EA039561B1 - Compounds for treating filoviridae virus infections - Google Patents

Abstract

Provided are compounds, uses of said compounds and pharmaceutical compositions for treating virus infections. The compounds and compositions provided are particularly useful for the treatment of Marburg virus and Ebola virus infections.

Description

Marburg.

State of the art

Filoviruses (such as Ebola virus (EBOV) and Marburg virus (MARV)) are among the most deadly and harmful viruses. They cause severe, often fatal viral hemorrhagic fevers in humans and non-human primates (eg, non-human apes, gorillas, and chimpanzees). Filoviruses are of particular concern as a potential bioweapon because they have the potential to disperse and be used as an aerosol for military purposes.

The incubation period for filovirus infection ranges from 2 to 21 days. The onset is acute and is characterized by high fever, headaches, joint and muscle pain, sore throat, fatigue, diarrhoea, vomiting, and abdominal pain. Some patients may experience a rash, red eyes, hiccups, and internal and external bleeding. Within one week of being infected with the virus, most patients experience chest pain and multiple organ failure, go into shock, and die. Some patients also experience blindness and massive bleeding before death.

Filoviridae is a family of RNA viruses. The existence of two representatives of the Filoviridae family, EBOV and MARV, has been established. The existence of two main pathogenic types of the virus of the Filoviridae family has also been established: Ebolavirus (Ebola virus, ebolavirus) and MARV. One variant of MARV and five species of ebolavirus have been identified: Zaire ebolavirus (also called Ebola virus, EBOV), Sudan ebolavirus, Thai forest ebolavirus, Bundibugyo ebolavirus, and Reston ebolavirus. The exact origin, distribution areas, and natural habitat of the Filoviridae are unknown. However, based on the available data and the nature of related viruses, it is assumed that viruses of the Filoviridae family are zoonotic (i.e., carried by animals) and usually remain in animal hosts living on the African continent.

For more than 30 years, ebolaviruses have been associated with recurrent outbreaks of hemorrhagic fever in Central Africa, which cause severe illness in infected patients. Mortality rates during such outbreaks ranged from 50% for Sudan ebolavirus (SEBOV) up to 90% for Zaire ebolavirus (EBOV, ZEBOV) (Sanchez et al., Filoviridae: Marburg and Ebola Viruses//Fields Virology (eds Knipe, D.M. & Howley, P.M.) 1409-1448 (Lippincott Williams & Wilkins, Philadelphia)). An outbreak in late 2007 in Uganda, apparently caused by new ebolavirus species, resulted in a mortality rate of approximately 25% (Towner et al., PLoS Pathog., 4: e1000212 (2008)). ZEBOV has also wiped out wild great ape populations in the same region of Africa (Walsh et al., Nature, 422:611-614 (2003)).

Prevention and treatment of filovirus infections, including ebolaviruses (i.e. EBOV), comes with many challenges. To date, there are no vaccines or post-exposure prophylaxis techniques suitable for preventing or controlling EBOV infections. Instead, patients receive only supportive care, i.e. maintenance of water-salt balance, oxygen, maintenance of blood pressure and treatment of any secondary infections.

Thus, there is a need for compositions and methods for the treatment of EBOV infections. The present invention is intended to meet this and other needs.

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Brief description of the invention

The proposed compound is selected from the group consisting of

or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention provides a pharmaceutical composition for the treatment or prevention of Filoviridae infections, comprising a therapeutically effective amount of said compound, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.

In another embodiment, the present invention provides the use of said compound, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, for the treatment of a Filoviridae viral infection in a human.

Detailed description of the invention

I. Definitions.

Unless otherwise indicated, the following terms and expressions are intended to have the following meanings in the context of this specification.

When trademarks are used in this specification, Applicants are intended to mean that such indications independently include the trademarked product and the active pharmaceutical ingredient(s) of the trademarked product.

As used herein, a compound of the present invention means a compound or a pharmaceutically acceptable salt thereof. Similarly, with respect to isolated intermediates, the expression compound of formula (number) means a compound of the indicated formula and its pharmaceutically acceptable salts.

The term prodrug as used herein refers to any compound which, when introduced into a biological system, produces a drug substance, i.e. an active ingredient, by spontaneous chemical reaction(s), enzyme-catalyzed chemical reaction(s), photolysis and/or metabolic chemical reaction. (reactions). Thus, a prodrug is a covalently modified analog or inactive form of a therapeutically active compound.

One skilled in the art will appreciate that substituents and other moieties of the compounds should be chosen to produce a compound that is sufficiently stable to provide a pharmaceutically useful compound that can be formulated into an acceptably stable pharmaceutical composition. Compounds that exhibit such stability are considered to be within the scope of the present invention.

Unless otherwise indicated, the carbon atoms of the compounds are meant to have a valence of four. In some representations of the chemical structure, where the carbon atoms do not have enough attached substituents to achieve a valency of four, the remaining substituents on the carbon atom necessary to provide a valency of four should be considered to be hydrogen. For example,

^R8

R ³ R ² means the same as

^R8

H

R ³ R ² .

A protecting group refers to a fragment of a compound that masks or modifies the properties of a functional group or the properties of the compound as a whole. The chemical structure of the protecting group varies widely. One of the functions of the protecting group is to serve as an intermediate in the synthesis of the parent drug. Chemical protecting groups and strategies for introducing/deprotecting groups are well known in the art. Cm.

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Protective Groups in Organic Chemistry, Theodora W. Greene (John Wiley & Sons, Inc., New York, 1991). See also Protective Groups in Organic Chemistry, Peter GM Wuts and Theodora W. Greene, 4th ^Ed ., 2006 communications in an orderly and pre-planned manner. Protecting the functional groups of a compound also changes other physical properties, in addition to the reactivity of the protected functional group, such as polarity, lipophilicity (hydrophobicity) and other properties that can be measured using commonly used analytical tools. Chemically protected intermediates may themselves be biologically active or inert. The term hydroxyl protecting groups refers to protecting groups suitable for protecting hydroxyl groups (-OH).

Protected compounds may also exhibit altered and in some cases optimized in vitro and in vivo properties such as passage through cell membranes and resistance to enzymatic degradation or sequestration. As such, protected compounds with the desired therapeutic effect can be considered as prodrugs. Another function of the protecting group is to convert the parent drug into a prodrug, whereby said parent drug is released upon in vivo conversion of the prodrug. Since active prodrugs may be absorbed more efficiently than the parent drug, the prodrugs may be more potent in vivo than the parent drug. The protecting groups are removed either in vitro for chemical intermediates or in vivo for prodrugs. In the case of chemical intermediates, it is not so important that the products obtained after deprotection, such as alcohols, be physiologically acceptable, although in general it is more desirable that these products be pharmacologically harmless.

The term chiral refers to molecules that do not match their mirror image when superimposed, while the term achiral refers to molecules that can be superimposed on their mirror image.

The term stereoisomers refers to compounds that have the same chemical structure but differ from each other in the arrangement of atoms or groups in space.

A diastereomer refers to a stereoisomer with two or more centers of chirality, and the diastereomer molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, reactivity, and biological properties. For example, compounds may contain a chiral phosphorus atom when R ⁷ is

and Z ¹ and Z ² are different. When at least one of Z ¹ or Z ² also contains a chiral center, for example Z ¹ or Z ² is a nitrogen-attached chiral ester of a naturally occurring α-amino acid, then the compound will exist as diastereomers because there are two center of chirality. All such diastereomers and methods of their use described in the present description are covered by the present invention. Mixtures of diastereomers can be separated using high resolution analytical techniques such as electrophoresis, crystallization and/or chromatography. Diastereomers may have different physical characteristics, such as, but not limited to, solubility, chemical stability, and crystallinity, and may also have different biological properties, such as, but not limited to, enzyme resistance, absorbability, and metabolic stability.

The term enantiomers refers to two stereoisomers of a compound that are mirror images of each other that do not match when superimposed.

A definition of approximately when applied to a quantity includes a specified quantity and is meaningful according to the context (eg, includes a fraction of the error associated with measuring a particular quantity).

Unless otherwise indicated, the term "treatment" as used herein means causing regression, alleviation, inhibition of progress, or prevention of the disorder or condition to which the term applies, or one or more of the symptoms of such disorder or condition. The term treatment in the context of the present description refers to the treatment process, which is defined immediately before the specified definition.

The term therapeutically effective amount in the context of the present description is the amount of the compound present in the composition described in the present description, which

- 4 039561 swarm is required to ensure that the content of the drug in the secretions and tissues of the respiratory tract and lungs, or alternatively in the bloodstream of the subject to be treated, is necessary to obtain the expected physiological response or the desired biological effect when such a composition is administered by the selected method. The exact amount will depend on a variety of factors, such as the particular compound, the particular potency of the composition, the delivery device used, the physical characteristics of the composition, its intended use, as well as patient-related considerations such as state of disease severity, contact with the patient, etc. ., and can be easily determined by a person skilled in the art based on the information provided in the present description.

The term saline solution means an aqueous solution containing 0.9% (w/v) NaCl.

The term hypertonic saline solution means an aqueous solution containing more than 0.9% (w/v) NaCl. For example, a 3% hypertonic saline solution would contain 3% (w/v) NaCl.

The formation of a reaction mixture refers to the process of bringing into contact at least two different compounds in such a way that they mix and can react. However, it should be understood that the resulting reaction product can be obtained directly from the reaction between the added reactants or from an intermediate that can be obtained in the reaction mixture from one or more added reactants.

A binding agent refers to an agent capable of binding two incompatible compounds. Coupling agents may be catalytic or stoichiometric. For example, the coupling agents may be a lithium-based coupling agent or a magnesium-based coupling agent such as a Grignard reagent. Illustrative binding agents include, but are not limited to, n-BuLi, MgCl2, iPrMgCl, tBuMgCl, PhMgCl, or combinations thereof.

Non-nucleophilic base refers to an electron donating Lewis base such as nitrogen bases including triethylamine, diisopropylethylamine, N,N-diethylaniline, pyridine, 2,6lutidine, 2,4,6-collidine, 4-dimethylaminopyridine and quinuclidine.

Leaving group refers to groups that retain a bonding electron pair upon heterolytic bond cleavage. For example, the leaving group is readily substituted during a nucleophilic substitution reaction. Suitable leaving groups include, but are not limited to, chloride, bromide, mesylate, tosylate, triflate, 4-nitrobenzenesulfonate, 4-chlorobenzenesulfonate, 4-nitrophenoxy, pentafluorophenoxy, etc. The person skilled in the art will be able to suggest other leaving groups suitable for use in the present invention.

Deprotecting agent refers to any agent capable of removing a protecting group. The deprotecting agent will depend on the type of protecting group used. Typical deprotecting agents are known in the art and can be found in Protective Groups in Organic Chemistry, Peter GM Wuts and Theodora W. Greene, 4th ^Ed ., 2006.

II. Compounds according to the present invention.

The following will be detailed references to some embodiments of the present invention, examples of which are illustrated by the accompanying description, structures and formulas. Although the present invention will be described in conjunction with the present embodiments, it should be understood that these embodiments are not intended to limit the present invention. On the contrary, the present invention is intended to cover all alternatives, modifications and equivalents that may be included within the scope of the present invention.

In another embodiment, the present invention provides a compound which is

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or a pharmaceutically acceptable salt or ester of said compound.

In other embodiments, the present invention provides a compound that is

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or a pharmaceutically acceptable salt or ester of said compound.

In other embodiments, the present invention provides a compound that is

or a pharmaceutically acceptable salt, hydrate or ester of said compound.

In another embodiment, the present invention provides a compound which is

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or a pharmaceutically acceptable salt or ester of said compound.

In another embodiment, the present invention provides a compound which is

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or a pharmaceutically acceptable salt or ester of said compound.

In another embodiment, the present invention provides a compound which is

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or a pharmaceutically acceptable salt, hydrate or ester of said compound.

In another embodiment, the present invention provides a compound which is

or a pharmaceutically acceptable salt or ester of said compound.

In another embodiment, the present invention provides a compound which is

or a pharmaceutically acceptable salt or ester of said compound.

In another embodiment, the present invention provides a compound which is

- 13 039561 or a pharmaceutically acceptable salt or ester of said compound.

The names of compounds according to the present description are given using the ACD/Name software for naming chemical compounds (Advanced Chemistry Development, Inc., Toronto, Canada). Other compounds or radicals may be referred to by conventional names or systematic or non-systematic names. The naming and numbering system of compounds according to the present description is illustrated by a typical compound

which is called (2S)-2-ethylbutyl-2-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7- yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propanoate. Other compounds according to the present invention include:

which is called (S)-2-ethylbutyl-2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1Sch1,2,4]triazine-7- yl)-5-cyano-3,4-dihydroxytetrahy.drofuran-2yl)methoxy)(phenoxy)phosphoryl)amino)propanoate, and

which is called (S)-2-ethylbutyl-2-(((R)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1Sch1,2,4]triazine-7- yl)-5-cyano-3,4-dihydroxytetrahy.drofuran-2yl)methoxy)(phenoxy)phosphoryl)amino)propanoate.

