPLC

MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 μm, ХВ-С18 100А, 50x4.6 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, 2.4-2.5 min 2% ACN.

HPLC: t _r = 3.442 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 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=0.5 (UV)

Intermediate 1e is 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 Id (4.74 g, 8.58 mmol) and triethylsilane (3.56 mL, 22.3 mmol) in DCM (43 mL) was added boron trifluoride diethyl ether complex (boron trifluoride etherate) (1.59 mL, 12.9 mmol) slowly via syringe at 0°C under argon. After 2 h, the reaction mixture was slowly diluted with saturated aqueous sodium bicarbonate (100 mL), and the resulting mixture was extracted with ethyl acetate (2 x 150 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude residue was purified by SiO ₂ column chromatography (24 g SiO ₂ Combiflash HP Gold Column, 0-100% ethyl acetate/hexane) to afford intermediate 1e.

1H NMR (400 MHz, CDCl ₃ ) 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).

GC/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, 2.6 µm, HB-C18 100A, 50x4.6 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 2 µL/min.

HPLC: tR=3.596 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA; gradient: 0-5.0 min 2-98% ACN, 5.0 min 6.0 min 98% ACN at 2 mL/min.

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

Intermediate 1f is 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 slowly added benzoyl chloride (1.69 mL, 14.68 mmol) at room temperature under argon. After 1 h, additional benzoyl chloride (1.69 mL, 14.68 mmol) was slowly added. After 19 h, water (20 mL) was slowly added, and the reaction mixture became slightly cloudy. Ammonium hydroxide (10 mL) was then slowly added until the reaction mixture became basic with a pH of 10. After 1 h, water (150 mL) was added dropwise using a dropping funnel, and a white solid began to slowly precipitate from the reaction mixture as the addition continued. The resulting mixture was stirred for 24 h, and the white solid was collected by vacuum filtration and dried by azeotropic distillation from toluene to yield intermediate 1f.

1H NMR (400 MHz, CDCl ₃ ) δ 8.23 (br s, 1H), 7.62 (t, J=7.8 Hz, 1H), 7.53 (t, J=7.6 Hz, 2H), 7.38-7.21 (m, 18H), 7.17 (d, J=7.6 Hz, 1H), 5.69 (d, J=4.1 Hz, 1H), 4.71 (s, 2H), 4.63-4.44 (m, 4H), 4.43-4.39 (m, 1H), 4.22 (t, J=4.5 Hz, 1H), 4.15-4.10 (m, 1H), 3.79 (dd, J=10.8, 3.2 Hz, 1H), 3.65 (dd, J=10.7, 3.7 Hz, 1H).

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

MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µm, HB-C18 100A, 50x4.6 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, 2.2-2.4 min 100%-2% ACN, 2.4-2.5 min 2% ACN at 2 µL/min.

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

NHBz NHBz

Intermediate 1g is N-(7-((2S,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrrolo[1.2-1][1.2.4]triazin-4-yl)benzamide.

To a mixture of intermediate 1f (4.39 g, 6.85 mmol) and palladium on carbon (10% by weight, 2.2 g) at room temperature under argon were added sequentially ethanol (68.5 mL) and formic acid (51.7 mL, 1.37 mol). 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 yield intermediate 1g, which was used directly in the next step without further purification.

1H NMR (400 MHz, CD3OD) δ 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,

- 20 045066

J=12.1, 3.5 Hz, 1H), 3.71 (dd, J=12.0, 4.5 Hz, 1H).

GC/MS: tR=1.04 min, MS m/z=371.15 [M+1]; LC system: Thermo Accela 1250 UHPLC

MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 μm, ХВ-С18 100А, 50x4.6 mm

Solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 0-1.5 min 2-10 0% 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 2 µL/min.

HPLC: tR=2.055 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 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

GO

NO-L o ΟΌ DMTrCW o \ / Rug \__/

H0 bn NO HE

1 day ^{1 hour}

Intermediate 1h is N-(7-((2S,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)pyrrolo[1.2-f][1.2.4]triazin-4-yl)benzamide.

To a solution of intermediate 1g (2.44 g, 6.59 mmol) in pyridine (32.5 mL) at room temperature was added 4,4'-dimethoxytrityl chloride (2.23 g, 6.59 mmol) as a solid in one portion. After 5.5 h, the reaction mixture was diluted with ethyl acetate (300 mL), and the resulting mixture was washed with saturated brine (3 x 200 mL). The organic layer was concentrated under reduced pressure, and the crude residue was purified by _SiO2 column chromatography (80 g _SiO2 Combiflash HP Gold Column, 0-100% ethyl acetate/hexane) to afford intermediate 1h.

GC/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, 2.6 μm, ХВ-С18 100А, 50x4.6 m;

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 2 µL/min.

HPLC: t _R = 4.270 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 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 hexane, Rf=0.15 (UV)

Intermediate 1i is N-(7-((2S,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-bis(tert-butyldimethylsilyloxy)tetrahydrofuran-2-yl)pyrrolo[1.2-f][1.2.4]triazin4-yl)benzamide.

To a solution of intermediate 1h (1.84 g, 2.74 mmol) and imidazole (2.23 g, 32.8 mmol) in N,N-dimethylformamide (28.2 mL) was added tert-butyldimethylsilyl chloride (2.47 g, 16.4 mmol) at room temperature. After 17 h, saturated aqueous sodium bicarbonate solution (500 mL) was slowly added to the reaction mixture. The resulting mixture was extracted with ethyl acetate (500 mL), and the organic layer was washed with saturated brine (2 x 400 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude residue was purified by _SiO2 column chromatography (80 g _SiO2 Combiflash HP Gold Column, 0-100% ethyl acetate/hexane) to afford intermediate 1i.

GC/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, 2.6 μm, ХВ-С18 100А, 50x4.6 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-3.55 min 100% ACN, 3.55 min-4.2 min 100%-2% ACN at 2 µL/min

HPLC: t _R = 5.724 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 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 hexane, Rf=0.75 (UV)

- 21 045066

Intermediate 1j is N-(7-((2S,3S,4R,5R)-3,4-bis(tert-butyldimethylsilyloxy)-5(hydroxymethyl)tetrahydrofuran-2-yl)pyrrolo[1.2-f][1.2.4]triazin-4-yl)benzamide.

To a solution of intermediate 1i (2.41 g, 2.67 mmol) in dichloromethane (22.3 mL) at 0°C was slowly added a solution of p-toluenesulfonic acid monohydrate (509 mg, 2.67 mmol) in methanol (3.7 mL). 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 concentrated under reduced pressure. The crude residue was purified by SiO ₂ column chromatography (120 g SiO ₂ Combiflash HP Gold Column, 0-100% ethyl acetate/hexane) to afford intermediate 1j.

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

GC/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, 2.6 μm, ХВ-С18 100А, 50x4.6 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 2 µL/min.

HPLC: t _R = 5.622 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 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 hexane, Rf=0.55 (UV)

NHBz NHBz

ΗΟ^ o UM ι-у o UM Ύ dmf Υ y

TBsd Ьтвв tbso 6tbs

1j Ik

Intermediate 1k is 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.

To a solution of methyltriphenoxyphosphonium iodide (0.99 g, 2.19 mmol) in DMF (9.9 mL) was added intermediate 1j (1.19 g, 1.99 mmol) at room temperature. After 3 h, additional methyltriphenoxyphosphonium iodide (0.99 g, 2.19 mmol) was added. After 1 h, the reaction mixture was diluted with ethyl acetate (200 mL) and washed with saturated brine (3 x 100 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by SiO ₂ column chromatography (80 g SiO ₂ Combiflash HP Gold Column, 0-100% ethyl acetate/hexane) to afford intermediate 1k.

1H NMR (400 MHz, CDCl ₃ ) δ 8.21 (br s, 1H), 7.61 (br t, J=7.2 Hz, 1H), 7.53 (br t, J=7.5 Hz, 3H), 7.05 (br s, 1H), 5.44 (d, J=4.5 Hz, 1H), 4.52 (t, J=4.3 Hz, 1H), 4.08-3.99 (m, 2H), 3.55 (dd, J=10.7, 5.2 Hz, 1H), 3.38 (dd, J=10.7, 5.0 Hz, 1H), 0.93 (s, 9H), 0.85 (s, 9H), 0.16 (s, 3H), 0.11 (s, 3H), -0.01 (s, 3H), -0.11 (s, 1H).

GC/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, 2.6 μm, ХВ-С18 100А, 50x4.6 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 2 µL/min.

HPLC: t _R = 5.837 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 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% ethyl acetate in hexane, Rf=0.45 (UV)

- 22 045066

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.

To a solution of intermediate 1k (1.77 g, 2.5 mmol) in pyridine (25 mL) at room temperature was added potassium tert-butoxide (700 mg, 6.24 mmol). After 2 h, the reaction mixture was diluted with saturated aqueous sodium bicarbonate solution (25 mL) and saturated brine (200 mL). The resulting mixture was extracted with ethyl acetate (300 mL). The organic layer was then washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude residue was purified by _SiO2 column chromatography (40 g _SiO2 Combiflash HP Gold Column, 0-100% ethyl acetate/hexane) to afford intermediate 11.

GC/MS: tR=2.87 min, MS m/z=581.37 [M+1]; LC system: Thermo Accela 1250 UHPLC

MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 μm, ХВ-С18 100А, 50x4.6 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 2 µL/min.

HPLC: tR=5.750 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 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 hexane, Rf=0.20 (UV)

NHBz NHBz

0J DMDO ^^O^y^'N acetone HO <^O./^N yl/ HO

TBS0 6tbs tbso 6tbs

Im

Intermediate 1m is N-(7-((2S,3S,4S)-3,4-bis(tert-butyldimethylsilyloxy)-5hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrrolo[1.2-f][1.2.4]triazin-4-yl)benzamide.

DMDO (0.07 M 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 and dried by azeotropic distillation with toluene (2 x 1 mL) to yield 1m, which was used immediately in the next step without further purification.

GC/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, 2.6 μm, ХВ-С18 100А, 50x4.6 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 2 µL/min.

Intermediate 1n - N-(7-((2S,3S,4S)-3,4-bis(tert-butyldimethylsilyloxy)-5-cyano-5((trimethylsilyloxy)methyl)tetrahydrofuran-2-yl)pyrrolo[1.2-f][1.2.4]triazin-4-yl)benzamide.

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

GC/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 Ac

- 23 045066 cela 1250 SVEZHKh

MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 μm, ХВ-С18 100А, 50x4.6 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 2 µL/min.

In 1o

Intermediate 1o is N-(7-((2S,3R,4S)-5-cyano-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrrolo[1.2-f][1.2.4]triazin-4-yl)benzamide.

To a solution of crude intermediate 1n (668.23 mg, 0.96 mmol) in DMF (9.6 mL) at room temperature was added cesium fluoride (729 mg, 4.8 mmol). After 5 h, the reaction mixture was diluted with saturated 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 concentrated under reduced pressure to yield intermediate 1o (1:1 mixture of diastereomers), which was used directly in the next step without further purification.

GC/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, 2.6 μm, ХВ-С18 100А, 50x4.6 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 2 µL/min.

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.

To a solution of crude intermediate 1o in methanol (1 mL) was added methylamine (40% in water, 0.3 mL) at room temperature. After 2.5 h, the reaction mixture was concentrated under reduced pressure and immediately purified by preparative HPLC (Phenominex Luna, 5 μm, C18 100A, 100x30 mm column, gradient 5-15% acetonitrile/water, 25 min). Fractions containing the desired product and the 4' anomer were combined and concentrated under reduced pressure. The 4' anomers were then separated by preparative HPLC (Phenominex Luna, 5 μm, C18 100A, 100x30 mm column, gradient 5-15% acetonitrile/water, 25 min). Fractions containing the desired product were pooled and lyophilized to yield the compound of Example 1.

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

GC/MS: tR=0.29 min, MS m/z=2 92.16 [M+1]; LC system: Thermo Accela 1250 UHPLC

MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 μm, ХВ-С18 100А, 50x4.6 mm

Solvents: acetonitrile with 0.1% acetic acid, water with 0.1% acetic acid; gradient: 0 min-1.5 min 2-10 0% 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 2 µL/min.

HPLC: t _R = 0.377 min; HPLC System: Agilent 1100 Series; Column: Gemini C18, 5 μm, 110A, 50x4.6 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 Series; Column: Luna 5 μm C18(2) 110A, 250x4.6 mm; Solvents: acetonitrile, water; Gradient: 5-15% ACN for 10 min at 2 mL/min.

Intermediate 2b is N-(7-((2S,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrrolo[1.2-f][1.2.4]triazin-4-yl)benzamide.

- 24 045066

To a 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 co-evaporation with pyridine 3 times), then dissolved in pyridine (18 mL). At 0°C, chlorotrimethylsilane (1.54 mL, 13.13 mmol) was added in one portion, and the resulting mixture was stirred under N ₂ for 1 h. Benzoyl chloride (675 μL, 5.82 mmol) was added dropwise, and the reaction mixture was stirred for 1 h. Additional benzoyl chloride (100 μL) was added to consume the remaining starting material. A mixture of mono- and bis-Bz protected products was observed. The reaction mixture was quenched with H 2 O (5 mL), and the resulting mixture was stirred for 5 min. Concentrated NH ₄ OH( _BODH ) (8 mL) was then added in one portion and the mixture was allowed to stir for 15 min, during which time the bis-Bz product was converted to the desired product. The solvents were removed under reduced pressure, and the residue was then co-evaporated with CH ₃ OH. Intermediate 2b was isolated after purification by silica gel chromatography using a gradient eluent of 50%-100% EtOAc in hexane.

1H NMR (400 MHz, DMSO-d ₆ ) δ 8.26-7.91 (m, 2H), 7.88-7.79 (m, 1H), 7.61 (t, J=7.5 Hz, 1H), 7.56-7.36 (m, 2H), 7.37-7.24 (m, 1H), 7.10 (d, J=4.6 Hz, 1H), 7.01 (s, 1H), 5.53 (d, J=23.4 Hz, 1H), 5.45 (d, J=6.5 Hz, 1H), 5.16-4.91 (m, 1H), 4.86 (t, J=5.6 Hz, 1H), 4.21-4.04 (m, 1H), 3.82 (dd, J=8.0, 3.9 Hz, 1H), 3.71 (ddd, J=12.3, 5.6, 2.6 Hz, 1H), 3.52 (ddd, J=12.2, 5.7, 4.5 Hz, 1H).

¹⁹ F NMR (376 MHz, DMSO-d ₆ ) δ -196.33 (dt, J=55.1, 22.5 Hz).

GC/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, 2.6 µm, 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.

Intermediate 2c is N-(7-((2S,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)pyrrolo[1.2-f][1.2.4]triazin-4-yl)benzamide.

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 N ₂ . At room temperature, 4,4'-dimethoxytrityl chloride (1.71 g, 5.0 mmol) was added in one portion and the mixture was stirred for 2 h. Ethanol (2 mL) was added, and the resulting solution was stirred for 5 min. The solvents were removed under reduced pressure. The residue was purified by silica gel chromatography using a gradient eluent of 0% to 100% EtOAc in hexanes to afford intermediate 2c.

1H NMR (400 MHz, DMSO-d ₆ ) δ 8.28-8.02 (m, 2H), 7.61 (t, J=7.5 Hz, 1H), 7.51 (t, J=7.6 Hz, 2H), 7.36 (ddt, J=6.0, 4.7, 2.0 Hz, 2H), 7.31-7.04 (m, 7H), 6.90-6.73 (m, 4H), 5.62 (d, J=24.4 Hz, 1H), 5.49 (d, J=7.0 Hz, 1H), 5.27-5.01 (m, 1H), 4.32-4.13 (m, 1H), 4.06-3.95 (m, 1H), 3.69 (s, 4H), 3.28 (s, 3H), 3.12 (dd, J=10.4, 5.2 Hz, 1H).

¹⁹ F NMR (376 MHz, DMSO-d ₆ ) δ -195.58 (dt, J=52.1, 24.7 Hz).

GC/MS: t _R = 1.37 min, MS m/z = 675.29 [M+1]; LC system: Thermo Accela 1250 UHPLC

MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µm, 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.

NHBz NHBz

1. TBSCI, Imid, dmf

2. TsOH, MeOH, CHCl ₃

HO F TBSO F

2c 2d

Intermediate 2d is 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 N2, imidazole (251 mg, 3.70 mmol) was added, followed by tert-butylchlorodimethylsilane (492 mg, 3.27 mmol). The solution was stirred at room temperature for 16 h. The solution was diluted with H2O (5 mL), and the solvents were then 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.

- 25 045066

The organic products _were dried over _Na2SO4 , which was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude material was used as such in the next step.

The crude material was dissolved in CHCl ₃ (15 mL) and cooled to 0 °C. To the mixture was added dropwise p-toluenesulfonic acid hydrate (414 mg, 2.18 mmol) dissolved in CH ₃ OH (6 mL), and the mixture was stirred for 15 min. The reaction mixture was quenched with sat. NaHCO ₃ ( _aq ). The organics were washed with brine and dried over Na ₂ SO ₄ , filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography using a gradient of 0-50% EtOAc in hexanes to afford intermediate 2d.

1H NMR (400 MHz, DMSO-d ₆ ) 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, 1H), 5.07 (dt, J=54.6, 3.8 Hz, 1H), 4.86 (t, J=5.5 Hz, 1H), 4.28 (ddd, J=17.8, 7.1, 4.4 Hz, 1H), 3.83-3.72 (m, 1H), 3.63 (ddd, J=12.1, 5.2, 3.0 Hz, 1H), 3.43 (ddd, J=12.1, 6.0, 4.2 Hz, 1H), 0.80 (s, 9H), 0.01 (s, 3H), 0.00 (s, 3H).

¹⁹ F NMR (376 MHz, DMSO-d ₆ ) δ -198.05 (dt, J=54.2, 19.8 Hz).

GC/MS: tR=1.35 min, MS m/z=487.24 [M+1]; LC system: Thermo Accela 1250 UHPLC

MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µm, 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.

Intermediate 2e is 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 a solution of intermediate 2d (856 mg, 1.75 mmol) in toluene (4 mL) and DMSO (6 mL) prepared under N2 was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (504 mg, 2.63 mmol). To this mixture were added pyridine (150 μL) and TFA (70 μL), and the mixture was stirred at room temperature for 15 min. Additional EDCI (100 mg) and pyridine (100 μL) were added, and the mixture was stirred for another 45 min. The reaction mixture was quenched with _H2O (10 mL) and _CH2Cl2 ( ₁₀ mL). The organic products were washed _with saturated brine and dried over _Na2SO4 , filtered, and the solvent was removed under reduced pressure to give the crude aldehyde, which was used as such in the next step.

The crude aldehyde was dissolved in dioxane (5 mL) and 37% formaldehyde ( _aq ) (925 μL) was added, followed by 2N NaOH ( _aq ) (925 μL). After stirring at room temperature for 3 h, the reaction mixture was quenched with AcOH, diluted with EtOAc, and washed with H ₂ O. The organics were dried over Na ₂ SO ₄ , filtered, and the solvent was removed under reduced pressure to yield the crude aldol product, which was used as such in the next step.

Under N ₂ atmosphere, the crude aldol product was dissolved in EtOH (9 mL) and cooled to 0 °C. NaBH ₄ (80 mg, 2.1 mmol) was added in one portion, and the reaction mixture was stirred for 10 min. The reaction mixture was quenched with AcOH, diluted with CH ₂ Cl ₂ , and washed with a 1:1 solution of water and saturated NaHCO ₃ ( _aq ). The organic phase was dried over Na ₂ SO ₄ , filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography using an elution gradient of 0 to 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), 7.15-6.93 (m, 2H), 5.50 (d, J=14.1 Hz, 1H), 5.24 (dt, J=54.2, 5.4 Hz, 1H), 4.78 (t, J=5.6 Hz, 1H), 4.48 (dd, J=10.7, 4.8 Hz, 1H), 4.34 (dd, J=6.7, 4.9 Hz, 1H), 3.62 (dd, J=11.9, 4.9 Hz, 1H), 3.55-3.35 (m, 3H), 0.82 (s, 9H), 0.07 (s, 3H), -0.09 (s, 3H).

¹⁹ F NMR (376 MHz, DMSO-d ₆ ) δ -200.37 (d, J=51.1 Hz).

GC/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, 2.6 µm, 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.

Intermediate 2f is N-(7-((2S,3S,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy)methyl)-3-fluorotetrahydrofuran-2-yl)pyrrolo[1.2-f][1.2.4]triazin-4-yl)benzamide.

Intermediate 2e (370 mg, 0.717 mmol) was dissolved in _CH2Cl2 (10 mL) and TEA (200 µL) _{under N2} and then cooled to 0°C. 4,4'-Dimethoxytrityl chloride (0.364 g, 1.07 mmol) was added in one portion, and the reaction mixture was stirred for 30 min. _CH3OH (2 mL) was added, and the solution was diluted _{with CH2Cl2} and sat. NaHCO3 _(luull) . 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 a gradient eluent of 5-100% EtOAc in hexane to afford the crude product as a mixture of bis-DMTr and 4β products. This mixture was used further without further purification.