(S)-2-ethylbutyl-2-(((R)-(((2R,3 S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine -7-yl)-5-cyano-3,4dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate can also be represented as

Any reference to compounds of the present invention described herein also includes reference to their physiologically acceptable salts. Examples of physiologically acceptable salts of the compounds of the present invention include salts derived from the corresponding

- 14 039561 bases such as alkali or alkaline earth metal salts (eg Na ⁺ , Li ⁺ , K ⁺ , Ca ⁺² and Mg ⁺² ); ammonium and NR4 ⁺ (where R is defined in the present description). Physiologically acceptable salts formed at the nitrogen atom or amino group include (a) acid addition salts formed with inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acids, phosphoric acid, nitric acid, and the like; (b) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, isethionic acid, lactobionic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, malonic acid, sulfosalicylic acid, glycolic acid, 2-hydroxy-3- naphthoate, pamoate, salicylic acid, stearic acid, phthalic acid, mandelic acid, lactic acid, ethanesulfonic acid, lysine, arginine, glutamic acid, glycine, serine, threonine, alanine, isoleucine, leucine and the like; and (c) salts formed by elemental anions such as chlorine, bromine and iodine. Physiologically acceptable salts of a compound with a hydroxy group include the anion of said compound in combination with a suitable cation such as Na ⁺ and NR ₄ ⁺ .

The compound and its pharmaceutically acceptable salts may exist as various polymorphs or pseudopolymorphs. In the context of the present description, crystalline polymorphism means the ability of a crystalline compound to exist in different crystalline structures. Crystal polymorphism may result from differences in crystal packing (packing polymorphism) or differences in packing between different conformers of the same molecule (conformational polymorphism). In the context of the present description, crystalline pseudopolymorphism means the ability of a hydrate or solvate of a compound to exist in the form of various crystalline structures. The pseudopolymorphs of the present invention may exist due to differences in crystal packing (packing pseudopolymorphism) or due to differences in packing between different conformers of the same molecule (conformational pseudopolymorphism). The present invention includes all polymorphs and pseudopolymorphs of the compounds and their pharmaceutically acceptable salts.

The compound and its pharmaceutically acceptable salts may also exist as an amorphous solid. In the context of the present description, an amorphous solid is a solid in which there is no long-range order of the positions of atoms in the solid. This definition also applies to crystals of 2 nm or less. Additives, including solvents, may be used to prepare amorphous forms according to the present invention. The present invention includes all amorphous forms of the compounds and their pharmaceutically acceptable salts.

For therapeutic use, the salts of the active ingredients of the compounds of the present invention will be physiologically acceptable, that is, they will be salts derived from a physiologically acceptable acid or base. However, salts of acids or bases that are not physiologically acceptable may also find use, for example, in the preparation or purification of a physiologically acceptable compound. All salts, whether or not derived from a physiologically acceptable acid or base, are within the scope of the present invention.

Ultimately, it should be understood that the compositions herein comprise the compounds of the present invention in their non-ionized as well as zwitterionic form, as well as combinations with stoichiometric amounts of water, as in hydrates.

It should be noted that all enantiomers, diastereomers and racemic mixtures, tautomers, polymorphs and pseudopolymorphs of the compounds are within the scope of the claimed compounds and their pharmaceutically acceptable salts are covered by the present invention. All mixtures of such enantiomers and diastereomers are within the scope of the present invention.

Compounds of the present invention may have chiral centers, such as chiral carbon or phosphorus atoms. The compounds of the present invention thus include racemic mixtures of all stereoisomers, including enantiomers, diastereomers and atropisomers. In addition, the compounds of the present invention include enriched or separated optical isomers at any or all of the asymmetric chiral atoms. In other words, the chiral centers evident from the images are represented as chiral isomers or racemic mixtures. Racemic and diastereomeric mixtures, as well as individual optical isomers, isolated or synthesized, essentially free of their enantiomeric or diastereomeric partners, are within the scope of the present invention. Racemic mixtures are separated to obtain individual substantially optically pure isomers by well-known methods such as, for example, separation of diastereomeric salts formed by optically active agents, such as acids or bases, and then converting them back to optically active substances. In most cases, the desired optical isomer is synthesized via stereospecific reactions starting from the corresponding stereoisomer of the desired starting material.

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As used herein, definitions and transformations relating to stereochemistry are generally consistent with those given in S.P. references. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., CTepeochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., New York. A large number of organic compounds exist in optically pure forms, that is, they can rotate the plane of plane polarized light. When describing an optically active compound, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule about its chiral center(s). The prefixes d and l, D and L or (+) and (-) are used to indicate the sign of the rotation of the plane of plane polarized light by the connection, where S, (-) or l mean that the connection is left-handed, and the prefixes R, (+) or d mean that the connection is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. A particular stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is usually referred to as an enantiomeric mixture. A 50:50 mixture of enantiomers means a racemic mixture or a racemate that can occur when a chemical reaction or process is not stereoselective or stereospecific. The terms racemic mixture and racemate refer to an equimolar mixture of two enantiomeric compounds that lacks optical activity.

Compounds according to the present invention in some cases may also exist as tautomeric isomers. Although only one delocalized coupling resonant structure can be depicted, all such forms are included within the scope of the present invention. For example, en-amine tautomers may exist for purine, pyrimidine, imidazole, guanidine, amidine and tetrazole systems, and their possible tautomeric forms are within the scope of the present invention.

Any formula or structure given herein, including compounds, is also intended to represent unlabeled forms as well as isotopically labeled forms of compounds. Isotopically labeled compounds have the structures depicted by the formulas given in the present description, but in them one or more atoms are replaced by atoms having a certain atomic mass or mass number. Examples of isotopes that may be included in the compounds of the present disclosure include the isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine such as, but not limited to, ² H (deuterium, D), ³ H (tritium) , ⁿ C, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ F, ³¹ P, ³² P, ³⁵ S, ³⁶ Cl, and ¹²⁵ I. Various isotopically labeled compounds are contemplated herein, such as compounds in which radioactive isotopes, such as ³ H, ¹³ C and ¹⁴ C. Such isotopically labeled compounds may be suitable for use in metabolic studies, reaction kinetics studies, detection or imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), including distribution of the drug or substrate in tissues, or in the treatment of patients with the use of radioactive substances.

The present description also includes compounds in which from 1 to n hydrogen atoms attached to a carbon atom are replaced by deuterium, where n corresponds to the number of hydrogen atoms in the molecule. Such compounds have increased resistance to metabolism and are therefore suitable for increasing the half-life of any compound when administered to a mammal, in particular a human. See, for example, Foster, Deuterium Isotope Effects in Studies of Drug Metabolism, Trends Pharmacol. sci. 5(12):524-527 (1984). Such compounds are synthesized by means well known in the art, for example using starting materials in which one or more hydrogen atoms are replaced by deuterium.

Deuterium-labeled or substituted therapeutic compounds as described herein may have improved DMPK (drug metabolism and pharmacokinetics) characteristics in terms of distribution, metabolism and excretion (ADME)). Substitution with heavier isotopes, such as deuterium, may provide some therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life, reduced required dosage, and/or improved therapeutic index. The ¹⁸ F labeled compound may be suitable for use in PET or SPECT studies. The isotopically labeled compounds of the present disclosure and their prodrugs can generally be prepared by following the procedures described in the Schemes or in the examples and preparations described below by replacing the non-isotopically labeled reagent with a readily available isotopically labeled reagent. It is understood that deuterium in this context is considered to be a substituent in the compound.

The concentration of such a heavier isotope, in particular deuterium, can be determined from the isotope enrichment factor. It is assumed that in the compounds according to the present description, any atom not listed as a specific isotope means any stable isotope of the specified atom. Unless otherwise indicated, when H or hydrogen is specifically indicated at a particular position, it is to be understood that the position is hydrogen in its natural isotopic composition. Accordingly, in the compounds of the present disclosure, any atom specifically referred to as deuterium (D) is intended to mean deuterium.

Recursive substituents are an intended aspect of the present invention.

A specialist in the field of medicinal chemistry understands the versatility of such substituents. Where recursive substituents are present in an embodiment of the present invention, their total number will be determined as above.

Compounds according to the present invention can be obtained by methods known to the person skilled in the art. For example, compounds of the present invention can be prepared according to the methods described in US Pat. No. 8,008,264 and US Patent Publication US 2012/0027752.

A. Metabolites of the compounds of the present invention.

Also within the scope of the present invention are the in vivo derived metabolic products of the compounds described herein, if such products are new and not obvious from the prior art. Such products may be obtained, for example, from oxidation, reduction, hydrolysis, amidation, esterification, and the like. of the administered compound, primarily due to enzymatic processes. Accordingly, the present invention includes novel and non-obvious compounds obtainable by a process comprising contacting a compound of the present invention with a mammal for a period of time sufficient to produce a metabolic product of said compound. Such products are typically identified by preparing a radioactively labeled (eg, with ¹⁴ C or ³ H) compound of the present invention, administering it parenterally at a detectable dose (eg, greater than about 0.5 mg/kg) to an animal such as a rat. , mouse, guinea pig, monkey, or human, for a period of time sufficient to undergo metabolism (typically from about 30 seconds to 30 hours) and excrete metabolic products from urine, blood, or other biological samples. These products can be easily isolated because they are labeled (other products are isolated using antibodies capable of binding epitopes retained in the metabolite). The structures of the metabolites are determined in a conventional manner, for example by mass spectrometry (MS) or nuclear magnetic resonance (NMR) analysis. In general, metabolite assays are performed in the same manner as conventional drug metabolism studies known to those skilled in the art. The transformation products, provided they are not otherwise found in vivo, are suitable for use in diagnostic assays for therapeutic administration of the compounds of the present invention, even if the compounds themselves do not have activity against Filoviridae.

Recipes and methods for determining the stability of compounds in simulated secretion of the gastrointestinal tract are known. Compounds are defined herein as stable in the gastrointestinal tract if less than about 50 mole % of the protected groups become unprotected in simulated intestinal or gastric juice when incubated for 1 hour at 37°C. Just because compounds are stable in the gastrointestinal tract does not mean that they cannot be hydrolyzed in vivo. Prodrugs of the present invention will generally be stable in the digestive system, but may be substantially hydrolyzed to the parent drug in the lumen of the digestive system, the liver or other organ where metabolism may occur, or in cells in general.

III. pharmaceutical formulations.

The compounds of the present invention are formulated with conventional carriers and excipients to be selected in accordance with normal practice. Tablets will contain adjuvants, glidants, fillers, binders, and the like. Aqueous formulations are prepared in sterile form, and when intended to be delivered in a manner other than oral administration, they are generally isotonic. All formulations will optionally contain excipients such as those presented in the Handbook of Pharmaceutical Excipients (1986). Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextran, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid, and the like. The pH of the formulations ranges from about 3 to about 11, but typically from about 7-10. In some embodiments, the pH of the formulations is from about 2 to about 5, but typically it is from about 3 to about 4. In some embodiments, the pH of the formulations is from about 2 to about 10, but typically it is from about 3.5 to about 8.5.

While it is possible for the active ingredients to be administered separately, it may be preferable to present them as pharmaceutical formulations. The compositions, both for veterinary purposes and for use in humans, according to the present invention contain at least one active ingredient as defined above, together with one or more acceptable carriers therefor and optionally other therapeutic ingredients, in particular additional therapeutic ingredients, which are discussed in the present description. The carrier(s) must be

- 17 039561 acceptable in terms of compatibility with other ingredients of the composition and physiologically safe for its recipient.

These formulations include those that are suitable for the above routes of administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art. Methods and formulations can generally be found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers, or finely divided solid carriers, or both, and further shaping the product as necessary.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; in the form of powder or granules; 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, electuary, or paste.

The tablet is obtained by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing in a suitable device the active ingredient in a free-flowing form such as powder or granules, optionally in admixture with a binder, lubricant, inert diluent, preservative, surfactant or dispersing agent. Molded tablets can be obtained by molding in a suitable device a mixture of powdered active ingredient moistened with an inert liquid diluent. The tablets may optionally be coated or scored, and optionally the tablets may be formulated to provide slow or controlled release of the active ingredient from them.

For infections of the eyes or other external tissues, such as the mouth and skin, the formulations are preferably applied as a topical ointment or cream containing the active ingredient(s) in an amount of, for example, 0.075 to 20% w/w, (including the content of the active(s) ingredient(s) in the range from 0.1% to 20% in increments of 0.1% wt./wt., such as 0.6% wt./wt., 0.7% wt./ wt., etc.), preferably from 0.2 to 15% wt./wt. and most preferably from 0.5 to 10% wt./wt. When preparing the formulation as an ointment, the active ingredient(s) may be applied together with a paraffinic or water-miscible ointment base. Alternatively, the active ingredient(s) may be formulated as a cream with an oil-in-water cream base.

If necessary, the aqueous phase of the cream base may include, for example, at least 30% wt./wt. polyhydric alcohol, that is, 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 optionally include a compound that enhances the absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogues.

The oil phase of the emulsions according to the present invention can be obtained from known ingredients in a known manner. Although this phase may contain only an emulsifying agent (also known as an emulsifier), it optionally contains a mixture of at least one emulsifier with fat or oil, or both fat and oil. Preferably, a hydrophilic emulsifier is included in the oil phase along with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both oil and fat. The emulsifier(s) together with or without stabilizer(s) form a so-called emulsifying wax, and such wax together with oil and fat form a so-called emulsifying ointment base which forms the oily dispersion medium of cream formulations.

Emulsifiers and emulsion stabilizers suitable for use in the formulation of the present invention include Tween® 60, Span® 80, cetyl stearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearate, and sodium lauryl sulfate. Also suitable emulsifiers and emulsion stabilizers for use in the formulation of the present invention include Tween® 80.

The choice of suitable oils or fats for the composition is based on achieving the desired cosmetic properties. The cream should preferably be a non-greasy, non-staining rinse-off product with a suitable consistency to prevent leakage from tubes and other containers. Single or dibasic straight or branched chain alkyl esters such as diisoadipate, isocetyl stearate, propylene glycol diesters with coconut oil fatty acids, isopropyl myristate, decyloleate, isopropyl palmitate, butyl stearate, 2ethylhexyl palmitate or a mixture of branched chain esters known as Crodamol CAP can be used.

- 18 039561 (Crodamol CAP), with the last three esters being preferred. These esters may be used singly 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 can be used.

Pharmaceutical compositions in accordance with the present invention contain a combination in accordance with the present invention together with one or more pharmaceutically acceptable carriers or excipients and, optionally, other therapeutic agents. Pharmaceutical formulations containing the active ingredient may be presented in any form suitable for the intended route of administration. For oral administration, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs can be prepared. Compositions intended for oral administration may be prepared in accordance with any method known in the art of pharmaceutical formulation and such compositions may contain one or more agents, including sweetening agents, flavoring agents, coloring agents, and preservatives, to provide an attractive formulation. . Suitable tablets are those containing the active ingredient in admixture with a non-toxic pharmaceutically acceptable excipient suitable for the preparation of tablets. Said excipients may be, for example, inert diluents such as calcium or sodium carbonate, lactose, calcium or sodium phosphate; granulating and disintegrating agents such as cornstarch or alginic acid; binding agents such as starch, gelatin or gum arabic (acacia); and lubricants such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated by known methods, including microencapsulation, to delay degradation and absorption in the gastrointestinal tract and thereby provide a delayed action over a longer period of time. For example, a delay material such as glyceryl monosterate or glyceryl distearate may be used alone or with a wax.

Formulations for oral administration may 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 oil medium such as peanut butter. oil, liquid paraffin or olive oil.

Aqueous suspensions according to the present invention contain the active materials in admixture with excipients suitable for the preparation of aqueous suspensions. Such excipients include a suspending agent such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum arabic, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of alkylene oxide with a fatty acid. (eg polyoxyethylene stearate), ethylene oxide condensate with a long chain aliphatic alcohol (eg heptadecaethyleneoxycetanol), ethylene oxide condensate with a partial ester derived from fatty acid and hexitol anhydride (eg polyoxyethylene sorbitan monooleate). Other non-limiting examples of suspending agents include Captisol (Captisol®) (sulfobutyl simple ether of beta-cyclodextrin, SBE-e-CD). 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.

Oil suspensions can be prepared 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. Suspensions for oral administration may contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those mentioned above and flavoring agents may be added to provide an attractive oral formulation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

The dispersible powders and granules of the present invention, suitable for preparing an aqueous suspension by the addition of water, provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Examples of suitable dispersing or wetting agents and suspending agents are those discussed above. Additional adjuvants may also be present, such as sweetening, flavoring and coloring agents.

The pharmaceutical compositions of the present invention may also be presented in the form of oil-in-water emulsions. The oil phase may be a vegetable oil such as olive oil or peanut oil, a mineral oil such as liquid paraffin, or a mixture thereof. Suitable emulsifying agents include naturally occurring gums such as gum arabic and gum tragacanth, naturally occurring phosphatides such as soy lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents. Syrups and elixirs may be prepared with sweetening agents such as glycerin, sorbitol, or sucrose. Such formulations may also contain an emollient, preservative, flavoring or coloring agent.

The pharmaceutical compositions of the present invention may be presented in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oily suspension. Said suspension may be prepared in a manner known in the art using suitable dispersing and wetting agents, as well as suspending agents, as described above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic, parenteral acceptable diluent or solvent, such as a solution in 1,3-butane-diol, or it may be prepared as a lyophilized powder. The sterile injectable preparation may also be a sterile injectable solution or suspension in a parenterally acceptable diluent or solvent. Acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils can conventionally be used as the solvent or suspending medium. Any bland fixed oil can be used for this purpose, including synthetic mono- or diglycerides. In addition, fatty acid(s), such as oleic acid, can be used in a similar manner in the preparation of injectables. Acceptable vehicles and solvents that may be used include water, Ringer's solution, isotonic sodium chloride solution, hypertonic sodium chloride solution, and hypotonic sodium chloride solution.

The amount of active ingredient that may be combined with the carrier material to form a single dosage form will depend on the host being treated and the particular route of administration. For example, a sustained release formulation intended for oral administration to a human may contain from about 1 to 1000 mg of active material combined with a suitable and convenient amount of carrier, which can range from about 5 to about 95% w/w. from the total weight of the composition. The pharmaceutical composition can be formulated to provide easily measurable amounts for administration. For example, an aqueous solution intended for intravenous infusion may contain from about 3 mg to about 500 mg of the active ingredient per milliliter of solution to allow an appropriate volume to be infused at a rate of about 30 ml/hour.

Formulations suitable for topical application to the eye also include eye drops, in which the active ingredient is dissolved or suspended in a suitable carrier, in particular an aqueous solvent of 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%, in particular about 1.5% w/w.

Formulations suitable for topical use in the oral cavity include lozenges containing the active ingredient in a flavored base, usually sucrose and gum arabic or tragacanth; pastilles (pastilles) containing the active ingredient in an inert base such as gelatin and glycerin or sucrose and gum arabic; and mouthwashes containing the active ingredient in a suitable liquid carrier.

Formulations for rectal administration may be presented as suppositories with a suitable base containing, for example, coconut oil or salicylate.

Formulations suitable for intrapulmonary or nasal administration contain particles, for example, from 0.1 to 500 microns, such as 0.5, 1, 30, 35, etc., which is administered by rapid inhalation through the nasal canal or by inhalation through the mouth to reach the alveolar sacs. Suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for administration as an aerosol or dry powder may be prepared according to conventional methods and may be delivered with other therapeutic agents, such as compounds previously used to treat or prevent Filoviridae infection, as described below.

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

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

These formulations are presented in single-dose or multi-dose containers, such as sealed ampoules or vials, and may be stored in a freeze-dried (lyophilized) state requiring only the addition of a sterile liquid carrier, such as water for

- 20 039561 injections, immediately before use. Injectable solutions and suspensions prepared for immediate administration are prepared from sterile powders, granules and tablets of the type described above. Preferred unit dose formulations are formulations containing a daily dose or a single daily sub-dose of the active ingredient as defined herein above, or an appropriate portion thereof.

It should be understood that, in addition to the ingredients specifically mentioned above, the formulations of the present invention may contain other agents conventional in the art relating to the type of formulation required, for example, agents suitable for oral administration may include flavoring agents.

The present invention also provides veterinary compositions comprising at least one active ingredient as defined above, together with a suitable veterinary carrier therefor.

Carriers for veterinary use are materials suitable for the purpose of administering the composition and may be solid, liquid or gaseous materials that are otherwise inert or that are acceptable for veterinary use and are compatible with the active ingredient. Such veterinary compositions may be administered orally, parenterally, or by any other desired route.

The compounds of the present invention are used to provide controlled release pharmaceutical formulations containing as an active ingredient one or more compounds of the present invention (controlled release formulations) from which the active ingredient is released in a controlled manner and which are formulated in such a way as to allow dosing with less frequent and have an improved pharmacokinetic and toxicity profile of the specific active ingredient.

IV. Ways of introduction.

One or more compounds of the present invention (herein referred to as active ingredients) are administered by any route suitable for the condition being treated. Suitable routes include oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intrathecal and epidural), and the like. Obviously, the preferred path may be different, for example, depending on the condition of the recipient. The advantage of the compounds according to the present invention is that they have oral bioavailability and can be administered orally.

In the methods of the present invention for treating a Filoviridae infection, the compounds of the present invention may be administered at any time to a human who may come into contact with humans suffering from a Filoviridae infection or already suffering from a Filoviridae infection. In some embodiments, the compounds of the present invention may be administered prophylactically to people who come into contact with humans suffering from a Filoviridae infection. In some embodiments, administration of the compounds of the present invention may be administered to humans who have tested positive for Filoviridae infection but who are not yet symptomatic of Filoviridae infection. In some embodiments, administration of the compounds of the present invention may be administered to humans at the onset of symptoms of a Filoviridae infection.

The effective dose of the active ingredient depends at least on the nature of the condition being treated, toxicity, whether the compound is used prophylactically (at lower doses) or against an active viral infection, on the method of delivery and on the pharmaceutical composition, and will be determined by the physician using routine studies according to dose increase. A dose of about 0.0001 to about 100 mg/kg of body weight per day may be contemplated; typically from about 0.01 to about 10 mg/kg of body weight per day; more typically from about 0.01 to about 5 mg/kg of body weight per day; most typically from about 0.05 to about 0.5 mg/kg of body weight per day. For example, a daily intended dose for an adult human weighing about 70 kg would be 1 mg to 1000 mg, preferably 5 mg to 500 mg, and may be presented as a single dose or multiple doses.

An effective dose of a compound of the present invention for treating a Filoviridae infection may depend on whether it is administered prophylactically or to treat a person already suffering from a Filoviridae infection. In addition, the dose may depend on whether the person suffering from the Filoviridae infection is showing symptoms of the infection or not. For people who test positive for Filoviridae infection and people who show symptoms of Filoviridae infection, higher doses may be required compared to those given to people for prophylactic treatment.

Any suitable period of time may be contemplated for the administration of the compounds of the present invention. For example, the administration can be carried out over a period of 1 day to 100 days, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80 or 90 days. The administration can also be carried out over a period of 1 week to 15 weeks, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or

- 21 039561 weeks. Longer periods of administration are also contemplated. The timing of administration may depend on whether the compound is being administered prophylactically or to treat a human suffering from a Filoviridae infection. For example, prophylactic administration may be carried out during the period during which a person is in regular contact with other people suffering from infection with Filoviridae, and for an appropriate period of time after the last contact with a person suffering from infection with Filoviridae. For people already suffering from Filoviridae infection, the period of administration may include any time needed to treat the patient and a certain time after a negative test result for Filoviridae infection to ensure that Filoviridae infection does not re-occur.

V. Combination therapy.

The compositions of the present invention are also used in combination with other active ingredients. For the treatment of a Filoviridae viral infection, preferably said other therapeutic agent has activity against a Filoviridae viral infection, in particular Marburg virus, Ebola virus and Cuevavirus infections. Non-limiting examples of these other active therapeutic agents are ribavirin, palivizumab, motavizumab, RSVIGIV (RespiGam®), MEDI-557, A-60444, MDT-637, BMS-433771, amiodarone, dronedarone, verapamil, convalescent plasma from Ebola virus survivors. .: Ebola Convalescent Plasma (ECP)), TKM-100201, VCX4430 ((2S,3S,4R,5R)-2-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)- 5-(hydroxymethyl)pyrrolidine 3,4-diol), favipiravir (also known as T-705 or Avigan), T-705 monophosphate, T-705 diphosphate, T705 triphosphate, FGI-106 (1-N,7-N-bis [3-(dimethylamino)propyl]-3,9-dimethylquinoline[8,7-h]quinolone1,7-diamine), JK-05, TKM-Ebola, ZMapp, rNAPc2, VRC-EBOADC076-00-VP, OS- 2966, MVA-BN filo, brincidofovir, Vaxart adenovirus vector 5 Ebola vaccine, Ad26-ZEBOV, FiloVax vaccine (FiloVax), GOVX-E301, GOVX-E302, Ebola entry inhibitors (NPC1 inhibitors) and rVSV-EBOV , as well as their mixtures. The compounds and compositions of the present invention may also be used in combination with phosphoramidate morpholino oligomers (PMOs), which are synthetic antisense nucleotide analogs designed to interfere with translation processes by forming base pair duplexes. with specific RNA sequences. Examples of PMOs include AVI-7287, AVI-7288, AVI-7537, AVI-7539, AVI-6002 and AVI-6003. The compounds and compositions of the present invention are also intended to be used in conjunction with general care of patients with Filoviridae viral infections, including parenteral fluids (including dextrose-containing saline and Ringer's lactate) and nutrition, antibiotics (including metronidazole and cephalosporin antibiotics such as ceftriaxone and cefuroxime) and/or antifungal prophylaxis, fever and pain medications, antiemetic agents (such as metoclopramide) and/or antidiarrheal agents, vitamin and mineral supplements (including vitamin K and zinc sulfate), anti-inflammatory agents (such as ibuprofen), pain medicines, and medicines for other common diseases in the patient population, such as anti-malaria agents (including artemether and artesunate-lumefantrine combination therapy), anti-typhoid (including quinolone antibiotics such as ciprofloxacin, macrolide antibiotics such as like azithromycin, cephalosporin antibiotics such as ceftriaxone or aminopenicillins such as ampicillin) or against shigellosis.

It is also possible to combine any compound of the present invention with one or more additional active therapeutic agents in unit dosage form for simultaneous or sequential administration to a patient. Combination therapy can be carried out in a simultaneous or sequential mode of administration. When administered sequentially, the combination may be administered by two or more administrations.

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

Co-administration includes administering unit doses of the compounds of the present invention before or after the administration of unit doses of one or more other active therapeutic agents, for example administering the compounds of the present invention at intervals of seconds, minutes or hours relative to the administration of one or more other active therapeutic agents. For example, a single dose of a compound of the present invention may be administered first, followed by a single dose of one or more other active therapeutic agents over seconds or minutes. Alternatively, a single dose of one or more other therapeutic agents may be administered first, followed by a single dose of a compound of the present invention over seconds or minutes. In some instances, it may be desirable to administer a single dose of a compound of the present invention first, followed by a single dose of one or more other active therapeutic agents at intervals of hours (eg, 1-12 hours). In other cases, it may be desirable to administer a single dose of one or more other active therapeutic agents first, and then, at intervals of hours (eg, 1-12 hours), administer a single dose of a compound of the present invention.