Under N ₂ atmosphere, imidazole (143 mg, 2.10 mmol) was added to the crude product (57.4 mg, mixture) in DMF (3 mL), followed by tert-butylchlorodimethylsilane (158 mg, 1.05 mmol). The solution was allowed to stir at room temperature for 2 h. The solution was diluted with CH ₃ OH (1 mL) and EtOAc. The organics were washed with H ₂ O and then brine. The organic layer was dried over Na ₂ SO ₄ , filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography using a gradient eluent of 0-50% EtOAc in hexane to afford intermediate 2f, which contains some bis-DMTr product.

GC/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, 2.6 µm, Cl8, 100A, 50x3.00 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 is X-(7-((28,38,4K,5K)-4-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy)methyl)-3-fluoro-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrrolo[ 1.2f] [ 1.2.4]triazin-4-yl)benzamide.

Intermediate 2f was dissolved in _CHCl3 (5 mL) and cooled to 0°C. p-Toluenesulfonic acid hydrate (90 mg, 0.474 mmol) dissolved in _CH3OH (4 mL) was added dropwise to the mixture, and the reaction mixture was stirred for 5 min. The reaction mixture was quenched with sat. _XaHCO3 ( _HOD n). 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 a gradient eluent _of 0-40% EtOAc in hexane to afford intermediate 2g.

Ή NMR (400 MHz, CDC1 ₃ ) 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).

¹⁹ F NMR (376 MHz, CDC1 ₃ ) δ -196 (m).

GC/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, 2.6 µm, Cl8, 100A, 50x3.00 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.

TBSOНО-TBS0 F ^vg 7BSQ f

2d 2h

Intermediate 2h is X-(7-((28,38,4Я,5К)-5-((E)-2-bromovinyl)-4-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy)methyl)-3-fluorotetrahydrofuran-2-yl)pyrrolo[1.2-f][1.2.4]triazin-4-yl)benzamide.

To a solution of intermediate 2g (228 mg, 0.361 mmol) in toluene (0.75 mL) and DMSO (0.15 mL) under N ₂ was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (208 mg, 1.08 mmol). To this mixture were added pyridine (30 μL) and TFA (15 μL), and the mixture was stirred at room temperature for 30 min. The reaction mixture was diluted with EtOAc and washed with H ₂ O, then brine. The organics were dried over Na ₂ SO ₄ , filtered, and the solvent was removed under reduced pressure to yield the crude aldehyde. This product was used as such without further purification.

NHBz

1, DMSO, Tol, EDCI, Rug.tFU

XT

N'

2. bromomethyl triphenyl phosphonium bromide, KOtBu, THF

TBSO

NHBz

-27045066

To a suspension of bromomethyltriphenylphosphonium bromide (314 mg, 0.72 mmol) in THF (4 mL) at -40 °C was added KOtBu (1.0 M in THF, 1.08 mL, 1.08 mmol), and the reaction mixture was stirred for 2 h under N ₂ . The crude aldehyde was dissolved in THF (4 mL) and added dropwise. The reaction mixture was removed from the cold bath and allowed to warm to 10 °C over 1 h. The reaction mixture was cooled again to -40 °C, and the reaction mixture was quenched with saturated NH ₄ Cl ( _aq ). The layers were separated, and the organic products were dried over Na ₂ SO ₄ , filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography using 0%-50% EtOAc in hexane as eluent to give intermediate 2h.

1H NMR (400 MHz, CDCl ₃ ) 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, 1H), 4.69-4.46 (m, 1H), 3.97 (d, J=11.4 Hz, 1H), 3.53 (d, J=11.4 Hz, 1H), 0.84 (d, J=3.9 Hz, 18H), 0.13-0.10 (m, 12H).

¹⁹ F NMR (376 MHz, CDCl3) δ -190.60 (m).

GC/MS: tR=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, 2.6 μm, C18, 100A, 50x3.00 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 NHBz

Intermediate 2i is N-(7-((2S,3S,4R,5R)-4-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy)methyl)-5-ethynyl-3-fluorotetrahydrofuran-2-yl)pyrrolo[1.2-f ][1.2.4]triazin-4-yl)benzamide.

Intermediate 2h (204 mg, 0.289 mmol) was dissolved in THF (8 mL) under N2 and cooled to -40°C. KOtBu (1.0 M in THF, 1.08 mL, 1.08 mmol) was added slowly. The reaction mixture was stirred for 20 min and quenched with sat. _NH4Cl ( _aq ). The solution was diluted with EtOAc and washed with brine. The organics were dried _{over Na2SO4} , filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography using a gradient eluent of 0%-50% EtOAc in hexanes to afford intermediate 2i.

1H NMR (400 MHz, CDCl3) 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, 18N), 0.20-0.08 (m, 12N).

¹⁹ F NMR (376 MHz, CDCl ₃ ) δ -193.10 (br s).

GC/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, 2.6 µm, C18, 100A, 50x3.00 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 is 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) at room temperature under N ₂ was added TBAF (1.0 M in THF, 700 μL, 0.700 mmol), and the mixture was stirred for 30 min. The solvents were removed under reduced pressure. The residue was purified by silica gel chromatography using a gradient eluent of 40% to 100% EtOAc in hexanes to afford intermediate 2j.

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

MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µm, C18, 100A, 50x3.00 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.

- 28 045066

Example 2-(2R,3R,4R,5S)-5-(4-aminopyrrolo[1.2-f][1.2.4]triazin-7-yl)-2-ethynyl-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol.

To a solution of intermediate 2j (71 mg, 0.179 mmol) in CH ₃ OH (2 mL) was added conc. NH ₄ OH( _BODH ) (0.7 mL), and the resulting solution was stirred at room temperature for 16 h. The solvents were removed under reduced pressure. The residue was purified by silica gel chromatography using a gradient of 0% to 20% CH ₃ OH in CH ₂ Cl ₂ , then by reverse-phase HPLC using a gradient of 0% to 20% ACN in H ₂ O to afford the compound of Example 2.

1H NMR (400 MHz, CD3OD) δ 7.77 (s, 1H), 6.90-6.70 (m, 2H), 5.62 (dd, J=25.5, 2.6 Hz, 1H), 5.18 (ddd, J=56.0, 5.4, 2.7 Hz, 1H), 4.57 (dd, J=20.5, 5.4 Hz, 1H), 3.93-3.59 (m, 2H), 3.02 (d, J=0.7 Hz, 1H).

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

GC/MS: t _R = 0.45 min, MS m/z = 2 93.13 [M+1]; LC system: Thermo Accela 1250 UHPLC

MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µm, 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 series. Column: Phenomenex Kinetex C18, 2.6 µm, 100 A, 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.

TBscf ¥ TBsef ¥

2d For

Intermediate 3a is N-(7-((2S,3S,4R,5R)-4-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy)methyl)-3-fluoro-5-formyltetrahydrofuran-2-yl)pyrrolo[1.2-f ][1.2.4]triazin-4-yl)benzamide.

To a solution of intermediate 2g (1.13 g, 1.79 mmol) in DMSO (1 mL) and toluene (10 mL) prepared under N ₂ were added EDCI-HCl (1.02 g, 5.36 mmol) and pyridine (149 µL, 1.92 mmol). TFA (74 µL, 0.97 mmol) was added dropwise. After 1 h, the reaction mixture was monitored by GC/MS. A single peak was observed with a retention time similar to the starting materials, but with an M+1 peak equal to that expected for the product. An additional 50 µL of pyridine was added, and the reaction mixture was stirred for an additional 15 min. No change was observed by GC/MS. The reaction mixture was diluted with EtOAc and quenched with a 1:1 mixture of sat. NaHCO ₃ ( _BODH ) and H ₂ O. The mixture was partitioned between EtOAc and more H ₂ O. The organic layer was separated and washed with H ₂ O, brine, and then dried over Na ₂ SO ₄ . The drying agent was removed by vacuum filtration, and the filtrate was concentrated. The residue was treated with CH ₂ Cl ₂ , concentrated, and the resulting material was placed under high vacuum for 1 h. The product, intermediate 3a, was used as such in the next step.

GC/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, 2.6 µm, C18, 100A, 50x3.00 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 NHBz tbso- о ХАN ^H0NH ^% Тв° о AA gi pyridine _z N^V Г ьИ but )—( TBsef ¥ TBsef ¥

For ЗЬ

Intermediate 3b is N-(7-((2S,3S,4R,5R)-4-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy)methyl)-3-fluoro-5-((E)-(hydroxyimino)methyl)tetrahydrofuran-2-yl)pyrrolo[1.2-f][1.2.4]triazin-4-yl)benzamide.

To a solution of intermediate 3a (the crude product from the previous step, estimated 1.79 mmol) in pyridine (11 mL) prepared under _N2 , _HONH2 -HCl was added in one portion at room temperature. The reaction mixture was monitored by GC/MS 5 min later; the starting material was consumed. The reaction mixture was checked again 25 min later. There were no changes from the initial time. The reaction mixture was concentrated, and the residue was partitioned between EtOAc and _H2O . The organic layer was separated, washed with saturated brine, dried over Na2SO4, and filtered. The filtrate was concentrated, taken up in CH2Cl2, concentrated again, and the residue was placed under high vacuum. The product, intermediate 3b, was used as such in the next step.

GC/MS: tR=1.83 min, MS m/z=644.55 [M+1]; LC system: Thermo Accela 1250 UHPLC

MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µm, C18, 100A, 50x3.00 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 is N-(7-((2S,3S,4R,5R)-4-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy)methyl)-5-cyano-3-fluorotetrahydrofuran-2-yl)pyrrolo[1.2-1][1.2.4]triazin-4-yl)benzamide.

To a solution of intermediate 3b (the crude product from the previous step, presumably 1.79 mmol) in ACN (16 mL) was added CDI (436 mg, 2.69 mmol) in one portion. The reaction was carried out under N2. After 20 min, the reaction mixture was monitored by GC/MS. The peaks corresponding to the mass of the starting material and the product were barely separated in time. The reaction mixture was checked 1.5 h later. The UV peak corresponding to the starting material had almost disappeared, and the intensity of the mass peak was reduced. The reaction mixture was diluted _{with CH2Cl2} and quenched with a 1:1 mixture of sat. _NaHCO3 ( _aq ) and _H2O . The layers were separated, the aqueous layer was back-extracted _{with CH2Cl2} , and the combined organic 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 eluent gradient: 0% EtOAc in hexanes, increasing to 20% EtOAc in hexanes, pause at 20% EtOAc in hexanes, and then increasing 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).

¹⁹ F NMR (376 MHz, DMSO-Y6) δ -194.658 (dt, J=53, 21.4 Hz).

GC/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, 2.6 µm, C18, 100A, 50x3.00 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.

Zs 3d

Intermediate 3d - (2R,3R,4S,5S)-5-(4-aminopyrrolo[1.2-f][1.2.4]triazin-7-yl)-3(tert-butyldimethylsilyloxy)-2-((tert-butyldimethylsilyloxy)methyl)-4-fluorotetrahydrofuran-2-carbonitrile.

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 NH ₄ OH( _aq ) (3.74 mL). The cooling bath was removed, and the resulting heterogeneous solution was stirred overnight. The next day, the reaction was incomplete as determined by GC/MS. Additional concentrated NH ₄ OH( _aq ) (4 mL) and 2-MeTHF (12 mL) were added. The reaction mixture became homogeneous, but no further progress was observed after 20 min. The reaction mixture was concentrated, and the residue was dissolved in THF (15 mL). To this mixture were added concentrated NH ₄ OH( _aq ) (5 mL) and enough MeOH (1.9 mL) to make the solution homogeneous and monophasic. The reaction mixture was stirred at room temperature for 22 h. The reaction was nearly complete (approximately 5% _of starting material remained). The reaction mixture was diluted with _CH2Cl2 and _H2O . The layers were separated, and the aqueous layer was diluted with saturated _NaHCO3 ( _aq ) and extracted _{with CH2Cl2} . The aqueous layer was neutralized with 2N HCl and then extracted _{with CH2Cl2} . The combined organic layers were dried over Na2SO4, which was removed by filtration. The filtrate was concentrated, and intermediate 3d was isolated from the residue by silica gel column chromatography using the following eluent gradient: 0% EtOAc in hexane, increasing to 50% EtOAc in hexane, pause at 50% EtOAc in hexane, and then increasing to 100% EtOAc in hexane.

GC/MS: t _R = 1.59 min, MS m/z = 522.47 [M+1]; LC system: Thermo Accela 1250 UHPLC

- 30 045066

MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µm, 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-1.8 min 100% ACN, 1.8 min-1.85 min 100%-2% ACN,

1.85 min-2 min 2% ACN.

nh ₂ nh ₂ ^TBS %O HFPyr HO 0% ^N THF ^N tbsc/ ¥ ns/ ¥

3d 3

Example 3 - (2R,3R,4R,5S)-5-(4-aminopyrrolo[1.2-f][1.2.4]triazin-7-yl)-4-fluoro-3-hydroxy-2(hydroxymethyl)tetrahydrofuran-2-carbonitrile.

To a solution of intermediate 3d (100 mg, 0.191 mmol) in THF (2 mL) in a polypropylene tube under N2 at 0°C was added 70% HF-pyridine in pyridine (60 μL, 0.478 mmol). After 20 min, the reaction was checked; no reaction had occurred, so additional 70% HF-pyridine in pyridine (150 μL) was added and the cooling bath was removed. After 1 h and 50 min, additional 70% HF-pyridine in pyridine (150 μL) was added. After an additional 2 h, additional 70% HF-pyridine in pyridine (300 μL) was added. After an additional 2 h and 15 min, additional 70% HF-pyridine in pyridine (1 mL) was added. The reaction mixture became clear and homogeneous with the last addition of 70% HF-pyridine in pyridine. The reaction mixture was stirred overnight. The reaction was complete the next day. The reaction mixture was cooled in an ice bath and quenched with H ₂ O and a small amount of saturated NaHCO ₃ ( _aq ). The mixture was concentrated, and the residue was taken up in DMSO. The remaining insoluble material was removed by filtration, and the filtrate was partially purified by HPLC. The isolated material was dissolved in DMF and purified by HPLC. The compound from Example 3 was isolated with 0.5% as a salt with TFA.

1H NMR (400 MHz, DMF-d7) δ 7.92 (s, 1H), 6.99 (d, J=4.4 Hz, 1H), 6.89 (d, J=4.9 Hz, 1H), 6.64 (d, J=6 Hz, 1H), 5.92 (t, J=6.4 Hz, 1H), 5.83 (dd, J=25.2, 2 Hz, 1H), 5.40 (ddd, J=54.8, 4.8, 2.4 Hz, 1H), 4.75 (dt, J=22, 5.2 Hz, 1H), 4.02 (dd, J=12, 6.4 Hz, 1H), 3.87 (dd, J=12, 6.4 Hz, 1H).

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

GC/MS: tR=0.56 min, MS m/z=294.10 [M+1]; LC system: Thermo Accela 1250 UHPLC

MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µm, 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-1.8 min 100% ACN, 1.8 min-1.85 min 100%-2% ACN, 1.85 min-2 min 2% ACN.

HPLC: t _R = 3.220 min; HPLC system: Agilent 1100 series; column: Phenomenex Kinetex C18, 2.6 μm, 100 A, 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.

......about TEMPO ......Q, _H /~V< NBS ' /AL

4a 4b

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

A 10 L 4-necked round-bottomed flask was charged with a solution of intermediate 4a, (3aR,5S,6S,6aR)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydrofuro[3,2-d][1,3]dioxol-6-ol, (500 g, 1.90 mol) in dichloromethane/water (2.7 L/2.3 L) at room temperature. To it was added sodium carbonate (290 g, 3.42 mol). Then potassium carbonate (451 g, 3.24 mol) was added. Then 2,2,6,6-tetramethylpiperidinooxy (TEMPO, 15.2 g, 96.31 mmol) was added. Tetrabutylammonium bromide (31 g, 95.20 mmol) was added to the mixture. N-Bromosuccinimide (514 g, 2.86 mol) was added portionwise at 35°C. The resulting solution was stirred for 2 h at room temperature. The resulting solution was extracted with 2 x 1 L of dichloromethane, and the organic layers were combined. The resulting mixture was washed with 1 x 1.5 L of water. The mixture was dried over anhydrous sodium sulfate and concentrated in vacuo. This yielded (crude) intermediate compound 4b.

4b 4c

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

A solution of intermediate compound 4b (370

- 31 045066

g, 1.29 mol) in methanol (1300 ml). Sodium borohydride (26.4 g, 706.38 mmol) was added portionwise at room temperature. The resulting solution was allowed to react with stirring for 2 h at room temperature. The resulting mixture was concentrated in vacuo. The reaction was then quenched by adding 1000 ml of 5% aqueous ammonium chloride solution. The resulting solution was extracted with 3 x 500 ml dichloromethane, and the organic layers were combined. The resulting solution was washed with 2 x 300 ml water. The mixture was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by recrystallization from petroleum ether. This gives intermediate compound 4c.

'Ό NapBr _; ХХУ''У / Х\ dcm _map У ⁴ Ό\

BUT 50% NaOH NAPO

4s 4d

Intermediate compound 4d.

A solution of intermediate 4c (350 g, 1.34 mol) in dichloromethane (700 mL) was placed in a 5000 mL 4-necked round-bottomed flask. Tetrabutylammonium bromide (476.8 g, 1.48 mol) was added to it. 50% sodium hydroxide/water (700 g) was added to the mixture. 2-(bromomethyl)naphthalene (340 g, 1.54 mol) was added to it in several portions. The resulting solution was reacted with stirring for 4 h at room temperature. The reaction was then quenched by adding 1800 mL of dichloromethane/water (1:1). The resulting solution was extracted with 2 x 1 L dichloromethane, and the organic layers were combined. The resulting mixture was washed with 1 x 1000 mL water. The residue was dissolved in a 1000/1000 ml mixture of petroleum ether and water. The crude product was purified by recrystallization from petroleum ether. This afforded intermediate compound 4d.

4d 4e

Intermediate 4e is (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.

Intermediate 4d (500 g, 1.25 mol) was placed in a 5 L 4-necked round-bottomed flask. Acetic acid (1.8 L) was added. Water (600 mL) was added to the mixture. The resulting solution was stirred overnight at room temperature. The solids were filtered off. The resulting solution was extracted with 3 x 1 L petroleum ether, and the aqueous layers were combined. The resulting solution was diluted with 2 L ethyl acetate. The resulting mixture was washed with 2 x 2 L sodium chloride ( _aq ). The pH of the solution was adjusted to 8 with 50% sodium carbonate. The resulting solution was extracted with 2 x 1 L ethyl acetate, and the organic layers were combined and concentrated in vacuo. This gave intermediate 4e.

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

A 10 L 4-necked round-bottomed flask was charged with a solution of intermediate 4e (300 g, 833.33 mmol) in 1,4-dioxane (2100 mL) at room temperature. It was treated by adding a solution of sodium periodate (250 g) in water (4000 mL) at room temperature over 0.5 h. The resulting solution was reacted with stirring for 0.5 h at room temperature. The solids were filtered off. The resulting solution was extracted with 3 x 1000 mL ethyl acetate, and the organic layers were combined. The resulting mixture was washed with 2 x 1000 mL sodium chloride solution ( _aq ). The resulting mixture was concentrated in vacuo. This gave intermediate 4f.

Intermediate compound 4g is ((3aR,6S,6aR)-2,2-dimethyl-6-(naphthalen-2-ylmethoxy)tet- 32 045066 rahydrofuro[3,2-d] [ 1,3]dioxol-5,5-diyl)dimethanol.

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

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

A 5 L 4-necked round-bottomed flask, purged and maintained under an inert atmosphere of nitrogen, was charged with a solution of intermediate 4g (125 g, 346.84 mmol) in dichloromethane (2500 mL) at room temperature. Triethylamine (157.5 mL) was added to the mixture at room temperature. Tert-butyldiphenylsilyl chloride (157.5 mL) was added dropwise with stirring at 0-10 °C. The resulting solution was allowed to react with stirring overnight at room temperature. 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 solution ( _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 in vacuo. The crude product was purified by recrystallization from a dichloromethane/hexane mixture. This afforded intermediate compound 4h.

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

A 1000 mL 3-necked round-bottomed flask, purged and maintained under an inert atmosphere of nitrogen, was charged with a solution of intermediate 4h (20 g, 31.73 mmol) in toluene (320 mL), triphenylphosphine (35 g, 132.11 mmol), and imidazole (8.96 g, 132.26 mmol). Iodine (16.95 g, 66.8 mmol) was then added in several portions at 60 °C. The resulting solution was stirred overnight at 80 °C in an oil bath. The reaction mixture was cooled to room temperature in an ice/water bath. The resulting solution was diluted with 1000 mL ethyl acetate. The resulting mixture was washed

2x300 mL sodium thiosulfate ( _aq ). The resulting mixture was washed with 1x300 mL sodium chloride solution ( _aq ). The mixture was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was applied to a silica gel column using ethyl acetate/petroleum ether (1:10). This gave intermediate compound 4i.