Combination therapy can provide synergy and a synergistic effect, that is, the effect achieved when the active ingredients are used together is greater than the sum of the effects achieved when the compounds are used separately. A synergistic effect can be achieved when the active(s) ingredient(s): (1) are formulated together and administered or delivered simultaneously in a combined formulation; (2) delivered one after the other or in parallel in separate formulations; or (3) administered in some other mode. When delivered one after the other, a synergistic effect can be achieved when the compounds are administered or delivered sequentially, for example in separate tablets, pills or capsules, or by different injections in separate syringes. In general, in post-delivery therapy, an effective dose of each active ingredient is administered sequentially, i.e. serially, while in combination therapy, effective doses of two or more active ingredients are administered together. A synergistic antiviral effect means an antiviral effect that is greater than the expected purely additive effects of the individual compounds of the combination.

In yet another embodiment, the present invention provides methods for inhibiting a Filoviridae polymerase in a cell, comprising contacting a cell infected with a filovirus with an effective amount of a compound or a pharmaceutically acceptable salt, solvate and/or ester, thereby inhibiting the Filoviridae polymerase.

In yet another embodiment, the present invention provides methods for inhibiting Filoviridae polymerase in a cell, comprising contacting a cell infected with a filovirus with an effective amount of a compound, or a pharmaceutically acceptable salt, solvate, and/or ester thereof, and at least one additional active therapeutic agent with thereby ensuring inhibition of Filoviridae polymerase.

In yet another embodiment, the present invention provides methods for inhibiting Filoviridae polymerase in a cell, comprising contacting a cell infected with a Filoviridae virus with an effective amount of a compound or a pharmaceutically acceptable salt, solvate, and/or ester and at least one additional active therapeutic agent selected. .

In yet another embodiment, the present invention provides methods for treating a Filoviridae viral infection in a human, comprising administering to a patient a therapeutically effective amount of a compound or a pharmaceutically acceptable salt, solvate and/or ester thereof.

In yet another embodiment, the present invention provides methods for treating a Filoviridae viral infection in a human, comprising administering to a patient a therapeutically effective amount of a compound or a pharmaceutically acceptable salt, solvate and/or ester thereof, and at least one additional active therapeutic agent, thereby providing polymerase inhibition. filoviridae.

In yet another embodiment, provided herein are methods of treating a Filoviridae viral infection in a human, comprising administering to a patient a therapeutically effective amount of a compound or a pharmaceutically acceptable salt, solvate and/or ester thereof, and at least one additional active therapeutic agent.

Also provided is a kit that contains a compound or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable ester, a stereoisomer, a mixture of stereoisomers, or a tautomer. In certain implementations, separate kits are provided that contain a compound selected from the group presented in the present description, as well as each of its subgroups and implementation options, including separate compounds 1, 8, 9, 10, 12, 15, 17, 21, 22 , 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32 (compounds 1-32), or a pharmaceutically acceptable salt, pharmaceutically acceptable ester, stereoisomer, mixture of stereoisomers or tautomer. In one aspect, the kit contains a compound or a pharmaceutically acceptable salt thereof. Each of the individual kits described herein may contain a label and/or instructions for using the compound to treat a disease or condition in a subject (eg, human) in need thereof. In some embodiments, the disease or condition is a Filoviridae viral infection, including an Ebola virus infection, or a Marburg virus infection. In other embodiments, each individual kit may also contain instructions for using additional medical agents in combination with a compound to treat a disease or condition in a subject (eg, human) in need thereof. In some of these embodiments, the disease or condition is a human infection with a Filoviridae virus, including an Ebola virus infection or a Marburg virus infection. For each of the kits in accordance with the present description, there is an additional implementation option, according to which the kit contains individual dosage units of the compound as described in the present description, or its pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, tautomer, polymorph, pseudopolymorph , amorphous form, hydrate or solvate. Examples of individual dosage units may include pills, tablets, capsules, pre-filled syringes or cartridge syringes, intravenous bags, etc., each containing a therapeutically effective amount of the desired compound or its pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, tautomer, polymorph, pseudopolymorph, amorphous form, hydrate or solvate. In some embodiments, the kit may contain a single dose unit, and in other embodiments, multiple dose units are provided, such as the number of dose units required for a particular regimen or period.

Also provided are articles that contain a compound or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable ester, a stereoisomer, a mixture of stereoisomers, or a tautomer; and capacity. In one aspect, the article contains a compound and individual compounds 1, 8, 9, 10, 12, 15, 17, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32 (compounds 1- 32), or a pharmaceutically acceptable salt thereof, and a container. In certain embodiments, the product container may be a vial, a jar, an ampoule, a pre-filled syringe, a blister pack, a tin, a can, a bottle, a box, and an intravenous bag.

VI. Methods for inhibiting Filoviridae polymerase.

Another aspect of the present invention relates to methods for inhibiting Filoviridae polymerase activity, comprising the step of treating a sample suspected of having Filoviridae with a compound or composition of the present invention.

Filoviridae viruses that can be treated using the methods of the present invention are single-stranded, negative-polar RNA viruses that typically infect primates. Filoviruses can replicate in almost all cell types. Filovirus antigens and virions are found mainly in the fibroblasts and interstitium of an infected individual. There are three identified filovirus genera: Ebola virus (EBOV; five species); Marburg virus (MARV); and Cuevavirus, also known as Lloviu virus (LLOV). Virions (viral particles) have the characteristic shape of long cylindrical filamentous particles that can be straight, curved, coiled, or in a 6 or U configuration. nm) is practically constant. The filovirus genome contains seven genes that encode 4 virion structural proteins (VP30, VP35, nucleoprotein (NP) and polymerase protein (Lpol)) and 3 membrane-associated proteins (VP40, glycoprotein (GP) and VP24).

The genus Ebolavirus contains five known species: (1) Bundibugyo ebolavirus, also known as Bundibugyo virus (BDBV, formerly BEBOV); (2) Reston ebolavirus, also known as Reston or Ebola-Reston virus (RESTV, formerly REBOV); (3) Sudan ebolavirus, also known as Sudan or Ebola Sudan virus (SUDV, formerly SEBOV); (4) Tai Forest ebolavirus, also known as Tai Forest ebolavirus or Tai Forest Virus (TAFV, formerly CIEBOV); and (5) Zaire ebolavieus, also known as Ebola or Ebola-Zaire virus (EBOV, formerly ZEBOV).

The Marburg virus genus includes the Marburg marburgvirus species, also known as Marburg virus (MARV) or Ravn virus (RAW). The genus Cuevavirus includes the species Lloviu cuevavirus, also known as the Lloviu virus (LLOV).

Compositions of the present invention may function as Filoviridae polymerase inhibitors, as intermediates for such inhibitors, or have other uses as described below. Said inhibitors will bind to a site on the surface or cavity of the Filoviridae polymerase having a geometry unique to the Filoviridae polymerase. Filoviridae polymerase-binding compositions can bind with varying degrees of reversibility. Compounds that bind substantially irreversibly are ideal candidates for use in the method of the present invention. Once labeled, these substantially irreversibly binding compositions are suitable for use as probes for the detection of Filoviridae polymerase. Accordingly, the present invention relates to methods for detecting a Filoviridae polymerase in a sample suspected of having a Filoviridae polymerase, comprising the steps of: treating a sample suspected of having a Filoviridae polymerase with a composition containing a compound of the present invention bound to a label; and observing the effect of the sample on label activity. Suitable labels are well known in the diagnostic art and include stable free radicals, fluorophores, radioisotopes, enzymes, chemiluminescent groups, and chromogens. The label in the compounds according to the present description is introduced in the usual way, using functional groups such as hydroxyl, carboxyl, sulfhydryl or amino.

In the context of the present invention, samples suspected of having a Filoviridae polymerase include natural and artificial materials such as living organisms; tissue or cell cultures; biological samples, such as samples of biological materials (blood, serum, urine, cerebrospinal fluid, tears, sputum, saliva, tissue samples, and the like); laboratory samples; samples of food, water or air; samples of bioproducts, such as cell extracts, in

- 24 039561 particular recombinant cells that synthesize the desired glycoprotein; etc. Typically, the sample will be expected to contain an organism that produces Filoviridae polymerase, often a pathogenic organism such as a Filoviridae 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.

The processing step of the present invention includes adding the composition of the present invention to the sample and includes adding the composition precursor to the sample. The addition step includes any route of administration described above.

If necessary, the activity of the Filoviridae polymerase after application of the composition can be observed by any method, including direct and indirect methods of detecting the activity of the Filoviridae polymerase. The present invention covers quantitative, qualitative and semi-quantitative methods for determining the activity of the Filoviridae polymerase. 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 suitable.

Organisms that contain the Filoviridae polymerase include the Filoviridae virus. The compounds of the present invention are suitable for use in the treatment or prevention of Filoviridae infections in animals or humans.

However, when screening for compounds capable of inhibiting human Filoviridae viruses, be aware that enzyme assay results may not correlate with cell culture assay results. Thus, cell-based assay should be the main screening tool.

In another embodiment, the present invention provides methods for treating a Filoviridae viral infection in a human, comprising administering to a patient a therapeutically effective amount of a compound or a pharmaceutically acceptable salt, solvate and/or ester thereof. In some embodiments, the Filoviridae infection is caused by a Filoviridae virus. In some embodiments, the Filoviridae infection is caused by the Ebola virus. In some embodiments, the Filoviridae infection is caused by the Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, or Zaire ebolavirus. In some embodiments, the Filoviridae infection is caused by the Marburg virus. In some embodiments, the Filoviridae infection is caused by the Lloviu virus. In some embodiments, Filoviridae polymerase is inhibited.

The compounds of the present invention may be used in the treatment of a person already suffering from a Filoviridae infection, or may be administered prophylactically to reduce or lessen the likelihood of a Filoviridae infection. Filoviridae infections can be characterized by hemorrhagic fever, hematemesis, diarrhoea, chest pain in the abdomen, and prostration. The incubation period is about 21 days after exposure to a person suffering from a Filoviridae infection. As a rule, the outcome of Filoviridae infection is death.

Also provided as separate implementation options is a compound selected from each of the formulas provided herein, as well as each subgroup and implementation thereof, including a compound selected from the group or one of the specific compounds given herein as examples, including compounds 1-32, or a pharmaceutically acceptable salt, solvate and/or ester thereof, for use in a method of treating a Filoviridae infection in a human. Also provided as separate embodiments is a compound selected from each of the formulas provided herein, as well as each subgroup and implementation thereof, including a compound selected from the group, or one of the specific compounds exemplified herein, including compounds 1-32, or a pharmaceutically acceptable salt, solvate and/or ester thereof, for use in a method of treating human Ebola virus infection. Also provided as separate embodiments is a compound selected from each of the formulas provided herein, as well as each subgroup and implementation thereof, including a compound selected from the group, or one of the specific compounds exemplified herein, including compounds 1-32, or a pharmaceutically acceptable salt, solvate and/or ester thereof, for use in a method of treating human Marburg virus infection. In each of the embodiments herein wherein the Filoviridae infection is an Ebola virus, there are further separate embodiments wherein said Filoviridae infection is caused by a Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, or Zaire ebolavirus. In some embodiments, the Filoviridae infection is caused by the Marburg virus. In some embodiments, the Filoviridae infection is caused by the Lloviu virus.

The present invention also provides compounds of each of the formulas herein, as well as each of their subgroups and implementation options, including a compound selected from the group of formula (IV), or one of the specific compounds exemplified in this description, including compounds 1-32, or their pharmaceutically acceptable salt, solvate and/or ester, for use in any of the methods according to the present invention, as defined in the present description.

- 25 039561

Also provided as separate embodiments are the uses of a compound selected from each of the formulas herein, as well as each of their subgroups and embodiments, including a compound selected from the group, or one of the specific compounds exemplified herein. , including compounds 1-32, or a pharmaceutically acceptable salt, solvate and/or ester thereof, for the preparation of a medicament for the treatment of Filoviridae infection in humans. Also, as separate implementation options, the use of a compound selected from each of the formulas given in the present description, as well as each of their subgroups and implementation options, including a compound selected from the group, or one of the specific compounds exemplified in the present description, including compounds 1-32, or a pharmaceutically acceptable salt, solvate and/or ester thereof, for the preparation of a medicament for the treatment of human Ebola virus infection. Also, as separate implementation options, the use of a compound selected from each of the formulas given in the present description, as well as each of their subgroups and implementation options, including a compound selected from the group, or one of the specific compounds exemplified in the present description, including compounds 1-32, or a pharmaceutically acceptable salt, solvate and/or ester thereof, for the preparation of a medicament for the treatment of human Marburg virus infection.

VII. Screening for Filoviridae polymerase inhibitors.

The compositions of the present invention are screened for inhibitory activity against Filoviridae polymerase using any of the commonly used methods for determining enzyme activity. In the context of the present invention, as a rule, compositions are first screened for inhibition of Filoviridae polymerase in vitro, and then compositions demonstrating inhibitory activity are screened for activity in vivo.

Compositions having Ki (inhibition constants) in vitro less than about 5x10' ⁶ M and preferably less than about IxlO' ⁷ M are preferred for use in vivo.

Suitable in vitro screening methods are described in detail and will not be specified here. However, the following examples describe assays suitable for in vitro use.

VIII. Getting connections.

Examples.

Some abbreviations and acronyms are used in the detailed description of the experiments. Although most of them will be clear to a person skilled in the art, table. 1 contains a list of many of these abbreviations and acronyms.