Intermediate 4j is tert-butyldiphenyl(((3aR,5R,6S,6aR)-2,2,5-trimethyl-6-(naphthalen2-ylmethoxy)tetrahydrofuro[3,2-d][1,3]dioxol-5-yl)methoxy)silane.

A solution of intermediate 4i (66 g, 88.47 mmol) in a mixture of ethanol/ethyl acetate (600/600 mL), triethylamine (20.7 g, 202.52 mmol), and palladium on carbon (10% by weight, 24.8 g, 23.30 mmol) was placed in a 2000 mL round-bottomed flask, purged and maintained under an inert atmosphere of nitrogen. The resulting solution was stirred for 3 h at 40 °C. The solid products were filtered off. The resulting mixture was concentrated in vacuo. The resulting solution was diluted with 1500 mL of ethyl acetate.

The resulting mixture was washed with 1 x 500 mL of sodium chloride solution ( _aq ). The mixture was dried over anhydrous sodium sulfate and concentrated in vacuo. This gave intermediate compound 4j.

- 33 045066

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

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

......о TBAF ^{НС Х} ° О _ноч ^{оУ D} “ ' ns/

4k 41

Intermediate 41 - (3aR,5R,6S,6aR)-5-(hydroxymethyl)-2,2,5-trimethyltetrahydrofuro[3,2-d] [1,3]dioxol-6-ol.

A 50 mL round-bottomed flask was charged with a solution of intermediate 4k (520 mg, 1.12 mmol) in tetrahydrofuran (9 mL) and tetrabutylammonium fluoride (369 mg, 1.40 mmol). The resulting solution was stirred for 2 h at room temperature. The resulting mixture was concentrated in vacuo. The residue was applied to a silica gel column using dichloromethane/methanol (100:1). This afforded intermediate 41.

'H NMR (300 MHz, DMSO-d ₆ ): δ 5.64 (d, J=3.9 Hz, 1H), 4.96 (d, J=6.6 Hz, 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).

ВпО^К сх

BnBr.NaH ...... ⁰

Nc/ ”O \ ΊΦ ' _Вп( / уЛ

4m

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

A 50 mL 3-necked round-bottomed flask, purged and maintained under an inert atmosphere of nitrogen, was charged with a solution of intermediate 4L (180 mg, 0.84 mmol) in tetrahydrofuran (4 mL). Sodium hydride (60% wt, 140 mg, 3.50 mmol) was then added portionwise 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 temperature. To this solution, benzyl bromide (452 mg, 2.62 mmol) was added dropwise with stirring at 0 °C. The resulting solution was allowed to react with stirring for an additional 3 h at room temperature. 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, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was applied to a silica gel column using ethyl acetate/petroleum ether (1:30-1:20). This yielded intermediate compound 4m.

^Вп0 ......О h _; so ₄ ^Вп0

ВпО '°У АСО, Ас ₂ О _BnC J ^ _Ac

4m 4 η

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

In a 1000 mL round-bottomed flask was placed intermediate 4m (also prepared according to Biosci. Biotech. Biochem. 1993, 57, 1433–1438, 45 g, 111.19 mmol), acetic acid (270 mL), acetic anhydride (90 mL), and sulfuric acid (45 d). The resulting solution was stirred for 30 min at room temperature. The reaction was then quenched by adding 1000 mL of water/ice mixture. 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 solution ( _aq ). The resulting mixture was washed with 2 x 1000 mL of sodium chloride solution ( _aq ). The mixture was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was applied to a silica gel column using ethyl acetate/petroleum ether (1:30-1:20).

- 34 045066

This gave the intermediate compound 4n.

1H NMR (300 MHz, CDCl ₃ ): δ 7.28-7.38 (m, 10H), 6.13 (s, 1H), 5.37 (d, J=4.8 Hz, 1H), 4.44-4.68 (m, 4H),

4.33 (d, J=5.1 Hz, 1H), 3.33-3.45 (m, 2H), 2.15 (s, 3H), 1.88 (s, 3H), 1.35 (s, 3H).

MS m/z=451 [M+Na]

.....' ^0Ac 1) K ₂ CO ₃₁ MeOH BnO^f°y ⁰

Βηό bas ² ) ^NIS ' DCM BnO θ _Η

4n 4o

Intermediate compound 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. Acetonitrile was added and stirred for 5 min. Insoluble materials were filtered off and washed with acetonitrile. The filtrate was concentrated under reduced pressure. The resulting material was dissolved in anhydrous DCM (20 mL). Tetrabutylammonium iodide (1.66 g, 4.5 mmol) and N-iodosuccinimide (NIS, 1.69 g, 2.5 mmol) were added. The reaction mixture was stirred in the dark for 16 h. Additional NIS (0.85 g, 1.25 mmol) was added and the mixture was stirred for 4 h. The reaction mixture was diluted with EtOAc and washed twice with aqueous sodium thiosulfate and then with saturated aqueous sodium chloride. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purification on a silica gel column (0-30% EtOAc in hexanes) afforded intermediate 4o.

1H NMR (400 MHz, _CDCl3 ): δ 7.35-7.22 (m, 10H), 4.82 (br, 1H), 4.75-4.66 (m, 2H), 4.55-4.44 (m, 2H), 4.13 (d, J=8 Hz, 1H), 3.70-3.45 (m, 2H), 1.38 (s, 3H).

GC/MS: t _R = 2.58 min, MS m/z = 342.9 [M+1], 360.0 [M+ _H2O ]; GC/MS system: Thermo LCQ Advantage; Phenomenex Gemini, _C18 , 5 μm, 110A, 30x4.6 mm; buffer A: 0.1% acetic acid in water; buffer B: 0.1% acetic acid in acetonitrile 5-100% buffer B for 2.5 min, then 100% for 0.9 min at 2 mL/min.

HPLC: t _R =3.78 min; HPLC system: Agilent 1100; Phenomenex Gemini, C ₁₈ , 5 µm, 110A, 50x4.6 mm; buffer A: 0.05% TFA in water; Buffer B: 0.05% TFA in acetonitrile; 2-98% buffer B for 5 min at 2 ml/min.

ВпО·^^^ ^{0 a} 92О, ВпВг ВпО^^Г ⁰

Βηό OH ^EYAc Vpb OVp

4o 4r

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

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

1H NMR (400 MHz, _CDCl3 ): δ 7.35-7.16 (m, 15H), 5.03 (d, J=12 Hz, 1H), 4.79-4.71 (m, 2H), 4.52-4.40 (m, 4H), 4.06 (d, J=6 Hz, 1H), 3.49-3.39 (m, 2H), 1.38 (s, 3H).

GC/MS: tR=2.91 min, MS m/z=433.1 [M+1], 450.1 [M+H2O]; GC/MS system: Thermo LCQ Advantage; Phenomenex Gemini, C ₁₈ , 5 μm, 110A, 30x4.6 mm; buffer A: 0.1% acetic acid in water; buffer B: 0.1% acetic acid in acetonitrile; 5-100% buffer B for 2.5 min, then 100% for 0.9 min at 2 mL/min.

HPLC: t _R =4.54 min; HPLC system: Agilent 1100; Phenomenex Gemini, C ₁₈ , 5 µm, 110A, 50x4.6 mm; buffer A: 0.05% TFA in water; buffer B: 0.05% TFA in acetonitrile 2-98% buffer B for 5 min at 2 ml/min.

4p 1b 4q

Intermediate 4q is (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,

- 35 045066

0.570 mmol) was dissolved in anhydrous THF (20 mL) and stirred under Ar (g) at -70 °C. n-Butyllithium (2.5 M solution in hexane, 684 μL, 1.71 mmol) was added dropwise to the reaction mixture while maintaining the temperature below -65 °C. The reaction mixture was allowed to warm to -40 °C and held for 15 min. A solution of intermediate 4p (224 mg, 0.518 mmol) in THF (10 mL), pre-cooled 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 stirred mixture of EtOAc and citric acid ( _aq ). The mixture was stirred for 5 min. The organic layers were combined and washed with saturated NaCl ( _aq ). The organic layer was dried over _{anhydrous Na2SO4} and concentrated under reduced pressure. Purification by preparative HPLC gave intermediate 4q.

GC/MS: t _R = 2.60 min, MS m/z = 567.3 [M+1], 565.1 [M-1]; GC/MS system: Thermo LCQ Advantage; Phenomenex Gemini, C ₁₈ , 5 μm, 110A, 30x4.6 mm; buffer A: 0.1% acetic acid in water; buffer B: 0.1% acetic acid in acetonitrile; 5-100% buffer B for 2.5 min, then 100% for 0.9 min at 2 mL/min.

HPLC: tR=3.22 min; HPLC system: Agilent 1100; Phenomenex Gemini, C ₁₈ , 5 µm, 110A, 50x4.6 mm; buffer A: 0.05% TFA in water; Buffer B: 0.05% TFA in acetonitrile; 2-98% buffer B for 5 min at 2 ml/min.

Intermediate 4r is 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 N ₂ (g) in an ice bath. Triethylsilane (114 μL, 0.715 mmol) was added in one portion. Boron trifluoride diethyl etherate (27 μL, 0.215 mmol) was added dropwise. Stirred for 15 min and then the ice bath was removed. Stirred for 60 min. Triethylamine (100 μL, 0.715 mmol) was added and concentrated under reduced pressure. Dissolved in EtOAc and washed with saturated NaHCO ₃ ( _aq ) (2x) and then with saturated NaCl ( _aq ). The organic fractions were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. Purification on a silica gel column (0-80% EtOAc in hexane) yielded intermediate compound 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=4.4 Hz, 1H), 4.70-4.45 (m, 6H), 4.25-4.15 (m, 2H), 3.55-3.40 (m, 2H), 1.42 (s, 3H).

GC/MS: t _R = 2.75 min, MS m/z = 551.4 [M+1]; GC/MS system: Thermo LCQ Advantage Phenomenex Gemini, C ₁₈ , 5 μm, 110A, 30x4.6 mm; buffer A: 0.1% acetic acid in water; buffer B: 0.1% acetic acid in acetonitrile; 5-100% buffer B for 2.5 min, then 100% for 0.9 min at 2 mL/min.

HPLC: t _R =3.57 min; HPLC system: Agilent 1100; Phenomenex Gemini, C ₁₈ , 5 µm, 110A, 50x4.6 mm; buffer A: 0.05% TFA in water; Buffer B: 0.05% TFA in acetonitrile; 2-98% buffer B for 5 min at 2 ml/min. nh ₂ nh ₂

About Smeriv

Vpsg^+y ^NN n ₃ s \__/ HCO ₂ H n ₃ C \/ ³ II MeOHί I

VPO OVPNO HE

4g4

Example 4 - (2R,3S,4R,5S)-5-(4-aminopyrrolo[1.2-f][1.2.4]triazin-7-yl)-2-(hydroxymethyl)-2methyltetrahydrofuran-3,4-diol

Intermediate 4r (23 mg, 0.042 mmol) was dissolved in formic acid/MeOH (1:9, 10 mL). Palladium black was added and the mixture was stirred at 60°C for 90 min. The mixture was cooled to room temperature and filtered through Celite. The filtrate was concentrated under reduced pressure and purified by preparative HPLC. The mixture was concentrated under reduced pressure. It was dissolved in NaHCO ₃ ( _aq ) and purified by neutral HPLC to give the compound according to Example 4.

Preparative HPLC system: Gilson 215 Liquid Handler; Phenomenex Gemini, C ₁₈ , 4 µm, 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 at 20 ml/min.

1H NMR (400 MHz, CDCls): δ 8.01 (s, 1H), 7.41 (d, J=4.8 Hz, 1H), 7.02 (d, J=4.8 Hz, 1H), 5.33 (d, J=8 Hz, 1H), 4.53-4.49 (m, 1H), 4.15 (d, J=5.6 Hz, 1H), 3.50 (m, 2H), 1.27 (s, 3H).

GC/MS: t _R = 0.30 min, MS m/z = 2 81.3 [M+1], 279.0 [M-1]; GC/MS system: Thermo LCQ Advantage; Phenomenex Gemini, C ₁₈ , 5 μm, 110A, 30x4.6 mm; buffer A: 0.1% acetic acid in water; buffer B: 0.1%

- 36 045066 acetic acid in acetonitrile; 5-100% buffer B for 2.5 min, then 100% for 0.9 min at 2 ml/min.

HPLC: tR=0.42 min; HPLC system: Agilent 1100; Phenomenex Gemini, C ₁₈ , 5 µm, 110A, 50x4.6 mm; buffer A: 0.05% TFA in water; Buffer B: 0.05% TFA in acetonitrile; 2-98% buffer B for 5 min at 2 ml/min.

NHBz NHBz

ApN TMSN ₃ A^N

NO-. oUA | _лВГз 'TMSO-уоУ'1?

noA/ ch ₂ ci ₂ ν/ \_A

TBS0 OTBS TBSO OTBS

I'm 5a

Intermediate 5a is N-(7-((2S,3S,4S,5R)-5-azido-3,4-bis(tert-butyldimethylsilyloxy)5-((trimethylsilyloxy)methyl)tetrahydrofuran-2-yl)pyrrolo[1.2-f][1.2.4]triazin-4-yl)benzamide.

To a solution of crude intermediate 1m, N-(7-((2S,3S,4S)-3,4-bis(tert-butyldimethylsilyloxy)-5-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrrolo[1.2-f][1.2.4]triazin4-yl)benzamide, (-110 mg, -0.18 mmol) and azidotrimethylsilane (242 μL, 1.84 mmol) in dichloromethane (1.5 mL) at room temperature under argon was added indium(III) bromide (130 mg, 0.369 mmol). After 1 h, the reaction mixture was quenched with saturated aqueous sodium bicarbonate (1 mL). The resulting mixture was partitioned between dichloromethane (20 mL) and saturated aqueous sodium bicarbonate (20 mL). The phases were separated, and the aqueous layer was extracted with dichloromethane (20 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield intermediate 5a, which was used directly in the next step without further purification.

GC/MS: t _R = 3.52 min, MS m/z = 712.16 [M+1]; LC system: Thermo Accela 1250 UHPLC

MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 μm, ХВ-С18, 100A, 50x4.6 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-3.55 min 100% ACN, 3.55 min-4.2 min 100%-2% ACN at 2 µL/min.

NHBz NHBz

TMSO-xo A -D-— HO—S oW

N3^V/ Nf VJ

TBSO 0TBS H0 Ьн

5a 5b

Intermediate 5b is 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) at room temperature was added cesium fluoride (256 mg, 1.68 mmol). After 25 h, the reaction mixture was diluted with saturated 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 concentrated under reduced pressure to yield intermediate 5b, which was used directly in the next step without further purification.

GC/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, 2.6 µm, XB-C18, 100A, 50x4.6 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 2 µL/min.

HPLC: t _R = 2.46 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 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.

but he but he

5Ь 5

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 room temperature. After 2 days, the reaction mixture was concentrated under reduced pressure and immediately purified by preparative HPLC (Phenominex Synergi 4 µm Hydro-RR 80A, 150x30 mm column, 5-100% acetonitrile/water gradient). Fractions containing the desired products were combined and concentrated under reduced pressure. The residue was purified again.

- 37 045066 by column chromatography with SiO2 (4 g SiO2 Combiflash HP Gold Column, 0-20% methanol/dichloromethane) to obtain the compound according to 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, 1H), 4.63 (t, J=5.8 Hz, 1H), 4.37 (d, J=5.7 Hz, 1H), 3.69 (d, J=12.0 Hz, 1H), 3.59 (d, J=12.0 Hz, 1H).

GC/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, 2.6 µm, XB-C18, 100A, 50x4.6 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 2 µL/min.

HPLC: t _R = 1.287 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 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)

ns/ ¥

TR1

Example 6 (also referred to as TP-1) - ((2R,3R,4R,5S)-5-(4-aminopyrrolo[1.2-f][1.2.4]triazin-7yl)-2-cyano-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl tetrahydrofuran-2-yl)methyl tetrahydrotriphosphate.

The compound from Example 3 (15.0 mg, 0.05 mmol) was dried in a flask under vacuum overnight. Trimethyl phosphate (0.5 mL) and 1,8-bis(dimethylamino)naphthalene (25 mg, 0.12 mmol) were added to the flask, and the solution was allowed to stir under N2 while cooling in an ice/water bath. Distilled phosphorus oxychloride (10 µL, 0.11 mmol) was added, and the reaction mixture was allowed to stir for 4 h while cooling. Tributylamine (0.1 mL, 0.42 mmol) and tributylammonium pyrophosphate (0.8 mL of a 0.5 M solution in DMF, 0.4 mmol) were added, and the reaction mixture was allowed to stir for another 45 min while cooling. The reaction mixture was quenched with triethylammonium bicarbonate (0.5 M, 5 mL). The solvents were removed by rotary evaporation, and the remaining crude mixture was dissolved in 2 mL of water. The product was purified using a Sephadex DEAE A-25 column with a linear gradient of 0-1 M triethylammonium bicarbonate. The product-containing fractions were combined and concentrated to give the compound from Example 6 (TR1), then dissolved in 1 mL of water to yield a 10 mM solution.

MS m/z=532.0 [M-1]

Ion-exchange HPLC, retention time: 12.015 min; column: DNAPac PA-100 4x250 mm SN

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

Example 7 (also TP2) - ((2R,3R,4R,5S)-5-(4-aminopyrrolo[1.2-f][1.2.4]triazin-7-yl)-2-ethynyl-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl tetrahydrotriphosphate.

The compound from Example 2 (16.0 mg, 0.055 mmol) was dried in a flask under vacuum overnight. Trimethyl phosphate (0.5 mL) and 1,8-bis(dimethylamino)naphthalene (28 mg, 0.13 mmol) were added to the flask, and the solution was allowed to stir under N2 while cooling in an ice/water bath. Distilled phosphorus oxychloride (11 µL, 0.12 mmol) was added, and the reaction mixture was allowed to stir for 4 h while 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 mixture was allowed to stir for another 45 min while cooling. The reaction mixture was quenched with triethylammonium bicarbonate (0.5 M, 5 mL). The solvents were removed by rotary evaporation, and the remaining crude mixture was dissolved in 2 mL of water. The product was purified using a Sephadex DEAE A-25 column with a linear gradient of 0-1 M triethylammonium bicarbonate. The product-containing fractions were combined and concentrated to give the compound according to Example 7 (TR2), then dissolved in 1.4 mL of water to obtain a 10 M solution.

MS m/z=531.0 [M-1]

Ion-exchange HPLC, retention time: 19.829 min; column: DNAPac PA-100 4x250 mm SN

Solvent A: milliQ water; Solvent B: 0.5 M tetraethylammonium bromide; Solvent gradient program:

- 38 045066 equilibration with 100% A for 10 min, then gradient 0-80% B for 14 min; flow: 1 ml/min.

but-r°

NH ₂ HO ^z Y _p P Example ^H % o P(O)C1 ₃ ; ^but o^° , ^nh 2

ьЧ ^Βυ ₃ ΝΗ] ₂ Ρ ₂ Ο ₇ Η ₂ γ°^Λ~Ν Ν nЪ Ън ......\__j ^N not/ Ън 1 TP3

Example 8 (TP3) - ((2R,3S,4R,5S)-5-(4-aminopyrrolo[1.2-f][1.2.4]triazin-7-yl)-2-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl tetrahydrotriphosphate.

To a solution of the compound from Example 1 (5.0 mg, 0.017 mmol) in PO(OMe) ₃ (0.6 mL) was added POCl ₃ (45 mg, 0.29 mmol). The reaction mixture was stirred at 0 °C for 10 h, at which point ion-exchange HPLC showed approximately 50% conversion. A solution of tributylamine pyrophosphate 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 that the reaction was complete. The reaction mixture was quenched with triethylammonium bicarbonate buffer (1 M, 5 mL). The reaction mixture was stirred at room temperature for 0.5 h, then concentrated and co-evaporated twice with water. The residue was dissolved in _H2O (5 mL) and loaded onto an ion exchange column, eluting with _H2O , then 5-35% triethylammonium bicarbonate buffer (1 M) _-H2O . The fractions containing the product were combined, concentrated, and co-evaporated with _H2O . The residue was again purified by ion exchange to give the crude product. ^31P NMR showed that this product contained impurities, so the product was re-purified on a C18 column, eluting with 0-15% ACN-H2O containing 0.05% TEA, and the fractions containing the product were combined and concentrated to give 3.6 mg of product, which contained only 1.5 equiv of TEA, as shown by 1H NMR analysis. The product was dissolved in H ₂ O (1 mL) and buffered with triethylammonium bicarbonate (1 M, 0.1 mL). The resulting mixture was concentrated under reduced pressure and co-evaporated twice with H ₂ O under reduced pressure to yield the compound of Example 8 (TP3) as a tetra-TEA salt.

1H NMR (400 MHz, D2O): δ 7.78 (s, 1H), 6.85 (d, J=2.4 Hz, 1H), 6.82 (d, J=2.4 Hz, 1H), 5.51 (d, J=3.0 Hz, 1H), 4.65-4.55 (m, 2H), 4.20-4.08 (m, 2H), 3.15-3.00 (m, 24H), 1.18-1.08 (m, 36H).

³¹ P NMR (162 MHz, D2O): δ -6.25 (d, J=52 Hz), -12.21 (d, J=52 Hz), -22.32 (t, J=52 Hz).

MS m/z=530.2 [M-1], 532.1 [M+1]

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

To a solution of the compound from Example 5 (6.0 mg, 0.019 mmol) in PO(OMe)3 (0.6 mL) was added _POCl3 (45 mg, 0.29 mmol) at 0°C. The reaction mixture was stirred at 0°C for 10 h, at which point ion-exchange HPLC showed approximately 50% conversion. A solution of tributylamine pyrophosphate 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 mixture was quenched with triethylammonium bicarbonate buffer (1 M, 5 mL). The reaction mixture was stirred at room temperature for 0.5 h, then concentrated and coevaporated twice with water. The residue was dissolved in H2O (5 mL) and loaded onto an ion exchange column, eluting with H2O, then 5-35% triethylammonium bicarbonate buffer (1 M) _-H2O . The fractions containing the product were combined, concentrated, and coevaporated with _H2O . The residue was again purified by ion exchange to give the crude product. ^31P NMR showed that this product contained impurities, so the product was repurified by ion exchange again to give the crude product. The product was treated with _NaHCO3 (10 mg), and the mixture was concentrated under reduced pressure. The solid residue was dissolved in 0.5 mL _H2O , and 40 µL NaOH (1 N) was added. The resulting mixture was purified on a C18 column, eluted with H2O, and the product-containing fractions were combined and concentrated under reduced pressure to give the compound of Example 9 (TP4) as the tetrasodium salt.

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).

³¹ P NMR (162 MHz, D2O): δ -8.13 (d, J=19.8 Hz), -14.04 (d, J=18.9 Hz), -24.00 (t, J=19.3 Hz).

- 39 045066

MS m/z=546.l [M-1], 547.9 [M+1]

I ? _io TEA, CH ₂ CI ₂ 0

PD1a

Intermediate PD1a is 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 was cooled to -78°C was added pivalyl chloride (6.15 mL, 50.0 mmol) dropwise over 30 min. The reaction mixture was allowed to warm slowly to room temperature and the reaction progress was monitored by TLC. After 30 min, the _reaction was determined to be complete and was quenched with water. The layers were separated, and the aqueous layer was washed _{with CH2Cl2} . The organics were combined and dried over sodium sulfate. The solids were filtered, and the solvent was removed under reduced pressure. The crude product was purified by silica gel chromatography with 0-50% EtOAc/hexanes to afford intermediate PD1a.

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

Phosphorus oxychloride (281 μL, 3.08 mmol) was taken up in _{CH2Cl2 (} 5 mL) and the solution was cooled to -78°C. Thioester PD1a (1.00 g, 6.17 mmol) was taken _up in _CH2Cl2 (5 mL) and slowly added to the solution with _POCl3 . Then TEA (891 μL, 6.16 mmol) was added dropwise and the mixture was stirred cold for 30 min, then warmed to room temperature and stirred for 2 h. p-Nitrophenol (428 mg, 3.08 mmol) was added in one portion, then TEA (449 μL, 3.08 mmol) was added slowly. The mixture was stirred at room temperature for 30 min. TLC (70:30 hexane/EtOAC) showed only one spot, but GC/MS showed 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 purified again by HPLC to give intermediate PD1b, S,S'-2,2'-((4-nitrophenoxy)phosphoryl)bis(oxy)bis(ethane-2,1-diyl)bis(2,2-dimethylpropanethioate).

1H NMR (400 MHz, CDCl ₃ ) δ 8.29–8.21 (m, 2H), 7.46–7.36 (m, 2H), 4.23 (br q, J=7.7 Hz, 4H), 3.16 (br t, J=6.7 Hz, 4H), 1.23 (s, 18H).

³¹ P NMR (162 MHz, CDCl3) δ -7.72 (s).

Example 10 (also referred to as PD1) - S,S-2,2-((((2R,3S,4R,5S)-5-(4-aminopyrrolo[1.2f][1.2.4]triazin-7-yl)-2-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)phosphoryl)bis(oxy)bis(ethane-2,1-diyl)bis(2,2-dimethylpropanethioate).

The compound from Example 1 (6.0 mg, 0.02 mmol) was dissolved in NMP (0.1 mL) and THF (0.2 mL) was added. Then, tert-butyl magnesium chloride (1.0 M solution in THF, 0.031 mL, 0.031 mmol) was added at room temperature under argon. 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 heated to 50 °C. After 5 h, the resulting residue was purified by preparative HPLC (Phenominex Synergi 4 µm Hydro-RR 80A, 150x30 mm column, 40-100% acetonitrile/water gradient). Fractions containing the desired product were pooled and lyophilized to yield the compound of Example 10 (PD1).

1H NMR (400 MHz, _CDCl3 ) δ 7.82 (s, 1H), 6.69 (d, J=4.5 Hz, 1H), 6.64 (d, J=4.5 Hz, 1H), 5.56 (d, J=3.4 Hz, 1H), 4.61 (br s, 2H), 4.45-4.32 (m, 2H), 4.22-4.06 (m, 4H), 3.13 (dt, J=11.7, 6.7 Hz, 4H), 1.23 (s, 9H), 1.21 (s, 9H).

³¹ P NMR (162 MHz, CDCl ₃ ) δ -2.34 (s).

GC/MS: tR=1.70 min, MS m/z=660.02 [M+1]; LC system: Thermo Accela 1250 UHPLC

MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 μm, ХВ-С18, 100A, 50x4.6 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 2 µL/min.

HPLC: t _R = 3.204 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 mm; solvents: acetonitrile with 0.1% TFA, water with 0.1% TFA

- 40 045066

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

Example 11 (PD2) - S,S-2,2-((((2R,3R,4R,5S)-5-(4-aminopyrrolo[1.2-f][1.2.4]triazin-7-yl)-2-cyano-4fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)phosphoryl)bis(oxy)bis(ethane-2,1-diyl)bis(2,2-dimethylpropanethioate).

The compound from Example 3 (10.5 mg, 0.036 mmol) was dissolved in NMP (0.1 mL) and THF (0.1 mL) was added. Then, tert-butyl magnesium chloride (1.0 M solution in THF, 0.054 mL, 0.054 mmol) was added at room temperature under argon. 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 heated to 50 °C. After 24 h, the resulting residue was purified by preparative HPLC (Phenominex Synergi 4 μm Hydro-RR 80A, 150x30 mm column, 40-100% acetonitrile/water gradient). Fractions containing the desired product were pooled and lyophilized to yield the compound of Example 11 (PD2).

1H NMR (400 MHz, CDCl3) δ 7.94 (s, 1H), 6.75 (d, J=4.5 Hz, 1H), 6.67 (d, J=4.5 Hz, 1H), 5.77 (dd, J=27.8, 1.4 Hz, 1H), 5.43 (ddd, J=55.2, 4.9, 1.3 Hz, 1H), 4.93 (dd, J=21.2, 4.9 Hz, 1H), 4.49 (dd, J=11.3, 7.8 Hz, 1H), 4.40 (dd, J=11.3, 7.8 Hz, 1H), 4.10 (ddt, J=15.9, 8.0, 6.7 Hz, 4H), 3.16-3.04 (m, 4H), 1.23 (s, 9H), 1.21 (s, 9H).

³¹ P NMR (162 MHz, CDCl3) δ -2.10 (s).

¹⁹ F NMR (376 MHz, CDCl3) δ -191.64 (ddd, J=55.0, 27.8, 21.3 Hz).

GC/MS: t _R = 1.85 min, MS m/z = 662.03 [M+1]; LC system: Thermo Accela 1250 UHPLC

MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µm, HB-C18, 100A, 50x4.6 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 2 µl/min.

HPLC: t _R = 3.385 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 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 compound PD3c is (2S)-2-ethylbutyl 2-((4-nitrophenoxy)(phenoxy)phosphorylamino)propanoate.

Phenyl dichlorophosphate PD3a (1.5 mL, 10 mmol) was dissolved in 30 mL of anhydrous DCM and stirred under N ₂ (g) in an ice bath. The HCl salt of the amino ester PD3b, (S)-2-ethylbutyl 2-aminopropanoate hydrochloride (prepared according to Eur. J. Med. Chem. 2009, 44, 3765–3770, 2.1 g, 10 mmol), was added in one portion. TEA (3 mL, 22 mmol) was added dropwise. The mixture was stirred for 1 h at 0°C. p-Nitrophenol (1.4 g, 10 mmol) and TEA (1.5 mL, 11 mmol) were added in one portion. The reaction mixture was then stirred at room temperature for 16 h. It was diluted with DCM and washed with saturated _NaHCO3 ( _aq ). The organic fractions were dried over _{anhydrous Na2SO4} and concentrated under reduced pressure. Purification on a silica gel column (0-15% EtOAc in hexanes) gave intermediate PD3c.

1H NMR (400 MHz, CDCl ₃ ) 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=7.2 Hz, 6H).

³¹ P NMR (162 MHz, CDCl ₃ ) δ -3.04 (s), -3.10 (s).

GC/MS: tR=2.87 min, MS m/z=451.1 [M+1], 449.0 [M-1]; GC/MS system: Thermo LCQ Advantage; Phenomenex Gemini, C ₁₈ , 5 μm, 110A, 30x4.6 mm; buffer A: 0.1% acetic acid in water; buffer B: 0.1% acetic acid in acetonitrile; 5-100% buffer B over 2.5 min, then 100% for 0.9 min at 2 mL/min.

HPLC: t _R = 4.40 min; HPLC system: Agilent 1100; Phenomenex Gemini, C ₁₈ , 5 μm, 110A, 50x4.6 mm;

buffer A: 0.05% TFA in water; Buffer B: 0.05% TFA in acetonitrile; 2-98% buffer B for 5 min at 2 ml/min.

- 41 045066

Example 12 (PD3) - (2S)-2-ethylbutyl 2-((((2R,3R,4R,5S)-5-(4-aminopyrrolo[1.2-f][1.2.4]triazin-7yl)-2-cyano-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propanoate.

The compound from Example 3 (15 mg, 0.051 mmol) was dissolved in anhydrous DMF (1 mL) and stirred under N ₂ (g). p-Nitrophenyl phosphoamidate PD3c (35 mg, 0.077 mmol) was dissolved in anhydrous DMF (0.5 mL) and added to the reaction mixture in one portion. tBuMgCl in THF (1 M in THF, 77 μL, 0.077 mmol) was added dropwise. Stirring was carried out for 2 h. Additional p-nitrophenyl phosphoamidate (35 mg in 0.5 mL anhydrous DMF) and additional tBuMgCl (1 M in THF, 50 μL, 0.050 mmol) were added. Stirred for 2 h. Added additional p-nitrophenylphosphoamidate (35 mg in 0.5 mL anhydrous DMF) and additional tBuMgCl solution (1 M in THF, 50 µL, 0.050 mmol). Stirred for 16 h. Dilute with EtOAc and wash with saturated _NaHCO3 ( _aq ) (3x). Wash with saturated NaCl ( _aq ) and dry the organic fractions over anhydrous _Na2SO4 . Concentrate under reduced pressure. Purify on a silica gel column (0-5% MeOH in DCM). Combine the fractions and concentrate under reduced pressure. Purify by preparative HPLC with TFA as _a modifier to give the compound of Example 12 (PD3).

Preparative HPLC system: Gilson 215 Liquid Handler; Phenomenex Gemini, C ₁₈ 4 μm, 100 x 30.0 mm

Buffer A: 0.1% TFA in water; Buffer B: 0.1% TFA in acetonitrile; 5-100% buffer B for 13 min at 20 ml/min.

1H NMR (400 MHz, CDCl3) δ 7.89 (s, 1H), 7.31-7.13 (m, 6H), 6.80-6.75 (m, 1H), 5.80-5.70 (m, 1H), 5.355.20 (m, 1H), 4.80-4.62 (m, 1H), 4.60-4.45 (m, 2H), 4.35-4.10 (m, 1H), 4.06-3.96 (m, 3H), 1.49-1.28 (m, 8H), 0.90-0.82 (m, 6H).

³¹ P NMR (162 MHz, CDCl ₃ ) δ 2.36 (s), 2.22 (s).

HPLC: t _R =3.00 min; HPLC system: Agilent 1100; Phenomenex Gemini, C ₁₈ , 5 µm, 110A, 50x4.6 mm; buffer A: 0.05% TFA in water; Buffer B: 0.05% TFA in acetonitrile; 2-98% buffer B for 5 min at 2 ml/min.

GC/MS: t _R = 2.39 min, MS m/z = 605.1 [M+1], 603.0 [M-1]; GC/MS system: Thermo LCQ Advantage; Phenomenex Gemini, C ₁₈ , 5 μm, 110A, 30x4.6 mm; buffer A: 0.1% acetic acid in water; buffer B: 0.1% acetic acid in acetonitrile; 5-100% buffer B over 2.5 min, then 100% for 0.9 min at 2 mL/min.

Intermediate 6a - N-(7-((2S,3S,4R,5R)-4-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy)methyl)-3-fluoro-5-vinyltetrahydrofuran-2-yl)pyrrolo[1.2-1][1.2.4]triazin-4-yl)benzamide

Intermediate 2g, N-(7-((2S,3S,4R,5R)-4-(tert-butyldimethylsilyloxy)-5-((Ίtert-butyldimethylsilyloxy)methyl)-3-fluoro-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrrolo[1.2-f][1.2.4]triazin-4-yl)benzamide, (220 mg, 0.35 mmol) was dissolved in 5 mL of anhydrous DMSO and stirred under _N2 (g). EDCI (100 mg, 0.52 mmol) was added, followed by TFA-pyridine (34 mg, 0.18 mmol). The mixture was stirred for 1 h. Additional EDCI (100 mg, 0.52 mmol) was added and stirred for 1 h. GC/MS monitoring showed that the starting alcohol product remained. Additional EDCI (100 mg, 0.52 mmol) was added and stirred for 1 h. GC/MS monitoring showed that the reaction had reached complete conversion. The mixture was diluted with ethyl acetate and washed with saturated _NaHCO3 ( _aq ) (2x) and then saturated NaCl ( _aq ). The organic layer was dried over _{anhydrous Na2SO4} and concentrated under reduced pressure. Purified on a silica gel column (0-20% EtOAc in hexanes). The fractions were combined and concentrated under reduced pressure to give the aldehyde as a solid. Methyl triphenylphosphonium bromide (500 mg, 1.40 mmol) was suspended in 10 mL of anhydrous THF and stirred at -78 °C under Ar (g). A 2.5 M solution of n-butyl lithium in hexane (560 μL, 1.40 mmol) was added dropwise. The reaction mixture was stirred in an ice bath for 1 h to give a yellow mixture. The above aldehyde was diluted in 5 mL of anhydrous THF and added dropwise to the reaction mixture. The ice bath was removed, and the reaction mixture was allowed to warm to room temperature. It was stirred for 3 h at room temperature. A saturated aqueous solution of NH ₄ Cl was added and the mixture was extracted with ethyl acetate. The organic extract was washed with a saturated solution of NaHCO ₃ ( _aq ) and then with a saturated solution of NaCl ( _aq ). The organic fractions were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. Purification on a silica gel column (0-20% EtOAc in hexane) yielded intermediate compound 6a.

1H NMR (400 MHz, DMSO-d ₆ ) δ 8.22 (br s, 1H), 8.03 (br s, 2H), 7.58 (dt, J=40.4, 7.4 Hz, 3H), 7.12

- 42 045066 (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 (dd, J=55.4, 4.9, 2.7 Hz, 1H), 4.61 (dd, J=20.8, 4.8 Hz, 1H), 3.633.40 (m, 2H), 0.89 (s, 9H), 0.84 (s, 9H), 0.09 (d, J=8.4 Hz, 6H), 0.00 (d, J=14.1 Hz, 6H).

¹⁹ F NMR (376 MHz, DMSO-6) δ -191.86 (d, J=56, 8 Hz).

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

ba 13

Example 13 - (2R,3R,4R,5 S)-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 in THF (10 mL) and the resulting solution was stirred in an ice bath. 1 M TBAF in THF (700 μL, 0.70 mmol) was added and the mixture was stirred for 2 h. It was diluted with EtOAc and washed with a saturated NaCl solution ( _Bohn ) (5x). The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. It was dissolved in 7 M ammonia in MeOH (7 mL) and stirred for 18 h. The reaction mixture was concentrated under reduced pressure. Purification was carried out on a C ₁₈ preparative HPLC column with TFA as a modifier. The fractions were combined and concentrated under reduced pressure. The product was dissolved in NaHCO ₃ ( _BODH ) and purified again by preparative HPLC under neutral conditions. The fractions were combined and freeze-dried to yield the compound according to Example 13.

1H NMR (400 MHz, D2O) δ 7.54 (s, 1H), 6.62-6.49 (m, 2H), 5.98-5.79 (m, 1H), 5.55-5.36 (m, 2H), 5.31 (d, J=11.1 Hz, 1H), 5.11 (ddd, J=54.8, 5.2, 2.9 Hz, 1H), 4.42 (dd, J=20.6, 4.8 Hz, 1H), 3.62-3.43 (m, 2H).

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

MS m/z=295.2 [M+1]

NO F H0 F

14

Example 14 - (2R,3R,4R,5S)-5-(4-aminopyrrolo[1.2-f][1.2.4]triazin-7-yl)-2-ethyl-4-fluoro-2(hydroxymethyl)tetrahydrofuran-3-ol

The compound from Example 13 (5 mg, 0.017 mmol) was dissolved in methanol (2 ml). Degussa 10% Pd/C catalyst (2 mg) was then added, and the resulting mixture was stirred under a hydrogen gas atmosphere. 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 yield the compound from 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).

¹⁹ F NMR (376 MHz, D ₂ O) δ -197.62 (ddd, J=54.5, 20.6, 15.0 Hz).

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

PD1b PD4

Example 15 (PD4) - S,S-2,2-((((2R,3R,4R,5S)-5-(4-aminopyrrolo[1.2-f][1.2.4]triazin-7-yl)-4-fluoro-3hydroxy-2-vinyltetrahydrofuran-2-yl)methoxy)phosphoryl)bis(oxy)bis(ethane-2,1-diyl)bis(2,2-dimethylpropanethioate)

The compound from Example 13 (5 mg, 0.017 mmol) was dissolved in anhydrous DMF (0.5 mL). p-nitrophenoate (13 mg, 0.026 mmol) was added in one portion. 1 M solution of tert-butylmagnesium chloride in THF (25 μL, 0.026 mmol) was added dropwise. The mixture was stirred for 1 h. Heat was heated to 50°C and stirred for 2 h. More p-nitrophenoate (13 mg, 0.026 mmol) was added and stirred for 2 h. Another 1 M solution of tert-butylmagnesium chloride in THF (25 μL, 0.026 mmol) was added and the mixture was stirred for 16 h at 50°C. Cooled to room temperature. The resulting mixture was purified directly on a preparative HPLC column and eluted with a linear gradient of 0-100% ACN in water to give the compound of Example 15 (PD4).

1H NMR (400 MHz, CD.OD) δ 7.84 (s, 1H), 6.92 (d, J=4.5 Hz, 1H), 6.80 (d, J=4.5 Hz, 1H), 6.10 (dd,

- 43 045066

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=ll,l, 5.2 Hz, 1H), 4.08-3.95 (m, 5H), 3.06 (dd, J=7.0, 6.1 Hz,

4H), 1.21 (s, 9H), 1.18 (s, 9H).

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

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

PD3c PD5

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

The compound from Example 5 (5 mg, 0.016 mmol) was dissolved in anhydrous N-methyl-2-pyrrolidone (0.2 mL) and THF (0.1 mL) was added under argon. Tert-butyl magnesium chloride (1 M in THF, 24 μL, 0.024 mmol) was then added at room temperature, and a white solid precipitated. After 5 min, a solution of p-nitrophenyl phosphoamidate 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 at 50°C. After 3.5 h, the reaction mixture was allowed to cool to room temperature and stirred for 18 h. Then, PD3c p-nitrophenyl phosphoamidate (50 mg, 0.111 mmol) and tert-butyl magnesium chloride (1 M in THF, 24 μL, 0.024 mmol) were added and the reaction mixture was stirred for another 5 days. The resulting residue was then purified directly by preparative HPLC (Phenominex Synergi 4 μm Hydro-RR 80A, 150x30 mm column, 40-100% acetonitrile/water gradient). Fractions containing the desired product were combined and lyophilized to give the compound of Example 16 (PD5) (2:1 mixture of diastereomers).