Table 1. List of abbreviations and acronyms

Reduction Meaning ASGO acetic anhydride AIBN 2,2'-azobis(2-methylpropionitrile) Vp benzyl VpVg benzyl bromide BSA bis(trimethylsilyl)acetamide BzCl benzoyl chloride OED carbonyldiimidazole DABCO 1,4-diazabicyclo[2.2.2]octane DBN 1,5 - diazabicycle o[4,3.0]non-5-ene DDH 2,3-dichloro-5,6-dicyano-1,4-benzoquinone DBU 1,5 - diazabicycle o[5,4.0]undec-5-ene DHA dichloroacetamide DCC dicyclohexylcarbodiimide dhm dichloromethane DMAP 4-dimethylaminopyridine DME 1,2-dimethoxyethane DMTC1 dimethoxytrityl chloride DMSO dimethyl sulfoxide DMTr 4,4'-dimethoxytrityl DMF dimethylformamide EtOAc ethyl acetate IES electrospray ionization

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EtOAc ethyl acetate GMDS hexamethyldisilazane HPLC high performance liquid chromatography LDA lithium diisopropylamide ISNR low resolution mass spectrum MHPBC meta-chloroperbenzoic acid MeCN acetonitrile Meon methanol mmth monomethoxytrityl chloride m/z or w/e mass-to-charge ratio MH ⁺ mass plus 1 MN' mass minus 1 MSOH methanesulfonic acid ms or ms mass spectrum MTBE tert-butyl methyl ether NBS N-bromosuccinimide Ph phenyl kt or kt room temperature TBAF tetrabutylammonium fluoride THF tetrahydrofuran TMSC1 chlorotrimethylsilane TMSBr bromotrimethylsilane TMSI iodotrimethylsilane TMSOTf (trimethylsilyl)trifluoromethylsulfonate TEA triethylamine TBA tributylamine TB AP tributylammonium pyrophosphate TV SCI tert-butyldimethylsilyl chloride TEAB triethylammonium bicarbonate TFU trifluoroacetic acid TCX or those thin layer chromatography Tg triphenylmethyl tol 4-methylbenzoyl Turbo Grignard mixture of isopropylmagnesium chloride and lithium chloride 1:1 δ chemical shift relative to tetramethylsilane, ppm

A. Preparation of compounds.

Example 1 (2S)-ethyl-2-(chloro(phenoxy)(phosphorylamino)propanoate (chloridate A).

Alanine ethyl ester hydrochloride salt (1.69 g, 11 mmol) was dissolved in anhydrous CH ₂ Cl ₂ (10 ml) and the resulting mixture was stirred while cooling to 0° C. under N 2 atmosphere (g). Phenyl dichlorophosphate (1.49 mL, 10 mmol) was added followed by Et ₃ N dropwise over about 10 minutes. The reaction mixture was then slowly warmed to room temperature and stirred for about 12 hours. Anhydrous Et ₂ O (50 ml) was added and the resulting mixture was stirred for about 30 minutes. The resulting solid was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was chromatographed on silica gel eluting with 050% EtOAc in hexane to give intermediate A. 1H NMR (300 MHz, CDCl ₃ ) δ 7.39-7.27 (m, 5H), 4.27 (m, 3H), 1.52 (m, 3H), 1.32 (m, 3H). ³¹ P NMR (121.4 MHz, CDCl ₃ ) δ 8.2, 7.8.

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Example 2 (2S)-2-ethylbutyl-2-(chloro((phenoxy)(phosphorylamino)propanoate (chloridate B)

Alanine 2-ethylbutyl ester of chlorophosphoramidate B was prepared using the same procedure as chloridate A, except for replacing alanine ethyl ester with alanine 2-ethylbutyl ester. The resulting crude material was used for the next reaction. Treatment with methanol or ethanol resulted in a substituted product with the desired LC/MS signal.

Example 3 (2S)-isopropyl-2-(chloro((phenoxy)(phosphorylamino)propanoate (chloridate C)

With

Alanine chlorophosphoramidate isopropyl ester C was prepared using the same procedure as chloridate A except for replacing alanine ethyl ester with alanine isopropyl ester. The resulting crude material was used for the next reaction. Treatment with methanol or ethanol resulted in a substituted product with the desired LC/MS signal.

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

Preparation of (2R,3R,4S,5R)-2-(4-aminopyrrolo[1,2-^[1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran- 2-carbonitrile is described below.

Commercially available lactol (10 g, 23.8 mmol) was dissolved in anhydrous DMSO (30 ml) under N ₂ (g). Ac ₂ O (20 ml) was added and the resulting reaction mixture was stirred at room temperature for about 48 hours. The reaction mixture was poured into ice-cold H2O (500 ml) and the resulting mixture was stirred for 20 minutes. The mixture was subjected to extraction with EtOAc (3x200 ml) and then the combined organic extracts were washed with H2O (3x200 ml). The organic extract was dried over anhydrous MgSO ₄ , filtered and concentrated under reduced pressure. The residue was dissolved in CH ₂ Cl ₂ and chromatographed on silica gel eluting with 25% EtOAc in hexane to give the lactone. ¹ H NMR (400 MHz, DMSO) δ 7.30-7.34 (m, 13H), 7.19-7.21 (m, 2H), 4.55-4.72 (m, 6H), 4 .47 (s, 2H), 4.28 (d, J = 3.9 Hz, 1H), 3.66 (m, 2H). LC/MS m/z 436.1 [M+H2O], 435.2 [M+OH] ^- Tr=2.82 min. HPLC Tr=4.59 [2-98% ACN in H2] for 5 min at a flow rate of 2 ml/min.

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Bromopyrazole (prepared according to WO 2009/132135) (0.5 g, 2.4 mmol) was suspended in anhydrous THF (10 ml) under N2 (g). The suspension was stirred and TMSCl (0.67 ml, 5.28 mmol) was added. The resulting mixture was stirred for 20 min at room temperature and then cooled to about -78°C, after which a solution of n-BuLi (6 ml, 1.6 N in hexane, 9.6 mmol) was slowly added. The reaction mixture was stirred for 10 minutes at about -78° C. and then lactone (1 g, 2.4 mmol) was added via syringe. When the reaction was complete, as determined by LC/MS, AcOH was added to quench the reaction. The mixture was concentrated under reduced pressure and the residue was dissolved in a mixture of CH ₂ Cl ₂ and H2O (100 ml, 1:1). The organic layer was separated and washed with H2O (50 ml). Then the organic layer was dried over anhydrous MgSO ₄ , filtered and concentrated under reduced pressure. The residue was chromatographed on silica gel eluting with 0-50% EtOAc in hexane to give the product as a 1:1 mixture of anomers. LC/MS m/z 553 [M+H].

Hydroxynucleoside (1.1 g, 2.0 mmol) was dissolved in anhydrous CH ₂ Cl ₂ (40 ml) and the solution was cooled with stirring to about -78° C. under N 2 atmosphere (g). TMSCN (0.931 mL, 7 mmol) was added and the resulting mixture was stirred for an additional 10 min. TMSOTf (1.63 ml, 9.0 mmol) was slowly added to the reaction mixture and the resulting mixture was stirred for 1 h. The reaction mixture was then diluted with CH ₂ Cl ₂ (120 ml) and an aqueous solution of NaHCO ₃ (120 ml) was added to quench reactions. The reaction mixture was stirred for an additional 10 min and the organic layer was separated. The aqueous layer was subjected to extraction with CH ₂ Cl ₂ (150 ml) and the combined organic extracts were dried over anhydrous MgSO ₄ , filtered and concentrated under reduced pressure. The residue was dissolved in a minimum of CH ₂ Cl ₂ and chromatographed on silica gel eluting with a gradient of 0-75% EtOAc and hexane to give tribenzylcyanonucleoside as a mixture of anomers. 1H NMR (300 MHz, CD3CN) δ 7.94 (s, 0.5H), 7.88 (s, 0.5H), 7.29-7.43 (m, 13H), 7.11-7, 19 (m, 1H), 6.82-6.88 (m, 1H), 6.70-6.76 (m, 1H), 6.41 (br s, 2H), 5.10 (d, J=3.9Hz, 0.5H), 4.96(d, J=5.1Hz, 0.5H), 4.31-4.85(m, 7H), 4.09-4.18 (m, 2H), 3.61-3.90 (m, 2H). LC/MS m/z 562 [M+H].

Tribenzylcyanonucleoside (70 mg, 0.124 mmol) was dissolved in anhydrous CH ₂ Cl ₂ (2 ml) and cooled to about -20° C. under N 2 (g). A solution of BCl ₃ (1N in CH ₂ Cl ₂ , 0.506 ml, 0.506 mmol) was added and the reaction mixture was stirred for 1 h at -78°C. When the reaction was complete, as determined by LC/MS, MeOH was added to quench the reaction. The reaction mixture was allowed to warm to room temperature and the solvent was removed under reduced pressure. The residue was subjected to reverse phase HPLC on C18 silica gel eluting for 5 min using H2O (0.1% TFA) followed by a gradient of 0-70% MeCN in H2O (0.1% TFA) over 35 min to wash out α -anomer and β-anomer 1. (α-anomer) 1H NMR (300 MHz, D2O) δ 7.96 (s, 1H), 7.20 (d, J=4.8 Hz, 1H), 6.91 (d, J=4.8 Hz, 1H), 4.97 (d, J=4.4 Hz, 1H), 4.56-4.62 (m, 1H), 4.08-4.14 ( m, 1H), 3.90 (dd, J=12.9, 2.4 Hz, 1H), 3.70 (dd, J=13.2, 4.5 Hz, 1H). (β-anomer) 1H NMR (400 MHz, DMSO) δ 7.91 (s, 1H), 7.80-8.00 (br.

- 29039561 s, 2H), 6.85-6.89 (m, 2H), 6.07 (d, J=6.0 Hz, 1H), 5.17 (br s, 1H), 4, 90 (broad s, 1H), 4.63 (t, J=3.9 Hz, 1H),

4.02-4.06 (m, 1H), 3.94 (br s, 1H), 3.48-3.64 (m, 2H). LC/MS m/z 292.2 [M+H], 290.0 [M-H]. Tr=0.35 min. ¹³ C NMR (400 MHz, DMSO): 156.0, 148.3, 124.3, 117.8, 117.0, 111.2, 101.3, 85.8, 79.0, 74.7, 70.5, 61.4.

HPLC Tr=1.32 min.

Preparation of (3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy )methyl)tetrahydrofuran-2-ol using LaCl ₃ -2LiCl.

A solution of 7-iodopyrrolo[2,1-f][1,2,4]triazine-4-amine (7.5 g, 28.8 mmol, 1.0 eq.) in THF (67 ml) was obtained. The solution was cooled to about 0° C. and TMSCl (3.3 ml, 30.3 mmol, 1.05 eq.) was added. The reaction mixture was stirred for about 30 minutes and then PhMgCl (2M in THF; 28 ml, 56.8 mmol, 1.97 eq.) was added while maintaining the internal temperature below 5°C. The reaction mixture was shaken at about 0°C for about 35 min, and then cooled to about -15°C. Then added iPrMgCl (2M in THF, 14 ml, 30.2 mmol, 1.05 eq.) while maintaining the internal temperature below about -10°C. After about 15 min at about -15°C, LaCl ₃ -2LiCl (0.6 M in THF, 50 ml, 14.4 mmol, 0.5 eq.) was added while maintaining the internal temperature below about -15°C. The reaction mixture was shaken for about 25 minutes at about -20°C.

In a separate flask, a solution of (3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)dihydrofuran-2(3H)-one (10.0 g, 23.9 mmol, 0 .83 eq.) in THF (45 ml). The solution was cooled to about -20°C, and then transferred into a solution containing the Grignard reagent, while maintaining the internal temperature below about -15°C. The resulting reaction mixture was stirred at about -20° C. for about 30 minutes.

The reaction was quenched with 2 M HCl (53 ml) and the mixture was heated to about 15°C. Added iPrOAc (38 ml) and separated the organic and aqueous phases. The lower aqueous layer was decanted and the upper organic layer was washed successively with 2.5 wt.% NaHCO ₃ (53 ml), 2.5 wt.% NaHCO ₃ (53 ml) and 10 wt.% NaCl (53 ml).

The organic phase was concentrated to about 45 ml and then diluted with iPrOAc (75 ml). The solution was again concentrated to about 45 ml and then diluted with iPrOAc (23 ml). The solution was concentrated to about 45 ml and then filtered through a pad of celite. The filtered solution was concentrated to about 26 ml and then diluted with MTBE (75 ml). After 2 hours, heptane (23 ml) was slowly added and the suspension stirred at about 25°C for about 2 hours and then cooled to about -5°C for about 8 hours. Solids were isolated by filtration and the filter cake was washed with MTBE /heptane (4:1, 23 ml). The solids were dried in a vacuum oven at a temperature not exceeding about 35° C. to give (3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7yl)-3 ,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol.

Preparation of (3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy )methyl)tetrahydrofuran-2-ol using CeCl ₃ .

Iodopyrazole (5.02 g, 19.3 mmol) was dissolved in THF (45 g) and the solution was cooled to about 0°C with stirring. TMSCl (2.04 g, 18.7 mmol) was added and phenylmagnesium chloride (2.0 M in THF, 19.9 g, 38.2 mmol) was added after about 1 h. The reaction mixture was cooled to about -20° C. and isopropyl magnesium chloride (2.0 M in THF, 9.99 g, 20.5 mmol) was added slowly. After about 30 minutes, the reaction mixture was transferred to a mixture of anhydrous cerium chloride (4.75 g, 19.3 mmol) in THF (22 g) at about -20°C. After about 1.5 h, a solution of lactone (6.73 g, 16.1 mmol) in THF (22 g) was slowly added and the resulting reaction mixture was stirred for about 1 h. 2 M HCl (41 g) was added, the mixture was heated to about 15° C. and isopropyl acetate (35 g) was added. The layers were separated and the organic layer

- 30 039561 washed with 2.5% NaHCO ₃ (2x40 g), 10% NaCl (1x35 g) and concentrated to a volume of approximately 30 ml. Isopropyl acetate (44 g) was added and the solution was concentrated to a volume of about 30 ml. Isopropyl acetate (43 g) was added and the solution was concentrated to a volume of about 30 ml. The solution was filtered and the filtrate was concentrated to a volume leaving about 18 ml. tert-Butyl methyl ether (37 g) was added followed by seed crystals of the product (10.7 mg). After about 14 hours, n-heptane (10.5 g) was added and the mixture was cooled to about -5° C. and filtered. The solids were washed with tert-butyl methyl ether (9 g) at about -5°C and dried under vacuum at about 34°C for about 15 hours to obtain the title product.