1H NMR (400 MHz, CDCl ₃ ) δ 7.88 (br s, 1H), 7.33-7.22 (br m, 2H), 7.22-7.10 (br m, 3H), 6.69 (br d, J=4.4 Hz, 1H), 6.61 (br d, J=4.5 Hz, 1H), 5.64-5.56 (m, 1H), 4.54 (d, J=6.3 Hz, 1H), 4.50-4.20 (m, 3H), 4.11-3.94 (m, 3H), 3.90-3.76 (m, 1H), 1.49 (s, J=6.2 Hz, 1H), 1.40-1.24 (m, 7H), 0.86 (t, J=7.4 Hz, 6H).

³¹ P NMR (162 MHz, CDCl ₃ ) δ 2.68 (s), 2.56 (s).

GC/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, 2.6 µm, XB-C18, 100A, 50x4.6 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 2 µL/min.

HPLC: t _R = 3.010 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 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.

TR5

Example 17 (TP5) - ((2R,3R,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-4-fluoro-3-hydroxy2-vinyltetrahydrofuran-2-yl)methyl tetrahydrotriphosphate.

To a solution of the compound from Example 13 (6.0 mg, 0.020 mmol) in PO(OMe) ₃ (0.6 mL) was added POCl3 (50 mg, 0.32 mmol) at 0°C. The reaction mixture was stirred at 0°C for 6 h, at which point ion-exchange HPLC showed approximately 90% conversion. A solution of tributylamine pyrophosphate 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 mixture was quenched with triethylammonium bicarbonate buffer (1 M, 5 mL). The reaction mixture was stirred at room temperature for 0.5 h, then concentrated and coevaporated twice with water. The residue was dissolved in _H2O (5 mL) and placed on an ion exchange column, eluting with _H2O , then 5-35% triethylammonium bicarbonate buffer (1 M) _-H2O . The product-containing fractions were pooled, concentrated, and co-evaporated with _H2O . The solid residue was dissolved in 3 mL of _H2O , and 100 μL of NaOH (1 N) was added. The resulting mixture was purified on a C-18 column, eluting with _H2O , and the product-containing fractions were pooled and concentrated under reduced pressure to give the compound of Example 17 (TP5) as the tetrasodium salt.

1H NMR (400 MHz, D ₂ O): δ 7.74 (s, 1H), 6.89 (d, J=4.4 Hz, 1H), 6.81 (d, J=4.6 Hz, 1H), 6.00 (dd, J=17.4, 11.1 Hz, 1H), 5.72 (d, J=23.3 Hz, 1H), 5.49 (d, J=16.9 Hz, 1H), 5.32 (d, J=11.1 Hz, 1H), 5.14 (dd, J=54.0, 4.6 Hz, 1H), 4.72 (dd, J=23.7, 4.5 Hz, 1H), 4.09 (dd, J=11.3, 5.8 Hz, 1H), 3.79 (dd, J=11.6, 3.8 Hz, 1H).

³¹ P NMR (162 MHz, D ₂ O): δ -8.38 (d, J = 20.5 Hz), -13.67 (d, J = 19.3 Hz), -24.20 (t, J = 19.9 Hz).

- 44 045066 ¹⁹ F NMR (376 MHz, CDCl ₃ ) δ -194.58 (dt, J=55.0, 23.8 Hz). MS m/z=533.0 [M-1], 535.0 [M+1].

TP6

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

To a solution of the compound from Example 14 (5.0 mg, 0.017 mmol) in PO(OMe) ₃ (0.6 mL) was added _POCl3 (45 mg, 0.30 mmol) at 0°C. The reaction mixture was stirred at 0°C for 6 h, at which point ion-exchange HPLC showed approximately 90% conversion. A solution of tributylamine pyrophosphate 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 mixture was quenched with triethylammonium bicarbonate buffer (1 M, 5 mL). The reaction mixture was stirred at room temperature for 0.5 h, then concentrated and coevaporated twice with water. The residue was dissolved in _H2O (5 mL) and loaded onto an ion exchange column, eluting with _H2O and then 5-35% triethylammonium bicarbonate buffer (1 M) _-H2O . The product-containing fractions were pooled, concentrated, and co-evaporated with _H2O . The solid residue was dissolved in 3 mL of _H2O , and 100 μL of NaOH (1 N) was added. The resulting mixture was purified on a C-18 column, eluting with _H2O , and the product-containing fractions were pooled and concentrated under reduced pressure to give 18 (TP6) as the tetrasodium salt.

1H NMR (400 MHz, D ₂ O): 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, 1H), 5.23 (dt, J=55.2, 4.2 Hz, 1H), 4.65 (dd, J=20.6, 5.3 Hz, 1H), 4.08-3.84 (m, 3H), 1.83 (dq, J=14.4, 7.4, 6.9 Hz, 1H), 1.62 (dq, J=15.0, 7.5 Hz, 1H), 0.87 (t, J=7.5 Hz, 3H).

³¹ P NMR (162 MHz, D ₂ O): -5.72 (d, J = 20.2 Hz), -10.81 (d, J = 19.3 Hz), -21.60 (t, J = 19.8 Hz).

¹⁹ F NMR (376 MHz, CDCl3) δ -194.77 (dt, J=55.2, 21.2 Hz). MS m/z=535.1 [M-1], 536.9.0 [M+1].

2b 8a

Intermediate 8a is 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 a flask purged with argon was added 2b (68 mg, 0.183 mmol) in DMF (2 mL), followed by methyltriphenoxyphosphonium iodide (124 mg, 0.274 mmol). The reaction mixture was stirred at room temperature for 5 min, at which time complete conversion to the product was observed by LCMS. The reaction mixture was quenched with methanol and the solvents were removed under reduced pressure. The crude material was partitioned between EtOAc and H ₂ O. The organics were separated and washed with brine. The resulting material was dried over Na ₂ SO ₄ , filtered, and the solvent was removed under reduced pressure. The crude material was purified by silica gel chromatography (20-100% EtOAc/Hexane) to afford intermediate 8a.

1H NMR (400 MHz, DMSO-4) δ 8.17 (m, 3H), 7.63 (t, J=7.4 Hz, 1H), 7.53 (t, J=7.6 Hz, 2H), 7.17-6.96 (m, 2H), 5.74 (s, 1H), 5.60 (d, J=24.9 Hz, 1H), 5.19 (ddd, J=54.6, 4.5, 2.1 Hz, 1H), 4.09-3.92 (m, 1H), 3.72 (t, J=6.4 Hz, 1H), 3.63 (dd, J=11.0, 3.4 Hz, 1H), 3.44 (dd, J=11.0, 5.9 Hz, 1H).

¹⁹ F NMR (376 MHz, DMSO-66) δ -194.23 (m).

GC/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, 2.6 µm, 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.

G _N o F ^8a 8Ь

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

- 45 045066 methylenetetrahydrofuran-3-ol.

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

1H NMR (400 MHz, DMSO-66) δ 8.34-8.05 (m, 3H), 7.63 (t, J=7.4 Hz, 1H), 7.53 (t, J=7.6 Hz, 2H), 7.13 (d, J=4.7 Hz, 1H), 6.85 (d, J=4.4 Hz, 1H), 5.97-5.82 (m, 2H), 5.39-5.13 (m, 1H), 4.89-4.69 (m, 1H), 4.38 (d, J=2.1 Hz, 1H), 4.16 (t, J=1.8 Hz, 1H).

¹⁹ F NMR (376 MHz, DMSO-66) δ -198.14 (ddd, J=53.9, 24.7, 20.9 Hz).

GC/MS: tR=1.05 min, MS m/z=355.15 [M+1]

LC system: Thermo Accela 1250 UHPLC

MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µm, C18, 100A, 50x3.00 mm

Solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid

Gradient: 0 min-1.4 min 2-10 0% 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.

H0 F Hb F

8Ь 8с

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

Benzyltrimethylammonium chloride (55 mg, 0.296 mmol) and sodium azide (19.3 mg, 0.296 mmol) were dissolved in ACN (1 mL). The resulting mixture was stirred at room temperature overnight and then filtered and added via syringe to a solution of intermediate 8b (50 mg, 0.141 mmol) in THF (1 mL). N-Methylmorpholine (0.078 mL, 0.706 mmol) was then added, followed by a solution of iodine (65 mg, 0.25 mmol) in THF (1 mL) dropwise. After 15 min, N-acetyl cysteine was added until no more gas evolution was observed. Saturated aqueous sodium thiosulfate solution was then added until the solution turned light yellow. The crude mixture was partitioned between EtOAc and _H2O . The phases were separated, and the organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude material was purified by silica gel chromatography (0-60% EtOAc/Hex) to afford intermediate 8c.

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

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

GC/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, 2.6 µm, 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.

AsO F

8d

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

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

1H NMR (400 MHz, CDCl3) 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, 1H), 7.00 (d, J=4.6 Hz, 1H), 5.95-5.80 (m, 1H), 5.70-5.43 (m, 2H), 3.71 (d, J=11.3 Hz, 1H), 3.60 (d, J=11.3 Hz, 1H), 2.25 (s, 3H).

¹⁹ F NMR (376 MHz, CDCl3) δ -192.78 (ddd, J=55.7, 24.6, 18.5 Hz).

GC/MS: tR=1.35 min, MS m/z=566, 14 [M+1]; LC system: Thermo Accela 1250 UHPLC

MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µm, C18, 100A, 50x3.00 mm

- 46 045066

Solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid

Gradient: 0 min-1.4 min 2-10 0% 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 8e is ((2R,3R,4S,5S)-3-acetoxy-2-azido-5-(4-benzamidopyrrolo[2,1f][1,2,4]triazin-7-yl)-4-fluorotetrahydrofuran-2-yl)methyl benzoate.

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

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

¹⁹ F NMR (376 MHz, CDCl ₃ ) δ -192.57 (ddd, J=53.9, 25.1, 22.0 Hz).

GC/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, 2.6 µm, 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.

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

A 7N solution of NH ₃ in CH ₃ OH (2 mL) was added to intermediate compound 8e (24 mg, 0.043 mmol) at room temperature. 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 the compound according to Example 19.

1H NMR (400 MHz, methanol-d ₄ ) δ 7.81 (s, 1H), 6.85 (d, J=4.5 Hz, 1H), 6.80 (d, J=4.5 Hz, 1H), 5.80 (dd, J=24.7, 1.9 Hz, 1H), 5.22 (ddd, J=55.6, 5.1, 1.9 Hz, 1H), 4.63 (dd, J=22.9, 5.1 Hz, 1H), 3.81 (d, J=12.1 Hz, 1H), 3.70 (d, J=12.2 Hz, 1H).

¹⁹ F NMR (376 MHz, methanol-d ₄ ) δ -195.30 (ddd, J=55.5, 24.6, 22.9 Hz).

GC/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, 2.6 µm, 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.

9a 9Ь

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

To a solution of sodium hydride (60% by weight, 1.55 g, 38.7 mmol) in THF (100 mL) at 0 °C under argon was added ((3aR,6S,6aR)-6-(benzyloxy)-2,2-dimethyltetrahydrofuro[2,3d][1,3]dioxol-5,5-diyl)dimethanol (9a, purchased from Carbosynth, 10.0 g, 32.2 mmol). After 10 min, benzyl bromide (4.54 mL, 38.6 mmol) was added, and the reaction mixture was allowed to warm to room temperature. After 2 h, the reaction mixture was quenched with saturated aqueous ammonium chloride (500 mL). The resulting mixture was extracted with ethyl acetate (500 mL). The organic phase was then washed with saturated brine (400 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a colorless oil. The crude residue was purified by SiO2 column chromatography (220 g _SiO2 Combiflash HP Gold column, 0-100% ethyl acetate/hexane) to give intermediate 9a (9.49 g, 73%) as a colorless oil.

1H NMR (400 MHz, DMSO-d ₆ ) δ 7.38-7.19 (m, 10H), 5.68 (almost t, J=3.6 Hz, 1H), 4.73 (q, J=4.4 Hz, 1H), 4.63 (d, J=12.1 Hz, 1H), 4.49-4.36 (m, 3H), 4.24 (br s, 1H), 4.20-4.13 (m, 1H), 3.81 (d, J=11.9 Hz, 1H), 3.56 (d, J=11.9 Hz, 1H), 3.46 (q, J=10.3 Hz, 2H), 1.47 (s, 3H), 1.25 (s, 3H).

GC/MS: tR=1.88 min, MS m/z=423.31 [M+Na]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µ, XB-C18, 100A, 50x4.6 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 2 µL/min.

HPLC: tR=3.79 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 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 hexane, Rf=0.4 (UV)

VpO-d _o periodinan VpO—* _p

IV H.no Dessa-Martina V \.iP

BUT—'\ 1 Ч / / X/

CH ₂ C1 ₂ ;

BnO ^U BnO

9Ь9с

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

To a solution of intermediate 9b (1.95 g, 4.87 mmol) in dichloromethane (24.5 mL) at room temperature was added Dess-Martin periodinane (3.1 g, 7.3 mmol). After 1.5 h, the reaction mixture was purified by _SiO2 column chromatography (80 g _SiO2 Combiflash HP Gold column, 100% ethyl acetate/hexane) to afford intermediate 9c (1.94 g, 100%) as a colorless oil.

1H NMR (400 MHz, CDCl3) 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 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).

GC/MS: t _R = 1.99 min, MS m/z = 421.25 [M+Na]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µ, XB-C18, 100A, 50x4.6 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 2 µL/min.

HPLC: t _R = 4.09 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 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 hexane, Rf=0.6 (UV)

Es 9d

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

To a solution of methyltriphenylphosphonium bromide (5.38 g, 15.1 mmol) in tetrahydrofuran (20 mL) at -7 8 °C was added 2.5 M n-butyl lithium solution (6.02 mL). The reaction mixture was allowed to warm to 0 °C, and a solution of intermediate 9c (2.00 g, 5.02 mmol) in tetrahydrofuran (5 mL) was slowly added via syringe. The reaction mixture was allowed to warm to room temperature and stirred for 4 h. The reaction mixture was then quenched with saturated aqueous ammonium chloride solution (10 mL) and partitioned between water (200 mL) and ethyl acetate (200 mL). The layers were separated, and the organic layer was washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude residue was purified by SiO2 column chromatography (120 g SiO2 Combiflash HP Gold column, 0-50% ethyl acetate/hexane) to afford intermediate 9d (1.01 g, 51%) as a colorless oil.

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

GC/MS: tR=2.13 min, MS m/z=419.24 [M+Na]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µ, ХВ-С18, 100А, 50х4.6 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- 48 045066

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

HPLC: tR=4.37 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 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 hexane, Rf=0.55 (UV)

NS!

; y / MeOH ; Bηό ° dioxane BPO he

9d Ee

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

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

GC/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: Kinetex, 2.6 μm, ХВ-С18, 10θА, 50x4.6 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 2 µl/min.

HPLC: major anomer t _R = 4.01 min, minor anomer t _R = 3.955 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 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 hexane, major anomer Rf=0.30 (UV), minor anomer Rf=0.25 (UV)

Βηό ОН Βηό ОВп

Ee 9f

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

To a solution of intermediate 9e (1.0 g, 2.7 mmol) in THF (13.5 mL) at room temperature under argon was added NaH (60 wt%, 130 mg, 3.2 mmol) as a solid. 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 (5 mL) and partitioned between ethyl acetate (100 mL) and brine (100 mL). The phases were separated, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give crude intermediate 9f (1.57 g, 2:1 mixture of 1' anomers) as a colorless oil.

GC/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, 2.6 µm, HB-C18, 100A, 50x4.6 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 2 µl/min.

HPLC: major anomer t _R = 4.83 min, minor anomer t _R = 4.62 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 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.

ВпО ОВп Вηό ОВп

9f 9d

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

To intermediate compound 9f (1.5 g, 3.2 mmol) at 0°C, a solution of TFA (16 mL) and water (1.6 mL) were added, and the reaction mixture was allowed to warm to room temperature. After 9 h, water (1 mL) was added, and the reaction mixture was stirred for another 10 h. The reaction mixture was then

- 49 045066

The mixture was concentrated under reduced pressure. The crude residue was dissolved in ethyl acetate (200 mL) and washed with saturated aqueous sodium bicarbonate (2 x 150 mL) and saturated brine (150 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by _SiO2 column chromatography (24 g _SiO2 Combiflash HP Gold column, 0-100% ethyl acetate/hexane). Fractions containing the desired product were combined to give intermediate compound 9g (580 mg) as a colorless oil, which was a mixture with other impurities. The mixture was used directly in the next step.

GC/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, 2.6 µ, XB-C18, 100A, 50x4.6 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 2 µL/min.

HPLC: t _R = 4.34 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 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.

TRAR ON NMO . - CH ₂ C1 ₂ _

VPO OVP VPO OVP

9d 9h

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

To a solution of intermediate 9g (580 mg, 1.30 mmol) and 4A MS (100 mg) in DCM (6.45 mL) at room temperature were added tetrapropylammonium perruthenate (45.7 mg, 130 μmol) and 4-methylmorpholine N-oxide (457 mg, 3.89 mmol). After 1 h, silica gel (~500 mg) was added to the reaction mixture, and the resulting slurry was filtered through a pad of silica gel (~1 g). The filtrate was concentrated under reduced pressure. The crude residue was purified by SiO ₂ column chromatography (12 g SiO ₂ Combiflash HP Gold, 0-100% ethyl acetate/hexane) to afford intermediate 9h (254 mg, 18% over two steps) as a colorless oil.

1H NMR (400 MHz, _CDCl3 ) 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)

GC/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, 2.6 µ, XB-C18, 100A, 50x4.6 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 2 µL/min.

HPLC: t _R = 4.53 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 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 hexane, Rf=0.45 (UV)

ьпи иьп

9h 1b 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

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 argon was quickly added n-butyllithium (2.5 M in hexane, 1.0 mL, 2.5 mmol). 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 argon. After 20 min, the reaction mixture was allowed to warm to 0 °C and stirred for 15 min. The reaction mixture was quenched with saturated aqueous ammonium chloride (1 mL). The resulting mixture was diluted with ethyl acetate (100 mL) and washed with saturated aqueous sodium bicarbonate solution (100 mL) and saturated brine (100 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by _SiO2 column chromatography (12 g _SiO2 Combiflash HP Gold Column, 0-100% ethyl acetate/hexane) to afford intermediate compound 9i (11.1 mg, 5%, mixture of isomers) as a colorless oil.

GC/MS: tR=1.97 min, MS m/z=579.27 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 μm, ХВ-С18, 100А, 50х4.6 mm; solvents: acetonitrile with

- 50 045066

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 2 µL/min.

HPLC: tR=3.37 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 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) nh ₂ nh ₂

Et ₃ SiH

A κι Λ BF ₃ *Et ₂ OCAJ

ΒηΟ-Λ ₀ —A BnO^ _o — ⁰ 'у /OH CH2CI2 ...............^/

BnO OBn BnO OBn

9i 9j

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

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

1H NMR (400 MHz, CDCl3) 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).

GC/MS: t _R = 2.32 min, MS m/z = 563.33 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µ, XB-C18, 100A, 50x4.6 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 2 µL/min.

HPLC: t _R = 3.51 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 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)

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

Boron tribromide (1 M, 0.06 mL, 60 μmol) was added dropwise to a solution of intermediate 9j (7.7 mg, 13.7 μmol) in dichloromethane (1 mL) at -7 8 °C under argon. After 1 h, the reaction mixture was allowed to warm to 0 °C and stirred for an additional 1.5 h. The reaction mixture was cooled to -78 °C and quenched with a 2:1 methanol/pyridine solution (1.5 mL). The resulting mixture was allowed to warm to room temperature and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (Phenominex Synergi 4 µm Hydro-RR 80A, 150x30 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, 1H), 6.88 (d, J=4.5 Hz, 1H), 6.76 (d, J=4.5 Hz, 1H), 6.02 (dd, J=17.4, 11.0 Hz, 1H), 5.47 (dd, J=17.4, 2.0 Hz, 1H), 5.23 (dd, J=10.9, 2.1 Hz, 1H), 5.15 (d, J=8.3 Hz, 1H), 4.72 (dd, J=8.2, 5.7 Hz, 1H), 4.34 (d, J=5.7 Hz, 1H), 3.60 (d, J=11.8 Hz, 1H), 3.49 (d, J=11.8 Hz, 1N)

GC/MS: t _R = 0.84 min, MS m/z = 293.19 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µ, XB-C18, 100A, 50x4.6 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 2 µL/min

HPLC: tR=2.181 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 µm, 110A, 50x4.6 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.

- 51 045066

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

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

¹ H NMR (400 MHz, CD ₃ OD) δ 7.73 (s, 1H), 6.63 (s, 1H), 5.80 (dd, J=23.9, 1.7 Hz, 1H), 5.16 (ddd, J=55.3, 5.0, 1.7 Hz, 1H), 4.56 (dd, J=23.5, 5.0 Hz, 1H), 3.94-3.60 (m, 2H) ¹⁹ F NMR (376 MHz, CD ₃ OD) δ -161.76 (s), -195.42 (d, J=55.4 Hz)

MS m/z=328 [M+H]. MS system: Thermo LCQ Fleet

NH ₂ nh ₂ (BuNO ₂ ^ ^ ^N 'N^NH ₂ HF/Pyr

11a 11b

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

Intermediate 11a (2.0 g, 13.4 mmol) was placed in a polyTube. The reaction vessel was then placed in an ice bath and 70% HF/Pyr (18 mL) and pyridine (9 mL) were added sequentially. Immediately after the addition of pyridine, tBuNO ₂ (2.07 mL, 17.43 mmol) was then slowly added over 20 min. The solution turned from tan with the evolution of heat and gas. The reaction mixture was then stirred for another 20 min, and the reaction mixture was then diluted with water and concentrated under reduced pressure. The crude residue was partitioned between ethyl acetate and water. The organic products were separated, and the aqueous layer was washed with ethyl acetate three times. The organic products were combined and washed with brine. The crude product was dried over Na ₂ SO ₄ , 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 lib (1.68 g, 82%) as a tan solid.

1H NMR (400 MHz, DMSO-d ₆ ) δ 8.49–8.09 (m, 2H), 7.58 (t, J=2.0 Hz, 1H), 6.95 (d, J=4.5, 1H), 6.59 (d, J=4.5, 1H).

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

GC/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, 2.6 µ, XB-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 at 1.8 mL/min.

VG

11b 11c

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

A solution of 11b (3.36 g, 22.1 mmol) in DMF (50 mL) was cooled to 0 °C in an ice bath. A solution of 1,3-dibromo-5,5-dimethylhydantoin (3.16 g, 11.0 mmol) in DMF (50 mL) was added dropwise via an addition funnel over 40 min. After 1 h, the reaction mixture was quenched with _{a saturated Na2S2O3 solution} ( _BODH ), and the crude product was partitioned between EtOAc and 5% LiCl( _BODH ). The organics were extracted with 5% LiCl( _BODH ) (4x) then 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 in _CH2Cl2 and the solids were collected by filtration and dried under high vacuum to give intermediate 11c ( _3.93 g, 77%) as a yellow solid.

1H NMR (400 MHz, DMSO-a6) δ 8.49-8.44 (m, 2H), 7.08 (d, J=4.6 Hz, 1H), 6.76 (d, J=4.6 Hz, 1H).

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

GC/MS: t _R = 1.22 min, MS m/z = 232.98 [M+H]; LC system: Thermo Accela 1250 UHPLC; system

- 52 045066

MS: Thermo LCQ Fleet; column: Kinetex, 2.6 µm, HB-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 at 1.8 mL/min.

h ₂ n

11s 11e

Intermediate 11e is (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 1,2-bis(chlorodimethylsilyl)ethane (STABASE, 1.96 g, 9.08 mmol) in one portion, and the resulting mixture was allowed to stir at ambient temperature for 1 h. The reaction mixture was then cooled to -78 °C using a dry ice/methanol bath. nBuLi (2.5 M in hexane, 10.9 mL, 27.2 mmol) was added, maintaining the internal temperature at -65 °C. A solution of intermediate 11d (prepared according to WO 2012012776, 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 allowed to return to ambient temperature. The solvents were removed under reduced pressure, and the residue was treated with ethyl acetate. The organics were washed with water, then with saturated brine. The layers were separated, and the organics were dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to yield crude 11e as a mixture of isomers, which was used as such in the next step.

GC/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, 2.6 µ, XB-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 at 1.8 mL/min.

Vpb F BnO F

Not 11f

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

Intermediate 11e (1.99 g, 3.72 mmol) was dissolved in CH ₂ Cl ₂ (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). After 15 min, the reaction mixture was quenched with _saturated NaHCO3 ( _aq ) and the layers were separated. The aqueous layer was washed _{with CH2Cl2} . The organic layers were combined and washed with saturated _NaHCO3 ( _aq ). The organics were dried over _Na2SO4 , _filtered , and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (0-60% EtOAc/Hex) to afford intermediate 11f (1.12 g, 64%, 2:1 mixture of 1' anomers).

GC/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, 2.6 µ, XB-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 at 1.8 mL/min.

Intermediate 11g - (2R,3R,4R,5S)-5-(4-amino-2-fluoropyrrolo[2,1-f][1,2,4]triazin-7yl)-4-fluoro-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 filled with H ₂ ( _g ) (3x). After 1 h, the reaction vessel was purged with nitrogen. The resulting mixture was filtered through a pad of celite, and the filter pad was washed with CH ₃ OH. The filtrate was concentrated under reduced pressure and then coevaporated with ethyl acetate and then hexane to give intermediate 11g (503 mg, 98%, 2:1 mixture of 1' anomers).

GC/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, 2.6 μm, XB-C18, 100A, 50x3.00 mm; solvents: acetonitrile

- 53 045066 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 at 1.8 ml/min.

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

To a flask purged with argon was added a solution of 11 g (283 mg, 0.989 mmol) in DMF (10 mL), then a solution of methyl triphenoxyphosphonium iodide (0.536 g, 1.19 mmol) in DMF (4 mL). The reaction mixture was allowed to stir at 0 °C for 10 min and then warmed to ambient temperature. After 30 min, the reaction mixture was quenched with a saturated solution of Na ₂ S ₂ O ₃ ( _aq ). The crude material was partitioned between EtOAc and 5% LiCl ( _{BO dH} ). The organic products were separated and washed with saturated brine. The organic products were dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure. The crude residue was treated with ACN and purified by HPLC without acid modifier to give intermediate 11h (201 mg, 52%, 2:1 mixture of 1'anomers) as a white solid.

GC/MS: t _R = 1.08 min, MS m/z = 397.12 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µm, XB-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 at 1.8 ml/min.

Intermediate 11i is (3R,4R,5S)-5-(4-amino-2-fluoropyrrolo[2,1-f][1,2,4]triazin-7-yl)4-fluoro-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 at 60 °C. After 3 h, the reaction mixture was concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (40-100% EtOAc/Hex) to afford intermediate 11i (201 mg, 83%, 2:1 mixture of 1' anomers) as a white solid.

GC/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, 2.6 µ, XB-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 at 1.8 mL/min.

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

Benzyltrimethylammonium chloride (292 mg, 1.57 mmol) and sodium azide (102 mg, 1.57 mmol) were dissolved in ACN (4 mL), and the resulting mixture was stirred at ambient temperature for 4 h. The mixture was filtered, and the filtrate was added to a solution of 11i (0.201 g, 0.749 mmol) in THF (4 mL). NMM (0.412 mL, 3.75 mmol) was added, followed by a solution of iodine (0.342 g, 1.35 mmol) in THF (4 mL) dropwise. After 15 min _{, N-acetyl cysteine was added portionwise until gas evolution was observed. A saturated solution of Na2S2O3 (BODH ) was added} until the solution turned light yellow. The resulting mixture was partitioned between water and ethyl acetate. The layers were separated, and the organic layer was dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (20-100% EtOAc/Hex) to afford intermediate 11j (183 mg, 56%) as a single isomer.

1H NMR (400 MHz, DMSO-d ₆ ) δ 8.44 (d, J = 28.6 Hz, 2H), 6.98 (d, J = 4.6 Hz, 1H), 6.79 (d, J = 4.6 Hz, 1H), 6.32 (d, J = 6.9 Hz, 1H), 5.60 (dd, J = 23.8, 2.5 Hz, 1H), 5.36 (ddd, J = 54.9, 5.0, 2.6 Hz, 1H), 4.60 (ddd, J = 21.5, 6.9, 5.0 Hz, 1H), 3.63 (ABq, Δδ = 0.09 ppm, J = 8 Hz, 2H).

- 54 045066 ¹⁹ F NMR (376 MHz, DMSOM6) δ -71.74 (s), -194.57 (ddd, J=54.9, 24.0, 21.7 Hz)

GC/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, 2.6 µm, HB-C18, 100A, 50x3.00 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 1.8 mL/min.

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

To a solution of 11j (0.183 g, 0.419 mmol) in THF (10 mL) were added isobutanoic anhydride (0.083 mL, 0.502 mmol), TEA (0.118 mL, 0.837 mmol), and DMAP (10 mg, 0.084 mmol). The reaction mixture was allowed to stir at ambient temperature for 15 min and 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-a ₆ ) δ 8.48 (d, J=30.7 Hz, 2H), 6.99 (d, J=4.6 Hz, 1H), 6.84 (d, J=4.6 Hz, 1H), 5.77-5.47 (m, 3H), 3.69 (ABq, A5=0.05 ppm, J=12 Hz, 2H), 2.70 (p, J=7.0 Hz, 1H), 1.24-1.05 (d, J=7.0 Hz, 6H).

¹⁹ F NMR (376 MHz, DMSO-d ₆ ) δ -71.58 (s), -194.89 (ddd, J=55.0, 24.3, 16.8 Hz).

GC/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, 2.6 µ, XB-C18, 100A, 50x3.00 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 1.8 mL/min.

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

Intermediate 11k (0.153 g, 0.302 mmol) was dissolved in _{CH2Cl2 (} 10 mL) and H2O (6 mL). Dibasic potassium phosphate (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 temperature and stirred for 16 h. The reaction mixture _was then quenched with _{saturated Na2S2O3} ( _aq ) and concentrated under reduced pressure. The crude aqueous residue was diluted with ACN and purified by preparative HPLC without acid modifier to give intermediate 111 (20 mg, 13%) as a white solid.

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

¹⁹ F NMR (376 MHz, DMSO-d ₆ ) δ=-71.45 (s), -193.41 (ddd, J=54.4, 25.4, 21.2 Hz).

GC/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, 2.6 µ, XB-C18, 100A, 50x3.00 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 1.8 mL/min.

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

In a solution of intermediate compound 111 (22 mg, 0.041 mmol) in CH3OH (1 ml) at room temperature

- At room temperature, concentrated NH4OH (1 mL) was added. After 30 min, the reaction mixture was concentrated under reduced pressure. The crude residue was diluted with a minimal amount of H2O and purified by preparative HPLC without modifier to afford the compound of Example 22 (10 mg, 77%) as a white solid.

'H NMR (400 MHz, DMSO-d ₆ ) 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).

¹⁹ F NMR (376 MHz, DMSO-d ₆ ) δ -71.18 (s), -193.48 (dt, J=55.3, 23.8 Hz).

GC/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, 2.6 µ, XB-C18, 100A, 50x3.00 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 1.8 mL/min.

F nh ₂ Ji nh ₂

ITBSO-^ Selectfluor II TBSO-^/ +

N^' \_J ^N ACN\ /

TBSO' F TBSO ¥ ^3d 12a

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

To _{a solution of intermediate 3d (57 mg, 0.109 mmol) in ACN (3 mL) was added Selectfluor II (52 mg, 0.164 mmol) in one portion. After 1.5 h, the reaction mixture was quenched by the addition of saturated NaHCO3 (aq )} . Ethyl acetate (4 mL) was added, and the biphasic mixture was stirred vigorously for 5 min. The reaction mixture was then diluted with EtOAc and saturated NaHCO3 ( _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 eluent gradient: 0% EtOAc in hexane, increasing to 70% EtOAc in hexane, rapidly increasing to 100% EtOAc after the starting material had eluted from the column.

‘H 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, ΔδАв=0.141 ppm, J=11 Hz, 2H), 0.99 (s, 9H), 0.86 (s, 9H), 0.21 (s, 6H), 0.07 (s, 3H), -0.02 (s, 3H).

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

GC/MS: R _T = 2.06 min, MS m/z = 540.64 [M+1]; LC system: Thermo Accela 1250 UHPLC; MS: Thermo LCQ Fleet; column: Kinetex, 2.6 µ, C18, 100A, 50x3.00 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.

Example 23 - (2R,3R,4R,5S)-5-(4-amino-5-fluoropyrrolo[2,1-f][1,2,4]triazin-7-yl)-4-fluoro-3-hydroxy2-(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% HFpyridine in pyridine (51 μL, 1.97 mmol) at 0 °C under N2. After 1.5 h, the reaction mixture was removed from the ice bath. Additional 70% HFpyridine in pyridine was added after 3 h (150 μL), 5 h 45 min (200 μL), and 21 h 15 min (0.7 mL). The reaction mixture was then stirred for an additional 24 h at ℃, at which point the reaction mixture was cooled in an ice bath and then quenched with water and saturated _NaHCO3 ( _BODH ). 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). The filtrate was injected onto an HPLC column, and the semi-purified product was further purified by silica gel column chromatography using the following eluent gradient: 0% MeOH in DCM, increasing to 20% MeOH in DCM. The product-containing fractions were concentrated, and the residue was lyophilized to afford the compound of Example 23 (5 mg, 30%) as a white powder.

'H NMR (400 MHz, DMF-c1 ₇ ) 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, '.6 Hz, 1H), 4.64 (dd, J=22, 4.4 Hz, 1H), 3.90 (ABq, Δδ _AB =0.151 ppm, J=12 Hz, 2H).

‘9F NMR (376 MHz, DMF-dy) δ -161.727 (s), -193.726 (ddd, J=54.5, 22.9, 21.8 Hz).

- 56 045066

GC/MS: R _T =0.81 min, MS m/z=312.13 [M+l]; LC: Thermo Accela 1250 UHPLC; MS: Thermo LCQ

Fleet; column: Kinetex, 2.6 µm, C18, 100A, 50x3.00 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.

2g ^13a

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

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

MS m/z=742 [M+H].

MS system: Thermo LCQ Fleet.

NHBz NHBz

TBSO^ 1-N/ 10%Pd/C, H ₂ cylinder TBSO-^ +n

V°y No. ----------- Y7 }—ζ TEA, CH ₃ OH 7—

TBSO' F TBSO F

13a

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

Intermediate 13a (200 mg, 0.243 mmol) was dissolved in methanol (10 mL) and 10% Pd/C (100 mg, 0.094 mmol) and TEA (0.035 mL, 0.243 mmol) were added under nitrogen atmosphere. The resulting mixture was then stirred under _H2 (balloon) at room temperature for 40 min. The resulting mixture was filtered and concentrated in vacuo, and the residue was purified by silica gel column chromatography (0 to 40% EtOAc in hexane) to afford intermediate 13b (145 mg, 72%) as a white solid with 75% purity.

MS m/z=616 [M+H].

MC System: Thermo LCQ Fleet

NHBz NH ₂

TBSO^ Cj'N TBAF, THF; ^Ho ^ 0 Kn z ^N y°Y ------ W n+ \ / NH3, MeOH )—(

TBSC·' F HO' F

13b ²⁴

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 (1 M in THF, 0.53 mL, 0.531 mmol) was added. The resulting mixture was stirred at room temperature for 2 h and then methanolic ammonia (7 N, 10 mL) was added. The resulting mixture was stirred for 24 h and concentrated under reduced pressure. The crude residue was purified by preparative HPLC (0 to 35% acetonitrile in water, over 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, 1H), 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.

- 57 045066

Intermediate 14a is (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 H ₂ ( _g ) three times. The reaction mixture was stirred under H ₂ ( _g ). After 1 h, the flask was purged with nitrogen, and the reaction mixture was filtered through a pad of celite, rinsing with CH ₃ OH. The filtrate was concentrated under reduced pressure and then co-evaporated with EtOAc and then hexane. 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, 1H), 7.66 (s, 2H), 6.82 (d, J=4.4 Hz, 1H), 6.66 (d, J=4.4 Hz, 1H), 5.09 (d, J=6.5 Hz, 1H), 5.06-4.56 (m, 3H), 4.21 (t, J=5.9 Hz, 1H), 3.93 (t, J=4.9 Hz, 1H), 3.77 (q, J=4.5 Hz, 1H), 3.48 (ddd, J=38.9, 11.8, 4.4 Hz, 2H).

GC/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, 2.6 µm, HB-C18, 100A, 50x3.00 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-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8 mL/min.

Intermediate 14b is ((3aR,4R,6S,6aS)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methanol.

Intermediate 14a (3.13 g, 11.7 mmol) was dissolved in acetone (80 mL) and TsOH (6.00 g, 31.5 mmol) was added. Triethyl orthoformate (6.0 mL, 36.1 mmol) was added slowly over 10 min. The resulting mixture was allowed to stir at ambient temperature overnight. Saturated aqueous sodium carbonate solution was added until the reaction mixture reached pH 8. The solids were removed by filtration, and the filtrate was concentrated under reduced pressure. The crude residue was partitioned between EtOAc and brine. The phases were separated, and the organics were dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure. The crude product 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-d ₆ ) δ 7.83 (s, 1H), 7.71 (s, 2H), 6.83 (d, J=4.4 Hz, 1H), 6.73 (d, J=4.5 Hz, 1H), 5.21 (d, J=4.9 Hz, 1H), 5.01 (dd, J=6.6, 4.9 Hz, 1H), 4.84 (t, J=5.7 Hz, 1H), 4.71 (dd, J=6.7, 3.7 Hz, 1H), 3.99-3.85 (m, 1H), 3.46 (t, J=5.5 Hz, 2H), 1.48 (s, 3H), 1.29 (s, 3H).

GC/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, 2.6 µ, XB-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 at 1.8 mL/min.

Intermediate 14c is 7-((3aS,4S,6R,6aR)-6-(((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4^][1,3]dioxol-4-yl)pyrrolo[2,1-f][1,2,4]triazin-4-amine.

Intermediate 14b (2.55 g, 8.32 mmol) was dissolved in DCM (50 mL), and the mixture was cooled to 0°C. Imidazole (1.70 g, 24.9 mmol) was added, followed by TBSCl (1.88 g, 12.5 mmol). After 16

- 58 045066 h The reaction mixture 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 Na ₂ SO ₄ , filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (50-100% EtOAc/Hex) to afford intermediate 14c (2.60 g,

74%) in the form of a white solid.

1H NMR (400 MHz, DMSO-d ₆ ) 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).

GC/MS: tR=1.91 min, MS m/z=421.60 [M+H]; LC system: Thermo Accela 1250 UHPLC

MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µm, HB-C18, 100A, 50x3.00 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-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8 mL/min.

H ₂ N BocHN λ j Boc ₂ O, TEA 4 } j

TBSO_DMAP, THF TBSO^ ₀ U %

14c 14d

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

Intermediate 14c (2.59 g, 6.16 mmol) was dissolved in THF (60 mL), and the resulting solution was cooled to 0 °C. Boc ₂ O (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 room temperature. After 3 h, the reaction mixture was cooled to 0 °C and MeOH (10 mL), then conc. NH ₄ OH( _BODH ) (50 mL) was added. The resulting mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was concentrated under reduced pressure, and the crude residue was partitioned between EtOAc and water. The layers were separated and the organic layer was dried _{over Na2SO4} , filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (0-100% EtOAc/Hex) to afford intermediate 14d (2.82 g, 88%) as a white solid.

1H NMR (400 MHz, DMSO-d6) δ 10.46 (s, 1H), 8.20 (s, 1H), 7.19 (d, J=4.6 Hz, 1H), 6.90 (d, J=4.6 Hz, 1H), 5.33 (d, J=4.2 Hz, 1H), 5.02 (dd, J=6.5, 4.3 Hz, 1H), 4.72 (dd, J=6.5, 3.6 Hz, 1H), 4.01 (q, J=5.0 Hz, 1H), 3.64 (d, J=5.1 Hz, 2H), 1.49 (s, 9H), 1.32 (d, J=22.7 Hz, 6H), 0.82 (s, 9H), -0.03 (c, 6H).

GC/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, 2.6 µ, XB-C18, 100A, 50x3.00 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 1.8 mL/min.

Intermediate 14e is 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-4-yl)carbamate.

Intermediate 14d (2.8 g, 5.4 mmol) was dissolved in THF (50 mL) and TBAF (1.0 M in THF, 5.92 mL, 5.92 mmol) was added. After 30 min, additional TBAF (1.0 M in THF, 5.92 mL, 5.92 mmol) was added. After another 30 min, the reaction mixture was quenched with water, and the resulting mixture was extracted with EtOAc (2x). The combined organic layers were washed with brine, dried _{over Na2SO4} , filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (10-100% EtOAc/Hex) to afford intermediate 14e (2.19 g, 86%) as a white solid.

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, 1H), 5.03 (dd, J=6.6, 4.7 Hz, 1H), 4.85 (t, J=5.7 Hz, 1H), 4.72 (dd, J=6.6, 3.6 Hz, 1H), 4.05-3.90 (m, 1H), 3.46 (t, J=5.6 Hz, 2H), 1.50 (s, 12H), 1.29 (s, 3H).

GC/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, 2.6 µm, XB-C18, 100A, 50x3.00 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

- 59 045066 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8 ml/min.

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

Intermediate 14e (1.78 g, 4.38 mmol) was dissolved in DMSO (20 mL) and toluene (15 mL). Pyridine (0.35 mL, 4.38 mmol) and EDCI (1.26 g, 6.56 mmol) were added, followed by TFA (0.178 mL, 2.39 mmol). After 90 min, additional pyridine (0.35 mL, 4.38 mmol) and EDCI (1.26 g, 6.56 mmol) were added, and the reaction mixture was stirred for an additional 30 min. The reaction mixture was quenched with water, and the resulting mixture was extracted _{with CH2Cl2} . The aqueous layer was again extracted _{with CH2Cl2} . The organic layers were combined and washed with brine, dried over _Na2SO4 , _filtered , and concentrated under reduced pressure. The crude product was placed under high vacuum for 15 min and then used as such in the next step.