Preparation of (3 R,4R,5R)-2-(4-aminopyrrolo [2,1-f][1,2,4]triazin-7 -yl)-3,4-bis(benzyloxy)-5 -(( benzyloxy)methyl)tetrahydrofuran-2-ol using CeCl ₃ and iPrMgCl-LiCl.

Iodopyrazole (5.03 g, 19.3 mmol) was dissolved in THF (45 g) and the solution was cooled to about 0° C. with stirring under N2 (g). TMSCl (2.06 g, 19.0 mmol) was added and phenyl magnesium chloride (2.0 M in THF, 20.23 g, 38.8 mmol) was added after about 1 h. The reaction mixture was cooled to about -20° C. and isopropyl magnesium chloride-lithium chloride complex (2.0 M in THF, 15.37 g, 21.0 mmol) was added slowly. After about 1 hour, the reaction mixture was transferred to a mixture of cerium chloride (4.77 g, 19.4 mmol) in THF (22 g) at about -20°C. After about 1 h, a solution of lactone (6.75 g, 16.1 mmol) in THF (23 g) was slowly added and the resulting reaction mixture was stirred for about 1.5 h. 2 M HCl (40 g) was added, the mixture was heated to about 15° C. and isopropyl acetate (35 g) was added. The layers were separated and the organic layer was washed with 2.5% NaHCO ₃ (2x40 g), 10% NaCl (1x36 g) and concentrated to a volume of about 30 ml. Isopropyl acetate (44 g) was added and the solution was concentrated to a volume of about 30 ml. The solution was filtered and the filtrate was concentrated to a volume of about 18 ml. tert-Butyl methyl ether (37 g) was added followed by seed crystals of the product (10.5 mg). After about 14 hours, n-heptane (11 g) was added and the mixture was cooled to about -5° C. and filtered. The solids were washed with tert-butyl methyl ether (9 g) at about -5°C and dried under vacuum at about 34°C for about 15 hours to give the title product.

Preparation of (3 R,4R,5R)-2-(4-aminopyrrolo [2,1-f][1,2,4]triazin-7 -yl)-3,4-bis(benzyloxy)-5 -(( benzyloxy)methyl)tetrahydrofuran-2-ol using YCl ₃ .

Iodopyrazole (4.99 g, 19.2 mmol) was dissolved in THF (44 g) and the solution was cooled to about 0°C with stirring. TMSCl (2.45 ml, 19.4 mmol) was added and phenylmagnesium chloride (2.0 M in THF, 20.29 g, 39.0 mmol) was added after about 30 minutes. The reaction mixture was cooled to about -20°C and isopropyl magnesium chloride (2.0 M in THF, 9.85 g, 20.1 mmol) was added slowly. After about 30 minutes, the reaction mixture was transferred into a mixture of anhydrous yttrium chloride (3.76 g, 19.3 mmol) and lactone (6.68 g, 16.0 mmol) in THF (24 g) at about -20°C. After about 2.5 hours, 2 M HCl (30 g) was added, the mixture was heated to about 15° C. and isopropyl acetate (22 g) was added. The layers were separated and the organic layer was washed with 2.5% NaHCO ₃ (2x40 g), 10% NaCl (1x35 g) and concentrated to a volume of about 30 ml. Isopropyl acetate (44 g) was added and the solution was concentrated to a volume of about 30 ml. Isopropyl acetate (45 g) was added and the solution was concentrated to a volume of about 30 ml. The solution was filtered and the filtrate was concentrated to a volume of about 18 ml. tert-Butyl methyl ether (37 g) was added followed by seed crystals of the product (11.5 mg). After about 1 hour, n-heptane (15 ml) was added and the mixture was cooled to about -5°C and shaken for about 17 hours. The suspension was filtered and the solids washed with a mixture of tert-butyl methyl ether (8 g)/n-heptane (2 g) , pre-chilled to about -5°C. Received

- 31 039561 These solids were dried under vacuum at about 34°C for about 22 hours to obtain the specified product.

Preparation of (3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy )methyl)tetrahydrofuran-2-ol using NdCl ₃ .

_H2N

BnO OBn BnO OBn

Iodopyrazole (5.02 g, 19.3 mmol) was dissolved in THF (38 g) and the solution was cooled to about 0° C. with stirring under N ₂ (g). TMSCl (2.45 ml, 19.4 mmol) was added and phenylmagnesium chloride (2.0 M in THF, 19.75 g, 38.0 mmol) was added after about 1 h. The reaction mixture was cooled to about -20° C. and isopropyl magnesium chloride (2.0 M in THF, 9.40 g, 19.2 mmol) was added slowly. After about 1.5 hours, the reaction mixture was taken up in a mixture of anhydrous neodymium(III) chloride (4.03 g, 16.1 mmol) and lactone (6.70 g, 16.0 mmol) in THF (22 g) at about - 20°C. After about 1.5 hours the reaction mixture was heated to -10°C and after another 2 hours was added 2 M HCl (36 g). The mixture was heated to about 15° C. and isopropyl acetate (23 g) was added. The layers were separated and the organic layer was washed with 2.5% NaHCO ₃ (2x44 g), 10% NaCl (1x41 g) and concentrated to a volume of about 30 ml. Isopropyl acetate (44 g) was added and the solution was concentrated to a volume of about 30 ml. Isopropyl acetate (45 g) was added and the solution was concentrated to a volume of about 30 ml. The solution was filtered and the filtrate was concentrated to a volume of about 18 ml. tert-Butyl methyl ether (37 g) was added followed by seed crystals of the product (11.9 mg). After about 1 h, n-heptane (15 ml) was added and the mixture was cooled to about -5°C and shaken for about 15 h. , pre-chilled to about -5°C. The resulting solids were dried under vacuum at about 34° C. for about 25 hours to give the title product.

Preparation of (2R,3r,4R,5R)-2-(4-aminopyrrolo [2,1-f][1,2,4]triazin-7-yl)-3,4-bis(benzyloxy)-5 -((benzyloxy )methyl)tetrahydrofuran-2-carbonitrile.

In a pre-chilled (-40°C) solution of (3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7yl)-3,4-bis(benzyloxy )-5-((benzyloxy)methyl)tetrahydrofuran-2-ol (10.0 g, 18.1 mmol, 1.0 eq.) in DCM (100 ml) trifluoroacetic acid (6.19 g, 54.3 mmol, 3.0 eq.), followed by a pre-chilled (-30°C) solution of TMSOTf (24.1 g, 108.6 mmol, 6.0 eq.) and TMSCN (10.8 g, 108.6 mmol , 6.0 eq.) in DCM (50 ml) while maintaining the internal temperature below about -25°C. The reaction mixture was shaken at a temperature below about -30°C for at least 10 min and extinguished in a pre-chilled (about -10°C) solution of 20 wt.% KOH in water (120 ml). The biphasic mixture was heated to ambient temperature. The organic layer was separated and washed with 10 wt.% NaCl in water (3x50 ml). The organic phase was filtered, concentrated under vacuum to about 50 ml, diluted again with toluene (200 ml) and concentrated under vacuum to 140 ml at about 50°C. The solution was seeded with (2R,3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-bis(benzyloxy)- 5((benzyloxy)methyl)tetrahydrofuran-2-carbonitrile at about 55°C. The mixture was shaken at about 55°C for about an hour and cooled to about 0°C for about 6 hours. Solids were isolated by filtration and the filter cake was washed with toluene (30 ml). The solids were dried under vacuum at about 50°C.

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Preparation of (2R,3R,4R,5R)-2-(4-aminopyrrolo[2,1-:f|[1,2,4]triazin-7-yl)-3,4-bis(benzyloxy)-5( (benzyloxy)methyl)tetrahydrofuran-2-carbonitrile using continuous flow chemistry.

Solutions of (3R,4R,5R)-2-(4-aminopyrrolo[2,1-:f|[1,2,4]triazin-7-yl)-3,4-bis(benzyloxy)-5-(( benzyloxy)methyl)tetrahydrofuran-2-ol (23.0g in 460.07g DCM), TMSOTf (55.81g in 138.07g DCM) and TMSCN (25.03g in 138.10g DCM) with using a pump was sequentially fed into a tubular reactor at about -40°C. The reaction mixture was collected in a flask in an ice bath containing a 20% aqueous KOH solution (46.91 g KOH and 210 g water). The layers were separated and the organic phase was washed successively with 10% aqueous KOH (10 g KOH and 90 ml water) and 10% brine (2x100 g). The organic phase was concentrated in vacuo to about 4 volumes, isopropyl alcohol (162.89 g) was added and the mixture was concentrated in vacuo to about 10 volumes. The contents were heated to about 60°C, then brought to about 0°C over about 6.5 hours and shaken at about 0°C for about 15.5 hours. The resulting suspension was filtered, the solids were washed with isopropyl alcohol (61.79 d) and then dried at about 50° C. under reduced pressure overnight to give the title product.

Preparation of (2R,3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran -2-carbonitrile.

Bno bvp but 'he

Tribenzylcyanonucleoside (48.8 g, 86.9 mmol, 1.0 eq.) was dissolved in anhydrous CH ₂ Cl ₂ (244 ml) and cooled to about -20°C. A solution of BC13 (1M in CH ₂ Cl ₂ , 295 ml, 295 mmol, 3.4 eq.) was added dropwise while maintaining the internal temperature below about -15°C. After the addition, the reaction mixture was stirred for 1 hour at about -20°C. MeOH (340 ml) was added dropwise while maintaining the internal temperature below -15°C. The resulting solution was distilled to about 250 ml, then replenished again with about 250 ml of MeOH. The resulting solution was again distilled to about 250 ml, then replenished again with about 250 ml of MeOH, and finally distilled to about 125 ml. Water (125 ml) was added followed by a solution of K2CO3 (20 wt% in water, 125 ml). Checked the pH and found that the pH was ~3. A solution of K2CO3 (20 wt% in water, 50 ml) was added and the pH was found to be ~8. The resulting suspension was stirred overnight, then filtered and washed with water (50 ml) and MeOH (50 ml). The wet filter cake of the product was dried at about 40° C. overnight. 1H NMR (300 MHz, D2O) δ 7.96 (s, 1H), 7.20 (d, J=4.8 Hz, 1H), 6.91 (d, J=4.8 Hz, 1H), 4.97 (d, J=4.4 Hz, 1H), 4.56-4.62 (m, 1H), 4.08-4.14 (m, 1H), 3.90 (dd, J= 12.9, 2.4 Hz, 1H), 3.70 (dd, J=13.2, 4.5 Hz, 1H).

Example 11 (2S)-isopropyl-2-((((2R,3R,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl) -5-cyano3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propanoate (compound 8).

Nucleoside 1 (45 mg, 0.15 mmol) was dissolved in anhydrous trimethyl phosphate (0.5 ml) and the solution was stirred under N ₂ (g) at about 0°C. Methylimidazole (36 μl, 0.45 mmol) was added to the solution. Chlorophosphoramidate C (69 mg, 0.225 mmol) was dissolved in anhydrous THF (0.25 ml) and added dropwise to the mixture containing the nucleoside. When the reaction was complete, which was determined by

- 33 039561

LC/MS, the reaction mixture was diluted with EtOAc and washed with saturated aqueous NaHCO ₃ , saturated with NaCl, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was subjected to silica gel chromatography eluting with 0-5% MeOH in CH2Cl2 followed by preparative HPLC to give the title product. 1H NMR (300 MHz, CD ₃ OD) δ 7.95 (m, 1H), 7.31-6.97 (m, 7H), 4.94 (m, 1H), 4.78 (m, 1H) , 4.43 (m, 3H), 4.20 (m, 1H), 3.80 (d, 1H), 1.30-1.18 (m, 9H). ³¹ P NMR (121.4 MHz, CD ₃ OD) δ 3.8. LC/MS m/z 561.0 [M+H], 559.0 [M-H].

Example 12 (2S)-2-ethylbutyl-2-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7- yl)-5cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphopylamino)propanoate (compound 9).

Compound 9 can be obtained in several ways, described below.

Method 1.

The title compound was obtained from compound 1 and chloridate B according to the same procedure as for compound 8. 1H NMR (300 MHz, CD3OD) δ 7.87 (m, 1H), 7.31-7.16 (m, 5H ), 6.92-6.89 (m, 2H), 4.78 (m, 1H), 4.50-3.80 (m, 7H), 1.45-1.24 (m, 8H), 0.95-0.84 (m, 6H). ³¹ P NMR (121.4 MHz, CD ₃ OD) δ 3.7. LC/MS m/z 603.1 [M+H], 601.0 [M-H].

Method 2.