The crude residue was dissolved in dioxane (15 mL) and formaldehyde (37% in water, 5.0 mL, 37.2 mmol) and 2N NaOH (5.34 mL, 10.7 mmol) were added sequentially. After 10 min, the reaction mixture was quenched with AcOH _, and the resulting mixture was partitioned between _saturated NaHCO3 ( _aq ) and _{CH2Cl2 .} The aqueous layer was back-extracted _{with CH2Cl2} . The organic layers were combined and washed with saturated brine, dried over _Na2SO4 , filtered, and concentrated under reduced pressure. The crude product was placed under high vacuum for 15 min, then used directly in the next reaction.

The crude residue was dissolved in EtOH (50 mL), and NaBH ₄ (0.324 g, 8.76 mmol) was added in small portions. After 20 min, the reaction mixture was quenched with AcOH and concentrated under reduced pressure. The crude residue was partitioned between EtOAc and saturated NaHCO ₃ ( _aq ). The organic layer was separated, dried over Na ₂ SO ₄ , filtered, and 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.

¹ H NMR (400 MHz, DMSO-d ₆ ) 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, 1H), 3.73-3.46 (m, 3H), 3.40-3.30 (m, 1H), 1.50 (s, 12H), 1.27 (s, 3H).

GC/MS: tR=1.45 min, MS m/z=437.09 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µ, ХВ-С18, 100А, 50х3.00 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 1.8 mL/min.

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

Intermediate 14f (1.15 g, 2.63 mmol) was dissolved in _{CH2Cl2 (} 50 mL) and TEA (0.73 mL, 5.27 mmol) was added. The resulting solution was cooled to 0 °C and DMTrCl (1.35 g, 3.95 mmol) was added. After 10 min, the reaction mixture was quenched with _CH3OH and then diluted _{with CH2Cl2} . The resulting mixture was washed _with sat. _NαHCO3 ( _aq ) and brine. The organic layer was dried over _Na2SO4 , filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (0-100% EtOAc/Hex) to afford 14g (1.95 g, 79%) as an off-white solid.

1H NMR (400 MHz, DMSO-d ₆ ) δ 10.46 (s, 1H), 8.23 (s, 1H), 7.56-7.07 (m, 10H), 7.07-6.70 (m, 5H), 5.24 (d, J=5.2 Hz, 1H), 5.04 (t, J=5.9 Hz, 1H), 4.93-4.71 (m, 2H), 3.80-3.59 (m, 7H), 3.52 (dd, J=10.9, 4.8 Hz, 1H), 3.25 (d, J=9.9 Hz, 1H), 3.09 (d, J=9.9 Hz, 1H), 1.50 (s, 9H), 1.25 (s, 3H), 1.21 (s, 3H).

GC/MS: tR=2.54 min, MS m/z=739.28 [M+H]; LC system: Thermo Accela 1250 UHPLC

MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µm, HB-C18, 100A, 50x3.00 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-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8 mL/min.

- 60 045066

Intermediate 14h is tert-butyl (7-((3aS,4S,6R,6aS)-6-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-6-(((tert-butyldimethylsilyl)oxy)methyl)-2,2-dimethyltetrahydrofuro[3,4d][1,3]dioxol-4-yl)pyrrolo[2,1-f][1,2,4]triazin-4-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 TBSCl (0.47 g, 3.11 mmol). After 1 h, the reaction mixture was quenched with methanol and partitioned between EtOAc and 5% LiCl ( _BODH ). The phases were separated and the organic layer was washed with brine, dried over _Na2SO4 , filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (0-50% EtOAc/Hex) to afford 14h (1.77 g, 78%) as _an off-white solid.

¹ H NMR (400 MHz, DMSO-d6) 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, 1H), 3.72 (s, 6H), 3.53 (d, J=9.7 Hz, 1H), 3.31 (m, 1H), 3.08 (d, J=9.8 Hz, 1H), 1.50 (s, 9H), 1.25 (s, 3H), 1.22 (s, 3H), 0.75 (s, 9H), -0.04 (s, 3H), -0.08 (s, 3H).

GC/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, 2.6 µ, XB-C18, 100A, 50x3.00 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0 min-1.0 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 1.8 mL/min.

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

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 CH ₃ OH (16 mL) was then slowly added. After 30 min, the reaction mixture was quenched with saturated NaHCO ₃ solution ( _{BO dH} ) and the resulting mixture was partitioned between EtOAc and brine. The layers were separated, and the organic layer was dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (0-100% EtOAc/Hex) to afford intermediate 14i (0.84 g, 94%) as a white solid.

1H NMR (400 MHz, DMSO-d ₆ ) δ 10.42 (s, 1H), 8.20 (s, 1H), 7.19 (s, 1H), 6.89 (s, 1H), 5.37 (d, J=4.8 Hz, 1H), 5.05 (dd, J=6.2, 4.8 Hz, 1H), 4.71 (d, J=6.2 Hz, 1H), 4.51 (t, J=5.5 Hz, 1H), 3.70 (d, J=10.2 Hz, 1H), 3.59 (d, J=5.5 Hz, 2H), 3.49 (d, J=10.2 Hz, 1H), 1.49 (s, 12H), 1.28 (s, 3H), 0.82 (s, 9H), -0.01 (s, 3H), -0.02 (s, 3H).

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

MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µm, HB-C18, 100A, 50x3.00 mm; solvents: acetonitrile with 0.1% formic acid, water with 0.1% formic acid; gradient: 0 min-1.0 min 2100% 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 1.8 mL/min.

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

Intermediate 14i (0.838 g, 1.52 mmol) was dissolved in DMSO (5 mL) and toluene (3 mL). Pyridine (0.14 mL, 1.67 mmol) and EDCI (0.438 g, 2.28 mmol) were added, followed by TFA (0.057 mL, 0.761 mmol). After 30 min, additional pyridine (0.14 mL, 1.67 mmol) and EDCI were added.

- 61 045066 (0.438 g, 2.28 mmol). 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 _1/2 sat. _NaHCO3 ( _aq ) and partitioned between EtOAc and _1/2 sat. _NaHCO3 ( _aq ). The layers were separated and the organic layer was dried over _Na2SO4 , _filtered and concentrated under reduced pressure. The residue was dissolved in CH2Cl2, concentrated under high vacuum for 1 h to give 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 partitioned between EtOAc and water. The organic layer was dried over Na ₂ SO ₄ , filtered, and concentrated under reduced pressure. The crude residue was placed under high vacuum for 30 min and used as is in the third step.

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 mixture was quenched with _1/2 sat. _NaHCO3 ( _aq ). The crude product was partitioned between EtOAc and _1/2 sat. _NaHCO3 ( _aq ). The layers were separated, and the organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (0-50% EtOAc/Hex) to afford intermediate 14j (0.72 g, 87%) as a white solid.

1H NMR (400 MHz, DMSO-06) δ 10.53 (s, 1H), 8.25 (s, 1H), 7.21 (s, 1H), 7.00 (d, J=4.6 Hz, 1H), 5.62 (d, J=3.6 Hz, 1H), 5.28 (dd, J=6.6, 3.7 Hz, 1H), 4.93 (d, J=6.6 Hz, 1H), 3.83 (s, 2H), 1.62 (s, 3H), 1.50 (s, 9H), 1.33 (s, 3H), 0.83 (s, 9H), 0.00 (s, 6H).

GC/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, 2.6 µ, XB-C18, 100A, 50x3.00 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 1.8 mL/min.

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

Intermediate 14j (0.688 g, 1.26 mmol) was dissolved in _{CH2Cl2 (} 15 mL). Zinc bromide (0.567 g, 2.52 mmol) was added in one portion, and the reaction mixture was stirred at ambient temperature. After 3 h, the reaction mixture was loaded onto a silica gel cartridge and purified by silica gel chromatography (40-100% EtOAc/Hex) to afford intermediate 14k (0.56 g, 99%) as a white solid.

1H NMR (400 MHz, DMSO-D6) δ 7.86 (s, 1H), 7.80 (s, 2H), 6.85 (d, J=4.5 Hz, 1H), 6.79 (d, J=4.5 Hz, 1H), 5.55 (d, J=3.7 Hz, 1H), 5.25 (dd, J=6.6, 3.8 Hz, 1H), 4.92 (d, J=6.6 Hz, 1H), 3.82 (s, 2H), 1.61 (s, 3H), 1.33 (s, 3H), 0.83 (s, 9H), -0.13 (s, 6H).

GC/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, 2.6 µ, XB-C18, 100A, 50x3.00 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 1.8 mL/min.

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

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

- 62 045066 color logo.

1H NMR (400 MHz, DMSO-6) δ 10.87 (s, 1H), 8.31 (s, 1H), 7.24 (d, J=4.7 Hz, 1H), 7.05 (d, J=4.7 Hz, 1H), 5.65 (d, J=3.6 Hz, 1H), 5.29 (dd, J=6.6, 3.6 Hz, 1H), 4.93 (d, J=6.6 Hz, 1H), 3.84 (s, 2H), 2.36 (s, 3H), 1.62 (s, 3H), 1.33 (s, 3H), 0.83 (s, 9H), 0.00 (s, 3H), -0.01 (s, 3H).

GC/MS: tR=1.14 min, MS m/z=488.38 [M+1]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µ, XB-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 at 1.8 mL/min.

Intermediate 14m is N-(5-bromo-7-((3aS,4S,6R,6aS)-6-(((tert-butyldimethylsilyl)oxy)methyl)-6-cyano-2,2-dimethyltetrahydrofuro[3,4^][1,3]dioxol-4-yl)pyrrolo[2,1f][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 mixture 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-6 ₆ ) δ 10.13 (s, 1H), 8.42 (s, 1H), 7.29 (s, 1H), 5.65 (d, J=3.2 Hz, 1H), 5.29 (dd, J=6.6, 3.2 Hz, 1H), 4.91 (d, J=6.5 Hz, 1H), 3.84 (d, J=1.6 Hz, 2H), 2.27 (s, 3H), 1.62 (s, 3H), 1.33 (s, 3H), 0.83 (s, 9H), 0.02 (s, 3H), 0.00 (s, 3H).

GC/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, 2.6 µ, XB-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 at 1.8 mL/min.

14m 14η

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

Intermediate 14m (50 mg, 0.088 mmol) was dissolved in THF (2 mL) and the solution was cooled to -78°C. nBuli (2.5 M in hexane, 0.071 mL, 0.18 mmol) was added. After 5 min, N-fluorobenzenesulfonimide (NSFI, 33.4 mg, 0.106 mmol) was added and the reaction mixture was stirred for 5 min. The reaction mixture 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-d ₄ ) δ 8.13 (s, 1H), 6.80 (s, 1H), 5.65 (d, J=3.5 Hz, 1H), 5.23 (dd, J=6.7, 3.6 Hz, 1H), 4.97 (d, J=6.7 Hz, 1H), 3.92 (d, J=1.7 Hz, 2H), 2.37 (s, 3H), 1.70 (s, 3H), 1.38 (s, 3H), 0.90 (s, 9H), 0.08 (s, 6H).

¹⁹ F NMR (376 MHz, methanol-d4) δ -156.43 (s).

GC/MS: tR=1.65 min, MS m/z=506.18 [M+H]; LC system: Thermo Accela 1250 UHPLC; MS system: Thermo LCQ Fleet; column: Kinetex, 2.6 µ, ХВ-С18, 100А, 50х3.00 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 1.8 mL/min.

- 63 045066

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

Intermediate 14n (11 mg, 0.022 mmol) was taken up in 50% TFA in water at ambient temperature. After 2 h, the reaction mixture was quenched _with solid _Na2CO3 until pH=8. The solvent was removed under reduced pressure, and the crude residue was purified by reverse-phase HPLC. Fractions containing the compound from Example 25 were pooled and set aside, and fractions containing N6-Acyl were pooled and concentrated under reduced pressure. The residue of the N6-Acyl intermediate was treated with conc. _NH4OH ( _aq ) (1 mL), and the mixture was allowed to stir at ambient temperature. After 30 min, the resulting mixture was concentrated under reduced pressure, and the crude residue was purified by HPLC. Fractions containing the compound of Example 25 were combined with the previously set aside fractions containing the compound of Example 25 to give the compound of Example 25 (4 mg, 58%) as a white solid.

1H NMR (400 MHz, methanol-d ₆ ) 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=0.05 ppm, J=12 Hz, 2H).

¹⁹ F NMR (376 MHz, methanol^) δ -161.81 (s).

GC/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, 2.6 µ, XB-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 at 1.8 mL/min.

Sun _ch Sun

NH NH TVSO-^ _o V _{υ TfC1 Ru} TBSO-do U ^NO-< UCH 2) iS1,DMF ^C1 “'' UCH 14i 15a

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

Intermediate 14i (100 mg, 0.18 mmol) was dissolved in anhydrous pyridine (5 mL). Trifluoromethanesulfonyl chloride (23 μL, 0.22 mmol) was added in one portion, and the reaction mixture was stirred for 45 min at room temperature. An additional amount of trifluoromethanesulfonyl chloride (100 μL) was then added. After 30 min, an additional amount of trifluoromethanesulfonyl chloride (100 μL) was added. After another 30 min, an additional amount of trifluoromethanesulfonyl chloride (100 μL) was added, and the reaction mixture 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 room temperature for 16 h. The reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated aqueous sodium chloride solution (3 x 20 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography (0-20% ethyl acetate in hexane) to yield intermediate compound 15a.

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

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

- 64 045066 was stirred for 16 h. The reaction mixture was then concentrated under reduced pressure.

The crude residue was dissolved in aqueous sodium bicarbonate and acetonitrile and purified by preparative HPLC to afford the compound of Example 26 (19 mg, 34%) as a white powder.

1H NMR (400 MHz, D2O) δ 7.61 (s, 1H), 6.72-6.64 (m, 2H), 5.19 (d, J=9.1 Hz, 1H), 4.73-4.66 (m, 1H), 4.28 (d, J=5.2 Hz, 1H), 3.78 (s, 2H), 3.72-3.57 (m, 2H).

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

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

The compound of Example 27 was prepared as the tetrasodium salt in a manner similar to that described in Example TP4, starting from the compound of Example 19.

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

¹⁹ F NMR (376 MHz, D ₂ O) δ -193.74–194.02 (m).

³¹ P NMR (162 MHz, D ₂ O) 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).

28

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

The compound of Example 28 was prepared as the tetrasodium salt in a manner similar to that described in Example TP4, starting from the compound of Example 21.

1H NMR (400 MHz, D ₂ O) 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).

¹⁹ F NMR (37 6 MHz, D2O) δ -161.00 (s), -196.39-196.69 (m).

³¹ P NMR (162 MHz, D2O) δ -8.24 (d, J=50.4 Hz), -14.20 (d, J=46.0 Hz), -24.08 (t, J=48.4 Hz).

MS m/z=567.87 [M+1]. MS system: Thermo LCQ Advantage

29

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

The compound of Example 29 was prepared as the tetrasodium salt in a manner similar to that described in Example TP4, starting from the compound of Example 22.

1H NMR (400 MHz, D2O) δ 6.81 (d, J=4.4 Hz, 1H), 6.75 (d, J=4.8 Hz, 1H), 5.81 (d, J=24.4 Hz, 1H), 5.16 (dd, J=54.4, 4.8 Hz, 1H), 4.70 (dd, J=26.8, 4.4 Hz, 1H), 4.02-4.12 (m, 2H).

¹⁹ F NMR (37 6 MHz, D2O) δ -75.95 (s), -196.51-196.80 (m).

³¹ P NMR (162 MHz, D2O) δ -8.29 (d, J=53.2 Hz), -14.22 (d, J=48.4 Hz), -24.09 (t, J=48.4 Hz).

MS m/z=567.59 [M+1]. MS system: Thermo LCQ Advantage

- 65 045066

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

The compound of Example 30 was prepared as the tetrasodium salt in a manner similar to that described in Example TP4, starting from the compound of Example 23.

1H NMR (400 MHz, D ₂ O) 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).

¹⁹ F NMR (37 6 MHz, D ₂ O) δ -161.05 (s), -194.92-195.19 (m).

³¹ P NMR (162 MHz, D ₂ O) 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

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

The compound of Example 31 was prepared as the tetrasodium salt in a manner similar to that described in Example TP4, starting from the compound of 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).

¹⁹ F NMR (376 MHz, D ₂ O) δ -195.74–196.01 (m).

³¹ P NMR (162 MHz, D2O) δ -8.24 (d, J=50.4 Hz), -13.54 (d, J=45.6 Hz), -24.11 (t, J=48.0 Hz).

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

The compound of Example 32 was prepared as the tetrasodium salt in a manner similar to that described in Example TP4, starting from the compound of Example 25.

1H NMR (400 MHz, D ₂ O) δ 7.59 (s, 1H), 6.57 (s, 1H), 5.44 (d, J=6.0 Hz, 1H), 4.56 (d, J=5.2 Hz, 1H), 4.48 (dd, J=5.6 Hz, 1H), 4.16 (dd, J=11.6, 6.0 Hz, 1H), 4.08 (dd, J=11.2, 5.2 Hz, 1H).

¹⁹ F NMR (376 MHz, D ₂ O) δ -161.25 (s).

³¹ P NMR (162 MHz, D ₂ O) 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

ΗΟ^ρ'θ

ΝΗ ₂ Ηθ Vp'' ⁰ Example 33

And ⁰ ' 0-^° but-, ₀ V' j P(O)Cl ₃ ; ^but _o WS a—ci—X / ^u { [nBu ₃ NH] ₂ P2O ₇ H2 X but' he but ⁴ 'he

TR13

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

- 66 045066

The compound of Example 33 was prepared as the tetra-triethylamine salt in a manner similar to that described in Example TP3, starting from the compound of Example 26.

'H NMR (400 MHz, D ₂ O) δ 7.76 (s, 1H), 6.92 (br s, 1H), 6.85 (br s, 1H), 5.32 (d, J = 9.6 Hz, 1H), 4.78 (dd, J = 8, 6.4 Hz, 1H), 4.53 (d, J = 5.6 Hz, 1H), 4.08 (dd, J = 10.0, 4.0 Hz, 1H), 3.83-3.95 (m, 3H), 3.07 (q, J = 7.6 Hz, 24 H), 1.16 (t, J = 7.6 Hz, 36H).

³¹ P NMR (162 MHz, D2O) δ -9.44 (d, J=45.6 Hz), -11.51 (d, J=48.8 Hz), -22.95 (t, J=48.4 Hz).

MS m/z=555.06 [M+1]. MS system: Thermo LCQ Advantage

PD3c PD6

Example 34 (also PD6) - (2S)-2-ethylbutyl 2-(((((2R,3R,4R,5S)-5-(4-aminopyrrolo[2,11][1,2,4]triazin-7-yl)-2-cyano-4-fluoro-3-hydroxytetrahydrofuran-2yl)methoxy)(phenoxy)phosphoryl)amino)propanoate.

The compound from Example 1 (3.8 mg, 0.013 mmol) was dissolved in anhydrous N-methyl-2-pyrrolidone (0.2 mL) and THF (0.1 mL) was added under argon. Tert-butyl magnesium chloride (1 M in THF, 20 μL, 0.024 mmol) was then added at room temperature, and a white solid precipitated. After 5 min, a solution of pnitrophenyl phosphoramidate 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 at 50°C. After 20 h, the reaction mixture was allowed to cool to room temperature and then directly purified by preparative HPLC (Phenominex Synergi 4 µm Hydro-RR 80A, 150x30 mm column, 40-100% acetonitrile/water gradient). Fractions containing the desired product were combined and lyophilized to afford the compound of Example 34 (2.9 mg, 37%, 3:2 mixture of diastereomers) 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 (dd br, J=4.7, 2.2 Hz, 1H), 6.75-6.71 (m, 1H), 5.54-5.46 (m, 1H), 4.65-4.58 (m, 1H), 4.53-4.31 (m, 3H), 4.07-3.84 (m, 3H), 1.541.39 (m, 1H), 1.38-1.19 (m, 7H), 0.92-0.81 (m, 6H) 29Н ³¹ Р NMR (162 MHz, CD3OD) δ 3.25 (broad s).

GC/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, 2.6 µ, XB-C18, 100A, 50x4.6 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 2 µL/min.

HPLC: t _R = 2.98 min; HPLC system: Agilent 1100 series; column: Gemini C18, 5 μm, 110A, 50x4.6 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 35 (also PD7) - (2S)-ethyl 2-(((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7yl)-2-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate.

The compound from Example 1 (19 mg, 65.3 μmol) was dissolved in NMP (0.2 mL). THF (0.1 mL) was added under argon at room temperature, followed by tert-butyl magnesium chloride (1.0 M solution in tetrahydrofuran, 0.098 mL). After 5 min, a solution of intermediate PD7a (prepared according to US 20120009147A1, 51.4 mg, 130 μmol) in THF (0.1 mL) was added and the resulting mixture was heated to 50 °C. After 1 h, the reaction mixture was allowed to cool to room temperature and directly purified by preparative HPLC (Phenominex Synergi 4 μm Hydro-RR 80A, 150x30 mm column, 5-100% acetonitrile/water gradient). The product-containing fractions were combined and concentrated, and the resulting residue was repurified by preparative HPLC (Phenominex Luna, 5 μm, C18 column, 100x30 mm, gradient 5-100% acetonitrile/water) to give Example 35 (12 mg, 34%, 3:2 mixture of diastereomers) as a white solid.