(2S)-2-ethylbutyl-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl) -5-cyano-3,4dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate. (2S)-2-ethylbutyl-2(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (1.08 g, 2.4 mmol) was dissolved in anhydrous DMF (9 ml) and stirred under nitrogen at room temperature. temperature. (2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5 (hydroxymethyl)tetrahydrofuran-2-carbonitrile (350 mg, 1.2 mmol). A solution of tert-butyl magnesium chloride in THF (1 M, 1.8 mL, 1.8 mmol) was then added dropwise to the reaction mixture over about 10 minutes. The reaction mixture was stirred for about 2 hours, after which the reaction mixture was diluted with ethyl acetate (50 ml) and washed with saturated aqueous sodium bicarbonate (3x15 ml) and then with saturated aqueous sodium chloride (15 ml). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting oil was purified using silica gel column chromatography (0-10% MeOH in DCM) to give (2S)-2-ethylbutyl-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo [2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (311 mg, 43 %, 1:0.4 diastereomeric mixture by phosphorus atom) in the form of a white solid. ¹ H NMR (400 MHz, CD3OD) δ 7.85 (m, 1H), 7.34-7.23 (m, 2H), 7.21-7.09 (m, 3H), 6.94-6 .84 (m, 2H), 4.78 (d, J=5.4 Hz, 1H), 4.46-4.33 (m, 2H), 4.33-4.24 (m, 1H), 4.18 (m, 1H), 4.05-3.80 (m, 3H), 1.52-1.39 (m, 1H), 1.38-1.20 (m, 7H), 0. 85(m, 6H). ³¹ P NMR (162 MHz, CD ₃ OD) δ 3.71, 3.65. LC/MS m/z 603.1 [M+H], 600.9 [M-H]. HPLC (gradient 2-98% MeCN-H ₂ O with 0.1% TFA as modifier over 8.5 min, 1.5 ml/min, column: Phenomenex Kinetex C18, 2.6 µm 100 A, 4, 6x100 mm) t _R =5.544 min, 5.601 min.

Separation of (S) and (R) diastereomers. (2S)-2-ethylbutyl-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,11][1,2,4]triazin-7-yl)-5 -cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate was dissolved in acetonitrile. The resulting solution was loaded onto a Lux Cellulose-2 chiral column equilibrated with acetonitrile and eluted with isocratic acetonitrile/methanol (95:5 v/v). The first eluting diastereomer had a retention time of 17.4 minutes and the second eluting diastereomer had a retention time of 25.0 minutes.

The first diastereomer eluting is (S)-2-ethylbutyl-2-(((R)-(((2R,3S,4R,5R)-5(4-aminopyrrolo[2,1-f][1,2, 4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate:

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¹ H NMR (400 MHz, CD3OD) δ 8.05 (s, 1H), 7.36 (d, J=4.8 Hz, 1H), 7.29 (brt, J=7.8 Hz, 2H), 7.19-7.13 (m, 3H), 7.11 (d, J=4.8 Hz, 1H), 4.73 (d, J=5.2 Hz, 1H), 4, 48-4.38 (m, 2H), 4.37-4.28 (m, 1H), 4.17 (t, J=5.6 Hz, 1H), 4.08-3.94 (m, 2H), 3.94-3.80 (m, 1H), 1.48 (sep, J=12.0, 6.1 Hz, 1H), 1.34 (n, J=7.3 Hz, 4H ), 1.29 (d, J=7.2 Hz, 3H), 0.87 (t, J=7.4 Hz, 6H). ³¹ R NMR (162 MHz, CD3OD) δ 3.71 (s). HPLC (gradient 2-98% MeCN-H ₂ O with 0.1% TFA as modifier over 8.5 min, 1.5 ml/min, column: Phenomenex Kinetex C18, 2.6 µm 100 A, 4, 6x100 mm) t _R =5.585 min.

The second eluting diastereomer is (S)-2-ethylbutyl-2-(((S)-(((2R,3S,4R,5R)-5-(4aminopyrrolo[2,1-f][1,2,4 ]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate:

¹ H NMR (400 MHz, CD3OD) δ 8.08 (s, 1H), 7.36-7.28 (m, 3H), 7.23-7.14 (m, 3H), 7.08 (d , J=4.8 Hz, 1H), 4.71 (d, J=5.3 Hz, 1H), 4.45-4.34 (m, 2H), 4.32-4.24 (m, 1H), 4.14 (t, J=5.8 Hz, 1H), 4.08-3.94 (m, 2H), 3.933.85 (m, 1H), 1.47 (sep, J=6 .2 Hz, 1H), 1.38-1.26 (m, 7H), 0.87 (t, J=7.5 Hz, 6H). ³¹ R NMR (162 MHz, CD3OD) δ 3.73 (s). HPLC (gradient 2-98% MeCN-H ₂ O with 0.1% TFA as modifier over 8.5 min, 1.5 ml/min, column: Phenomenex Kinetex C18, 2.6 µm 100 A, 4, 6x100 mm) t _R =5.629 min.

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

Preparation of (2S)-ethyl-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f|[1,2,4]triazin-7-yl)- 5-cyano-3,4dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate is described below.

Method 1. Preparation using chloridate A.

The title compound was prepared from compound 1 and chloridate A using the same procedure as for compound 8. ¹ H NMR (300 MHz, CD ₃ OD) δ 7.95 (m, 1H), 7.32-6, 97 (m, 7H), 4.78 (m, 1H), 4.43-4.08 (m, 6H), 3.83 (m, 1H), 1.31-1.18 (m, 6H) . ³¹ P NMR (121.4 MHz, CD3OD) δ 3.7. LC/MS m/z 547.0 [M+H], 545.0 [M-H].

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Method 2: Preparation using nitrobenzene compound L.

2' ^L 10

Compound 1 (50 mg, 0.17 mmol) was dissolved in NMP-THF (1:1 ml) and cooled in an ice bath. Then tBuMgCl (0.257 mL, 0.257 mmol) was added over about 5 minutes. The resulting mixture was allowed to warm to room temperature and the mixture was stirred for about 30 minutes. Then a solution of compound L (prepared according to US 20120009147, 74.6 mg, 0.189 mmol) in THF (2 ml) was added. After about 30 minutes, the reaction mixture was purified by HPLC (10% to 80% acetonitrile in water) to give compound 29 as a yellow solid. The solid was further purified by silica gel chromatography (0 to 20% MeOH in DCM) to give compound 29. 1H NMR (400 MHz, CD ₃ OD) δ 7.76 (d, J=6.0 Hz, 1H) , 7.25-7.14 (m, 2H), 7.116.99 (m, 3H), 6.87-6.72 (m, 2H), 4.70 (d, J=5.4 Hz, 1H ), 4.39-4.24 (m, 2H), 4.20 (dddd, J=9.7, 7.9, 5.1, 2.8 Hz, 1H), 4.10 (dt, J =12.8, 5.5 Hz, 1H), 4.06-3.91 (m, 2H), 3.72 (ddc, J=14.3, 9.3, 7.1 Hz, 1H), 1.17 (dd, J=7.1, 1.0 Hz, 1H), 1.14-1.06 (m, 5H). ³¹ P NMR (162 MHz, CD3OD) δ 3.73, 3.68. MS m/z=547 (M+1)+.

Example 15 (2S,2'S)-diethyl-2,2'-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine -7-yl)-5-cyano3,4-dihydroxytetrahydrofuran-2-yl)methoxy)phosphoryl)bis(azanediyl)dipropanoate (compound 12).

Nucleoside 1 (14.6 mg, 0.05 mmol) was dissolved in anhydrous trimethyl phosphate (0.5 ml) and stirred under N2 (g) at room temperature. POCl ₃ (9.2 μl, 0.1 mmol) was added and the resulting mixture was stirred for about 60 minutes. Alanine ethyl ester hydrochloride (61 mg, 0.4 mmol) was added followed by Et ₃ N (70 μl, 0.5 mmol). The resulting mixture was stirred for about 15 min, and then additional Et ₃ N (70 μl, 0.5 mmol) was added to give the pH of the solution was 910. The resulting mixture was stirred for about 2 h, and then diluted with EtOAc, washed with saturated aqueous NaHCO ₃ solution and then saturated aqueous NaCl solution. The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The residue was subjected to preparative HPLC (C ₁₈ phase column) to give the title product 12. 1H NMR (400 MHz, CD3OD) δ 8.13 (s, 1H), 7.41 (d, J=4.8 Hz, 1H) , 7.18 (d, J=4.8 Hz, 1H), 4.78 (d, J=5.6 Hz, 1H), 4.36 (m, 1H), 4.25-4.08 ( m, 7H), 3.83 (m, 2H), 1.33-1.23 (m, 12H). ³¹ R NMR (121.4 MHz, CD3OD) δ 13.8. LC/MS m/z 570.0 [M+H], 568.0 [M-H].

Example 18 S,S'-2,2'-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[1,2-f][1,2,4]triazine-7- yl)-5-cyano-3,4dihydroxytetrahydrofuran-2-yl)methoxy)phosphoryl)bis(oxy)bis(ethane-2,1-diyl)bis(2,2-dimethylpropanthioate) (compound 15).

Nucleoside 1 (0.028 g, 0.096 mmol) was dissolved in trimethyl phosphate (1 ml). The reaction mixture was stirred in N2 (g) and then treated with 1H-tetrazole (0.021 g, 0.29 mmol). The reaction mixture was cooled to 0°C and phosphane was added (Nucleoside, Nucleotides, Nucleic acids; 14; 3-5; 1995; 763-766. Lefebvre, Isabelle; Pompon, Alain; Perigaud, Christian; Girardet, Jean-Luc; Gosselin, Gilles; et al.) (87 mg, 0.192 mmol).

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The reaction mixture was stirred for 2 hours and then quenched with 30% hydrogen peroxide (0.120 ml). The resulting mixture was stirred for 30 minutes at room temperature and then treated with saturated aqueous sodium thiosulfate (1 ml). The resulting mixture was stirred for 10 minutes and then concentrated under reduced pressure. The residue was subjected to preparative HPLC to isolate the title product 15. 1H NMR (300 MHz, CD3CN) δ 7.98 (s, 1H), 6.92 (d, 1H), 6.81 (d, 1H), 6, 44 (br s, 2H), 4.82 (m, 2H), 4.47 (m, 1H), 4.24 (m, 2H), 4.00 (m, 4H), 3.80 (br s, 1H), 3.11 (m, 4H), 1.24 (s, 9H). ³¹ P NMR (121.4 MHz, CD3CN) δ -1.85 (s). LC/MS m/z 661 [M+H].

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

Compound 17 was obtained from compound 1 using a procedure similar to that described previously (WO 2012012776). The product was isolated as the sodium salt. 1H NMR (400 MHz, D2O) δ 7.76 (s, 1H), 6.88 (d, J=4.8 Hz, 1H), 6.73 (d, J=4.4 Hz, 1H), 4.86 (d, J=5.2 Hz, 1H), 4.43 (m, 1H), 4.39 (m, 1H), 4.05 (m, 1H), 3.94 (m, 1H ). ³¹ R NMR (121.4 MHz, D2O) δ -5.4 (d, 1P), -10.8 (d, 1P), -21.1 (t, 1P). LC/MS m/z 530 [MH], 531.9 [M+H] Tr = 0.22 min. Ion exchange HPLC: Tr=9.95 min.

Example 20-a. ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2- yl)methyl phosphate (compound 33).

A mixture of about 0.05 mmol of compound 1 and about 0.5 ml of trimethyl phosphate was sealed in a container for a period of from about one to about 48 hours. The mixture was cooled to a temperature of from about -10°C to about 10°C and about 0.075 mmol of oxychloride phosphorus. After a period of about one to about 24 hours, the reaction was quenched with about 0.5 ml of 1M tetraethylammonium bicarbonate and the desired fractions were isolated by anion exchange chromatography to give the title compound.

Compound 33 was obtained as a bis-triethylammonium salt from compound 1 as described previously (WO 2011150288). 1H NMR (400 MHz, D2O) δ 7.82 (s, 1H), 6.91-6.88 (m, 1H), 6.81-6.78 (m, 1H), 4.87-4, 84 (m, 1H), 4.40-4.30 (m, 2H), 3.95-3.77 (m, 2H), 3.10-3.00 (m, 6H), 1.20- 1.10 (m, 9H). ³¹ R NMR (162 MHz, D2O) δ 2.33. MS m/z 371.

Example 20-b. ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2- yl) methyltrihydrogen diphosphate (compound 34).

Compound 34 was obtained as a trilithium salt from compound 1 as previously described (WO 2002057425). ³¹ R NMR (162 MHz, D2O) δ -5.34 (d), -9.75 (d). MS m/z 451.

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Example 24 (2S)-ethyl-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl )-5-cyano-3,4dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate (21).

_NH2

Preparation of (2S)-ethyl-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)- 5-cyano-3,4dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate is described below.

Preparation of (S)-ethyl-2-amino-3-phenylpropanoate hydrochloride.

L-phenylalanine (5 g, 30 mmol) was added to EtOH (30 ml). TMSCl (6.915 mL, 54 mmol) was added to the reaction mixture at room temperature. The reaction vessel was supplied with a reflux condenser and the reaction mixture was placed in a bath with a temperature of 80°C. The reaction mixture was stirred overnight. The next day, the reaction mixture was cooled to room temperature, concentrated under reduced pressure, and the resulting residue was taken up in Et ₂ O. The resulting suspension was filtered and the isolated solids were further washed with Et ₂ O. The washed solids were placed in high vacuum to obtain an example of hydrochloride (S) -ethyl-2-amino-3-phenylpropanoate. 1H NMR (400 MHz, DMSO-d6) δ 8.52 (s, 3H), 7.30 (m, 5H), 4.24 (ABX, Jax=7.S Hz, Jbx=6.2 Hz, 1H ), 4.11 (m, 2H), 3.17, 3.05 (ABX, Jab=-14 Hz, Jbx=5.8 Hz, Jax=7.6 Hz, 2H), 1.09 (t, J=6.8 Hz, 3H).