1H NMR (400 MHz, CD ₃ OD) 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), 6.886.81 (m, 1H), 6.76-6.70 (m, 1H), 5.53-5.46 (m, 1H), 4.66-4.60 (m, 1H), 4.55-4.30 (m, 3H), 4.15-3.98 (m, 2H), 3.93-3.79 (m, 1H), 1.30-1.12 (m, 6H).

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

GC/MS: major diastereomer tR=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, 2.6 μm, ХВ-С18, 100А, 50х4.6 mm; solvents: acetonitrile with 0.1% acetic acid

- 67 045066 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 2 µl/min.

HPLC: major diastereomer tR=2.44 min, minor diastereomer tR=2.46 min; HPLC system:

Agilent 1100 series; column: Gemini C18, 5 microns, 110A, 50x4.6 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):

where in each case X ¹ represents an oxygen protecting group and X ² represents an amino protecting group.

Oxygen protecting groups used include the silyl ether protecting group or the benzyl type protecting group, including methoxybenzyl groups.

The silyl ether protecting groups used include trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylhexylsilyl (TDS), tert-butyldimethylsilyl (TBS or TBDMS), tert-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triisopropylsilyl (TIPS), diphenylmethylsilyl (DPMS), di-tert-butylmethylsilyl (DTBMS), triphenylsilyl (TPS), methyldiphenylsilyl (MDPS), tert-butylmethoxyphenylsilyl, tris(trimethylsilyl)silyl (sisyl), (2-hydroxystyryl)dimethylsilyl (HSDMS), (2-hydroxystyryl)diisopropylsilyl (HSDIS). tert-butylmethoxyphenylsilyl (TBMPS) and tert-butoxydiphenylsilyl (DPTBOS) protecting groups.

Benzyl type protecting groups that may be used include benzyl, halogenated benzyl, p-methoxybenzyl, benzyloxymethyl, 2,4-dimethoxybenzyl, 3,4-dimethoxybenzyl, 2,6-dimethoxybenzyl, pCF3- _benzyl , p-methylbenzyl, p-methoxybenzyl, 3,5-dimethylbenzyl, p-tert-butylbenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl including p-Br-benzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2,6-difluorobenzyl, 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), p,p'-dinitrobenzhydryl, triphenylmethyl, alpha-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4,4'4-tris(benzoyloxyphenyl)methyl and 2-naphthylmethyl protecting groups.

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

Examples of useful substituted ethyl carbamates include 1,1-dimethyl-2-cyanoethyl, 2-phosphonioethyl (Reoc), 2-methylthioethyl, 2-(p-toluenesulfonyl)ethyl, 2,2,2-trichloroethyl (Troc), 2(trimethylsilyl)ethyl (Teos), 2-phenylethyl (hZ), 1-(1-adamantyl)-1-methylethyl (Adpoc), 1,1-dimethyl-2-bromoethyl, 1,1-dimethyl-2-chloroethyl, 1,1-dimethyl-2,2-dibromoethyl (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl (BroC), 1-(3,5-di-tert-butylphenyl)-1-methylethyl (tBumeoc), 2-(2'-pyridyl)ethyl, 2-(4'-pyridyl)ethyl, 2, 2-bis (4'-nitrophenyl)ethyl (Bnpeoc), N-(2-pivaloylamino)-1,1-dimethylethyl, 2-[(2-nitrophenyl)dithio]-1-phenylethyl (NpSSPeoc), 2-(N,N-dicyclohexylcarboxamido)ethyl, tert-butyl (Boc or BOC), 1-adamantyl (1-Adoc), 2-adamantyl (2Adoc), vinyl (Voc), allyl (Aloc or alloc), l-isopropylallyl (Ipaoc), cinnamyl (Coc), 4-nitrocinnamyl (Noc), 3-(3'-pyridyl)prop-2-enyl (Paloc), 8-quinolyl and N-hydroxypiperidinyl, carbamates, and alkyl dithio carbamates, including methyl dithio, ethyl dithio, isopropyl dithio, tert-butyl dithio, and phenyl dithio carbamates.

- 68 045066

Also used are aryl and substituted aryl 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) (Pros), 2-dansylethyl (Dnseoc), 2-(4-nitrophenyl)ethyl (Npeoc), 4-phenylacetoxybenzyl (PhAcOZ), 4-azidobenzyl (ACBZ), 4-azidomethoxybenzyl, m-chloro-p-acyloxybenzyl, p-(dihydroxyboryl)benzyl, carbobenzyloxy (Cbz), 4-benzisoxazolylmethyl (Bic), 2-(trifluoromethyl)-6-chromonylmethyl (Tcroc), phenyl, and diphenylmethyl carbamates. Additional carbamates include butynyl, cyclopentyl, cyclohexyl, cyclopropylmethyl, 1-methylcyclobutyl, 1-methylcyclohexyl, 1,1-dimethylpropynyl, and 1-methyl-1-cyclopropylmethyl carbamates.

Useful amide protecting groups for amines include N-formyl, N-acetyl, N-chloroacetyl, N-trichloroacetyl, N-trifluoroacetyl (TFA), N-phenylacetyl, N-3-phenylpropionyl, N-4-pentoyl, N-picolinoyl, N-3-pyridylcarboxamido, T-benzoylphenylalanyl, N-benzoyl, and N-pphenylbenzoyl amides.

Antiviral activity

Another embodiment of the invention relates to methods for inhibiting viral infections, comprising the step of treating a sample or subject believed to be in need of such inhibition with a composition according to the present document.

Samples suspected of containing a virus as discussed herein include natural or artificial materials, such as living organisms; tissue or cell cultures; biological samples, such as samples of biological material (blood, serum, urine, cerebrospinal fluid, tears, sputum, saliva, tissue samples, and the like); laboratory samples; food, water, or air samples; samples of biopreparations, such as cell extracts, especially recombinant cells that synthesize the desired glycoprotein; and the like. Typically, a sample is suspected of containing an organism that causes a viral infection, often a pathogenic organism such as a tumor virus. Samples may be contained in any medium, including water and organic solvent/water mixtures. Samples include living organisms, such as humans, and artificial materials, such as cell cultures.

If desired, the antiviral activity of the compound after application of the composition can be tested using any method, including direct and indirect methods for detecting such activity. All quantitative, qualitative, and semi-quantitative methods for determining such activity are envisaged. Typically, one of the screening methods described above is used; however, any other method, such as observing the physiological properties of a living organism, is also applicable.

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

Respiratory syncytial virus (RSV) antiviral activity and cytotoxicity assay

Anti-RSV activity

Antiviral activity against RSV was determined using a cytopathic cell infection protection assay in the HEp-2 cell line. In this assay, compounds that inhibit viral infection and/or replication exert a cytoprotective effect against virus-induced cell killing, which can be quantified using a cell viability indicator reagent. The methods used herein are new adaptations of methods described in the published literature (Chapman et al., Antimicrob Agents Chemother. 2007, 51(9):3346-53).

Hep-2 cells were obtained from ATCC (Manassas, WI) and maintained in MEM supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cells were passaged twice weekly and maintained at a subconfluent stage. Commercial RSV strain A2 (Advanced Biotechnologies, Columbia, MD) was titrated prior to compound testing to determine the appropriate dilution of viral material that produced the desired cytopathic effect in Hep-2 cells.

For antiviral assays, Hep-2 cells were grown in large cell culture flasks to near but not complete confluence. Test compounds were prediluted in DMSO in 384-well compound dilution plates in a standardized dose-response format at either 8 or 40 samples per plate. Three-fold serial dilutions of each test compound were performed on the plates, and test samples were transferred using an acoustic transfer apparatus (Echo, Labcyte) at 100 nL per well to 384-well cell culture assay plates. Each compound dilution was transferred as single or quadruplicate samples to dry assay plates, which were stored until the assay was ready to run. Positive and negative controls were placed on opposite sides of the plate in vertical sections (1 column).

- 69 045066

An infection mixture was then prepared using an appropriate dilution of viral material, previously determined by titration with cells at a density of 50,000/mL, and 20 µL/well were added to assay plates with compound automation (uFlow, Biotek). Each plate included positive and negative controls (16 replicates each) to create 0 and 100% virus inhibition standards, respectively. Following infection, Cell TiterGlo cell viability control reagent (Promega, Madison, WI) was added to assay plates and incubated for 4 days at 37°C in a cell culture incubator. Following incubation, Cell TiterGlo cell viability control reagent (Promega, Madison, WI) was added to assay plates, briefly incubated, and the luminescence readings were determined on all assay plates (Envision, Perkin Elmer). RSV-induced cytopathic effect (percent inhibition) was determined based on the viability of the remaining cells. These quantitative parameters were calculated for each test concentration relative to 0 and 100% inhibition controls, and the _EC50 value for each compound was determined using nonlinear regression as the concentration inhibiting RSV-induced cytopathic effect by 50%. Various effective anti-RSV experimental compounds were used as positive controls for antiviral activity.

Cytotoxicity assay on HEp-2 cells

The cytotoxicity of the test compounds was determined on uninfected HEp-2 cells in parallel with the antiviral activity assay, using a cell viability control reagent in the same manner as described above for other cell types (Cihlar et al., Antimicrob Agents Chemother. 2008, 52(2):655-65). To determine compound cytotoxicity, the same protocol was used as for determining antiviral activity, except that the cells were not infected with RSV. Instead, an uninfected cell mixture at the same density was added at 20 μl/well to plates containing prediluted compounds, also at 100 nl/sample. The plates were then incubated for 4 days, followed by a cell viability assay using the same addition of CellTiter Glo reagent and determination of the recorded luminescence value. Untreated cells and cells treated with 2 μM puromycin (Sigma, St. Louis, MO) served as 100% and 0% cell viability controls, respectively. Percent cell viability was calculated for each test compound concentration compared to the 0% and 100% controls, and the _CC50 value was determined by nonlinear regression as the compound concentration that restored cell viability to 50%.

Cytotoxicity assay on 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 units/mL streptomycin, and 2 mM L-glutamine. MT-4 cells were passaged twice weekly to maintain cell density below 0.6 × 106 cells/mL. Complete RPMI-1640 medium containing a 100x concentration of 3-fold serially diluted compound, ranging from 26 nM to 530 μM, was punched in quadruplicate into black 384-well plates. After compound addition, 2 x 10 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. After incubation, the cells were equilibrated to 25°C, and cell viability was determined by adding 25 μl of Cell-Titer Glo cell viability control reagent. The mixture was incubated for 10 min 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. Data were analyzed using Pipeline Pilot Plate Data Analytics Collection software (version 7.0, Accelrys, San Diego, CA). _CC50 values were calculated by nonlinear regression analysis using a 4-parameter sigmoid dose-response equation: Y=Bottom+(Top-Bottom)/( ^{1+10 A [} (LogCC50-X)*HillSlope]), where Top and Bottom were fixed at 100% and 0% cell viability, respectively. _CC50 values were calculated as the mean±standard deviation of 3 independent experiments.

- 70 045066

Example EC ₅₀ /μm HEP-2 SS ₅₀ /μm MT-4 SS ₅₀ /mkm 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 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 positive effect is related to the advantage that compounds that have an R ⁴ substitution provide over compounds that do not have an R ⁴ substitution (i.e., those in which R ⁴ =H) with respect to MT-4 cytotoxicity. For example, the compound (2S,3R,4S,5R)-2-(4aminopyrrolo[1.2-f][1.2.4]triazin-7-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (Patil, SA; Otter, RA; Klein, RS Tetrahedron Lett. 1994, 35, 5339-5342), having the structure:

but* % exhibits MT-4 CC50=0.007 μm; while the compounds of Examples 1, 4, 5, 20 and 26 all exhibit MT-4 CC50 _> 53 μm. Further, the compound (2R,3R,4R,5S)-5-(4-aminopyrrolo[1.2f][1.2.4]triazin-7-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol (WO2012037038A1), having the structure:

НС^ exhibits MT-4 CC ₅₀ = 30 μm; whereas the compounds of Examples 2, 3, 13 and 14 all exhibit MT-4 CC ₅₀ > 106 μm.

Another positive effect is related to the advantage provided by ^R3 =F compounds that have an ^R4 substitution compared to compounds without an ^R4 substitution (i.e., those with ^R4 =H) with respect 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-ol (WO 2012037038A1) having the structure shown above has an HEp-2 _EC50 of >100 μM; whereas the compounds of Examples 2, 3, 13, 14, 19, 21, 22, 23, and 24 all exhibit an _EC50 of <100 μM.

Receiving RSV RNP RSV RNP Preparation

RSV ribonucleoprotein (RNP) complexes were prepared using a method modified by Mason et al.

- 71 045066 al (1). Hep-2 cells were seeded at a cell density of 7.1 ^x10 cells/ ^cm2 in MEM + 10% fetal bovine serum (FBS) and allowed to attach overnight at 37°C (5% _CO2 ). After attachment, cells were infected with RSV A2 (MOI = 5) in 35 ml MEM + 2% FBS. At 20 h postinfection, the medium was changed to MEM + 2% FBS supplemented with 2 μg/ml actinomycin D and returned to 37°C for 1 h. Cells were then washed once with PBS and treated with 35 ml PBS + 250 μg/ml lysolecithin for one minute, after which all fluid was aspirated. Cells were collected by scraping them into 1.2 mL of buffer A [50 mM TRIS acetate (pH 8.0), 100 mM potassium acetate, 1 mM DTT, and 2 μg/mL actinomycin D] and lysed by repeated passage through an 18-gauge syringe (10 times). The cell lysate was placed on ice for 10 min and then centrifuged at 2400g for 10 min at 4°C. The supernatant (S1) was discarded, and the pellet (P1) was reconstituted in 600 μL of buffer B [10 mM TRIS acetate (pH 8.0), 10 mM potassium acetate, and 1.5 mM _MgCl2 ] supplemented with 1% Triton X-100 by repeated passage through an 18-gauge needle (10 times). The resuspended pellet was placed on ice for 10 min and then centrifuged at 2400g for 10 min at 4°C. The supernatant (S2) was discarded, and the pellet (P2) was reconstituted in 600 μl of buffer B supplemented with 0.5% deoxycholate and 0.1% Tween 40. The resuspended pellet was placed on ice for 10 min and then centrifuged at 2400g for 10 min at 4°C. The supernatant (S3), a fraction containing RSV RNP-enriched complexes, was collected, and protein concentration was determined by UV absorbance at 280 nm. Aliquots of RSV RNP fraction S3 were stored at -80°C.

RSV RNP Analysis

The transcription reaction mixture contained 25 μg crude RSV RNP complexes in 30 μl reaction buffer [50 mM TRIS-acetate (pH 8.0), 120 mM potassium acetate, 5% glycerol, 4.5 mM

MgCl2, ₃ mM DTT, 2 mM ethyleneglycol bis(2-aminoethyl ether)tetraacetic acid (EGTA), 50 μg/ml BSA, 2.5 U RNasin (Promega), ATP, GTP, UTP, CTP, and 1.5 μCi [aaa- ^32P ]NTP (3000 Ci/mmol)]. The radiolabeled nucleotide used in the transcription assay was chosen to match the nucleotide analog being evaluated for inhibition of RSV-RNP transcription. Cold competitive NTP was added at a final concentration half its _Km (ATP = 20 μM, GTP = 12.5 μM, UTP = 6 μM, and CTP = 2 μM). The remaining three nucleotides were added at a final concentration of 100 μM.

To determine whether RSV RNP nucleotide analogs inhibit transcription, compounds were added using a 6-step serial dilution with 5-fold volume increases. After 90 min of incubation at 30°C, RNP reactions were stopped with 350 μl of Qiagen RLT lysis buffer, and RNA was purified using the Qiagen RNeasy 96 kit. Purified RNA was denatured in RNA sample loading buffer (Sigma) at 65°C for 10 min and run on a 1.2% agarose/MOPS gel containing 2 M formaldehyde. The agarose gel was dried and exposed to a Storm phosphor screen and analyzed using a Storm phosphorimager (GE Healthcare).

The concentration of compound that reduces total radiolabeled transcripts by 50% (IC ₅₀ ) was calculated using nonlinear regression analysis in duplicate.

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

Example ICso/μm 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 (TRIO) 31 (THREE) 32 (TP12) 0.022 33 (TP13)

Another consideration relates to the advantage that the compounds shown in the examples demonstrate potent inhibition of RSV RNP transcription compared to compounds with the substituted

Claims (30)

2СМе. For example, ((2R,3R,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy4-methyltetrahydrofuran-2- yl)methyl tetrahydrotriphosphate (WO 2008089105A2 and WO 2010002877A2), having the structure: demonstrates IC5 ₀ =8.5 µm; whereas the connection according to example TP3 shows IC5 ₀ =0.025 μm. Moreover, the compound ((2R,3R,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-4-fluoro-3-hydroxy4- methyltetrahydrofuran-2-yl)methyl tetrahydrotriphosphate (WO 2011035231 A1), having the structure: exhibits IC ₅₀ >100 µm; whereas the connection of example TP1 shows IC ₅₀ =0.086 μm and the connection of example TP2 shows IC ₅₀ =1 μm. CLAIM 1. A compound of formula (I) or a pharmaceutically acceptable salt thereof Where R ¹ represents H; R ² represents H or F; R ³ represents OH or F; R ⁴ is cyano, C1-C4 alkyl, C2-C4 is OH, R ⁴ is not C2- _C4 alkenyl; R ⁶ represents OH; R ⁵ is selected from the group consisting of H and Η£ _R 8_JL _s __ _(CH2) II____ o / _R 8, and alkenyl or C1-C4 haloalkyl, where when R ³ where n' is selected from 1, 2, 3 and 4; R ⁸ represents C _r C ₈ alkyl; R ⁹ is phenyl, R ¹⁰ is selected from H and CH ₃ ; R ¹¹ is selected from H and C ₁ -C ₆ alkyl and R ¹² represents C1-C8 alkyl. - 73 045066 2. A compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R ² represents H. 3. A compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R ² represents F. 4. A compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein R ³ represents OH. 5. A compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, where R ³ represents F. 6. A compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, wherein R ⁴ represents cyano. 7. A compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, wherein R ⁴ represents C1-C4 alkyl. 8. The compound according to claim 7 or a pharmaceutically acceptable salt thereof, wherein R ⁴ represents ethyl. 9. A compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, where R ⁴ represents C ₂ -C ₄ alkenyl. 10. The compound according to claim 9 or a pharmaceutically acceptable salt thereof, wherein R ⁴ represents ethenyl. 11. A compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, wherein R ⁴ represents a C ₁ -C ₄ haloalkyl. 12. The compound according to claim 11 or a pharmaceutically acceptable salt thereof, wherein R ⁴ represents a methyl halo. 13. The compound according to claim 12 or a pharmaceutically acceptable salt thereof, wherein R ⁴ represents chloromethyl. 14. A compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, where R ⁵ represents H. 15. A compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, where R ⁵ is selected from the group consisting of 16. The compound according to claim 15 or a pharmaceutically acceptable salt thereof, where R ⁵ represents 17. A compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, where R ⁵ represents 18. A compound according to any one of claims 1-13 or 17 or a pharmaceutically acceptable salt thereof, where R' represents C1-C8 alkyl. 19. A compound according to any one of claims 1 to 13, 17 or 18, or a pharmaceutically acceptable salt thereof, wherein R represents butyl. 20. A compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, where R ⁵ represents 21. A compound according to any one of claims 1-13 or 20 or a pharmaceutically acceptable salt thereof, where R ¹² represents C ₁ -C ₆ alkyl. 22. The compound according to claim 21 or a pharmaceutically acceptable salt thereof, wherein R ¹² represents hexyl. 23. A compound or a pharmaceutically acceptable salt thereof, selected from the group - 74 045066 but'' bn and 24. A pharmaceutical composition for the treatment of infection with the Pneumovirinae virus in humans, containing a therapeutically effective amount of a compound of any one of p. 1-23 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. 25. A pharmaceutical composition for the treatment of respiratory syncytial virus infection in humans, containing a therapeutically effective amount of a compound according to any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. 26. A pharmaceutical composition containing a pharmaceutically effective amount of a compound according to any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and excipient. 27. Use of a pharmaceutical composition containing a compound according to any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier and excipient for the treatment of Pneumovirinae virus infection in humans. 28. Use of a compound according to any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof for the preparation of a medicament intended for the treatment of Pneumovirinae virus infection in humans. 29. Use of a pharmaceutical composition containing a compound according to any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier and excipient for the treatment of respiratory syncytial virus infection in humans. 30. Use of a compound according to any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof for the preparation of a medicinal product intended for the treatment of respiratory syncytial virus infection in humans. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky lane, 2