Preparation of (2S)-ethyl-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate (compound D).

(S)-Ethyl-2-amino-3-phenylpropanoate hydrochloride (1.01 g, 4.41 mmol) was dissolved in DCM (50 ml). This solution was cooled to about 0°C and PhOP(O)Cl ₂ (0.656 ml, 4.41 mmol) was added followed by slow addition of Et ₃ N (1.62 ml, 11.5 mmol) over 5 minutes. The cold bath was removed and the reaction mixture was allowed to warm to room temperature and stir for a period of 80 minutes. p-NO ₂ PhOH (0.583 g, 4.19 mmol) was added followed by additional Et ₃ N (0.3 ml, 2.1 mmol). The progress of the reaction was monitored by LC/MS. After completion of the reaction, the reaction mixture was diluted with Et ₂ O and the resulting solids were removed by filtration. The filtrate was concentrated and compound D was isolated by silica gel column chromatography (25 g cartridge for dry entry, 120 g column; eluent: 100% hexane with a linear change to 55% EtOAc in hexane). 1H NMR (400 MHz, CD3OD) δ 8.17 (m, 2H), 7.33 (m, 2H), 7.09-7.25 (m, 10H), 4.17 (m, 1H), 4 .07 (m, 2H), 3.08 (m, 1H), 2.84 (m, 1H), 1.14 (m, 3H). ³¹ R NMR (162 MHz, DMSO^) δ -1.479 (s), -1.719 (s). MS m/z=471.01 [M+1].

Preparation of (2S)-ethyl-2-(((((2R,3 S,4R,5R)-5-(4-aminopyrrolo[2,1-f] [1,2,4]triazin-7-yl) -5-cyano-3,4dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate (compound 21).

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Compound 1 (0.030 g, 0.103 mmol) was dissolved in DMF (1 ml) and then THF (0.5 ml) was added. t-BuMgCl (1M/THF, 154.5 µl, 0.154 µmol) was added dropwise to the reaction mixture with vigorous stirring. The resulting white suspension was stirred at room temperature for about 30 minutes. A solution of compound D (0.058 g, 0.124 mmol) in THF (1 mL) was added dropwise to the reaction mixture at room temperature. The progress of the reaction was monitored by LC/MS. When the reaction reached a conversion of 50%, the reaction mixture was cooled in an ice bath and quenched with glacial acetic acid (70 μl). The reaction mixture was concentrated and compound 21 was isolated from the residue using reverse phase HPLC. 1 H NMR (400 MHz, DMSO-d ₆ ) δ 7.91 (d, J=4 Hz, 1H), 7.90 (br s, 2H), 7.09-7.30 (m, 8H), 7.01, (t, J=8.2 Hz, 2H), 6.89 (d, J=4.4 Hz, 1H), 6.82 (t, J=4.4 Hz, 1H), 6 .27 (m, 1H), 6.14 (m, 1H), 5.34 (m, 1H), 4.62 (t, J=5.6 Hz, 1H), 4.15 (m, 1H) , 3.78-4.01 (m, 6H), 2.92 (m, 1H), 2.78 (m, 1H), 1.04 (m, 3H). ³¹ R NMR (162 MHz, DMSO-do) δ 3.69 (s), 3.34 (s). MS m/z=623.0 [M+H].

Example 25 (2S)-ethyl-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl )-5-cyano-3,4dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-methylbutanoate (22).

NH ₂ op-crV ^N o I \___ 3 - ^{N 0} J BUT OH

Preparation of (2S)-ethyl-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)- 5-cyano-3,4dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-methylbutanoate is described below.

Preparation of (28)-ethyl-3-methyl-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)butanoate (compound E).

E(8)-ethyl-2-amino-3-methylbutanoate (0.351 g, 1.932 mmol) was dissolved in DCM (17 ml). This solution was cooled in an ice bath and PhOP(O)Cl ₂ (0.287 ml, 1.932 mmol) was added followed by slow addition of Et ₃ N (1.62 ml, 11.4 mmol) over about 5 minutes. The cold bath was removed and the reaction mixture was allowed to warm to room temperature and stir for a period of 1 hour. p-NO ₂ PhOH (0.255 g, 1.836 mmol) was added and the progress of the reaction was monitored by LC/MS. After completion of the reaction, the mixture was diluted with Et ₂ O and the resulting solids were removed by filtration. The filtrate was concentrated and compound E was isolated by silica gel column chromatography (12 g cartridge for dry entry, 80 g column; eluent: 100% hexane with a linear change to 55% EtOAc in hexane). 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.30 (d, J=9.2 Hz, 2H), 7.48 (t, J=9.6 Hz, 2H), 7.40 (t, J=7.8Hz, 2H), 7.20-7.27(m, 3H), 6.60(quart, J=11.6Hz, 1H), 4.01(m, 2H), 3, 61 (m, 1H), 1.93 (m, 1H), 1.11 (m, 3H), 0.79 (m, 6H). ³¹ R NMR (162 MHz, DMSO-d ₆ ) δ -0.342 (s), -0.578 (s). MS m/z=422.9 [M+H].

Preparation of (2S)-ethyl-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)- 5-cyano-3,4dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-methylbutanoate (compound 22).

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Compound 1 (0.040 g, 0.137 mmol) was dissolved in NMP (1.5 ml) and then THF (0.25 ml) was added. This solution was cooled in an ice bath and t-BuMgCl (1M/THF, 425.7 μl, 0.426 μmol) was added dropwise with vigorous stirring. The ice bath was removed and the resulting white suspension was stirred at room temperature for about 15 minutes. A solution of compound E (0.081 g, 0.192 mmol) in THF (0.5 ml) was added dropwise to the reaction mixture at room temperature. The progress of the reaction was monitored by LC/MS. When the reaction reached a conversion of 50%, the reaction mixture was cooled in an ice bath and quenched with glacial acetic acid (70 μl). The reaction mixture was concentrated and compound 22 was half purified from the residue using reverse phase HPLC. The half pure material was further purified by silica gel column chromatography (12 g dry run cartridge, 40 g column; eluent: 100% EtOAc with a linear change to 10% MeOH in EtOAc) to give compound 22. 1H NMR (400 MHz, DMSO -de) δ 7.91 (d, J=1.6 Hz, 1H), 7.88 (br s, 2H), 7.32 (m, 2H), 7.15 (m, 3H), 6 .90 (t, J=4.2Hz, 1H), 6.84 (d, J=4.8Hz, 1H), 6.26 (dd, J=13.4, 6.2Hz, 1H) , 5.87 (quart, J=11.2 Hz, 1H), 5.35 (m, 1H), 4.64 (m, 1H), 4.25 (m, 2H), 3.93-4, 15 (m, 4H), 3.45 (m, 1H), 1.87 (m, 1H), 1.09-1.16 (m, 3H), 0.70-0.83 (m, 6H) . ³¹ R NMR (162 MHz, DMSO-de) δ 4.59 (s), 4.47 (s). MS m/z=575.02 [M+H].

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

Preparation of (S)-isopropyl-2-(((R)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7 -yl)-5cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate is described below.

Compound 1 (60.0 mg, 206 μmol) was dissolved in NMP (0.28 ml). THF (0.2 ml) was added followed by tert-butyl magnesium chloride (1.0 M solution in tetrahydrofuran, 0.309 ml) at room temperature under argon. After 20 min, a solution of compound F (prepared according to Cho, A. et al., J. Med. Chem. 2014, 57, 1812-1825, 81 mg, 206 µmol) in THF (0.2 ml) was added and the resulting mixture was heated up to about 50°C. After 3 h, the reaction mixture was allowed to cool to room temperature and purified directly by preparative HPLC (Phenomenex Synergi 4 µm Hydro-RR 80A 150x30 mm column, 5-100% acetonitrile/water gradient) to give compound 23. 1H NMR (400 MHz, CD ₃ OD) δ 7.86 (s, 1H), 7.34-7.26 (m, 2H), 7.21-7.12 (m, 3H), 6.91 (d, J=4, 6 Hz, 1H), 6.87 (d, J=4.6 Hz, 1H), 4.92 (sep, J=6.3 Hz, 1H), 4.80 (d, J=5.4 Hz , 1H), 4.43-4.34 (m, 1H), 4.33-4.24 (m, 1H), 4.18 (t, J=5.6 Hz, 1H), 3.82 ( dk, J=9.7, 7.1 Hz, 2H), 1.27 (dd, J=7.1, 1.0 Hz, 3H), 1.18 (dd, J=6.3, 4, 8 Hz, 6H). ³¹ P NMR (162 MHz, CD ₃ OD) δ 3.72 (s). LC/MS: t _R = 1.39 min, MS m/z = 561.11 [M+H]; LC system: Thermo Accela 1250 for UHPLC; MS system: Thermo LCQ Fleet; column: Kinetex 2.6 µm XB-C18 100A, 50 x4.6 mm; solvents: ACN 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 = 2.523 min; HPLC system: Agilent 1100 series; column: Gemini 5 µm C18 110A, 50x4.6 mm; solvents: ACN 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.

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Example 27 (2S)-cyclobutyl-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl )-5cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphopyl)amino)propanoate (24).

Preparation of (2S)-cyclobutyl-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]Ίriazin-7-yl)- 5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate is described below.

Preparation of (2S)-cyclobutyl-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (compound G).

Phenyl dichlorophosphate (1.49 ml, 10 mmol) was dissolved in 10 ml of anhydrous DCM and stirred under nitrogen in an ice bath. Lalanine isobutyl ester hydrochloride (0.9 g, 5 mmol) was added in one portion. Triethylamine (765 μl, 5.5 mmol) was then added dropwise. The reaction mixture was stirred for about 1 hour. Additional triethylamine (765 µl, 5.5 mmol) was added dropwise and the reaction mixture was stirred for about 45 minutes. p-Nitrophenol (1.25 g, 9 mmol) was added in one portion and stirred for about 30 minutes. Triethylamine (765 µl, 5.5 mmol) was added and the reaction mixture was stirred for about 2 hours. Then additional p-nitrophenol (1.25 g, 9 mmol) and triethylamine (765 µl, 5.5 mmol) were added and the reaction mixture stirred for about another 2 hours. The reaction mixture was concentrated under reduced pressure. The resulting crude material was diluted with EtOAc and washed twice with 5% aqueous citric acid and then with saturated aqueous sodium chloride. Then, the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified on a silica gel column (0-20-50% EtOAc in hexane) to give compound G. ¹ H NMR (400 MHz, CD3OD) δ 8.33-8.23 (m, 2H), 7.52- 7.33 (m, 4H), 7.33-7.17 (m, 3H), 4.96-4.85 (m, 1H), 4.07-3.96 (m, 1H), 2, 27 (m, 2H), 2.07-1.91 (m, 2H), 1.83-1.70 (m, 1H), 1.70-1.55 (m, 1H), 1.32 ( m, 3H). ³¹ P NMR (162 MHz, CD ₃ OD) δ -1.36, -1.59. MS m/z=420.9 [M+H].

Preparation of (2S)-cyclobutyl-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)- 5-cyano3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (compound 24).

Compound 1 (58 mg, 0.2 mmol) was mixed with compound G (101 mg, 0.24 mmol) in 2 ml anhydrous DMF. Magnesium chloride (42 mg, 0.44 mmol) was added in one portion. The reaction mixture was heated to about 50°C. DIPEA (87 μl, 0.5 mmol) was added and the reaction mixture was stirred for about 2 hours at about 50°C. The reaction mixture was cooled to room temperature, diluted with EtOAc and washed with 5% aqueous citric acid solution and then with saturated aqueous sodium chloride solution. Then, the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified on a silica gel column (0-2-5% MeOH in DCM) to give compound 24. 1H NMR (400 MHz, methanol ₄ ) δ 7.85 (m, 1H), 7.34-7, 22 (m, 2H), 7.22-7.08 (m, 3H), 6.94-6.84 (m, 2H), 4.95-4.85 (m, 1H), 4.79 ( m, 1H), 4.46-4.34(m, 2H), 4.34-4.24(m, 1H), 4.19(m, 1H), 3.81(m, 1H), 2 .27 (m, 2H), 2.01 (m, 2H), 1.84-1.68 (m, 1H), 1.62 (m, 1H), 1.30-1.16 (m, 3H ). ³¹ P NMR (162 MHz, CD ₃ OD) δ 3.70, 3.65. MS m/z=573.0 [M+H].

Example 28 (2S)-isopropyl-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]Ίriazin-7-yl )-5-cyano3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate (25).

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Preparation of (2S)-isopropyl-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)- 5-cyano3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate is described

below.

Preparation of (2S)-isopropyl-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate (compound H).

H

Phenyl dichlorophosphate (718 μl, 4.8 mmol) was dissolved in 10 ml anhydrous DCM and stirred under nitrogen in an ice bath. L-phenylalanine isopropyl ester hydrochloride (1 g, 4.1 mmol) was added in one portion. An additional 10 ml of anhydrous DCM was added. Triethylamine (736 μl, 5.3 mmol) was added dropwise and the reaction mixture was stirred for about 30 minutes. Then additional triethylamine (736 μl, 5.3 mmol) was added dropwise and the reaction mixture was stirred for 30 minutes. Further triethylamine (736 μl, 5.3 mmol) was then added dropwise and the reaction mixture was stirred for about 15 minutes. Then p-nitrophenol (600 mg, 4.32 mmol) was added. The ice bath was then removed and the reaction mixture was allowed to warm to room temperature and stirred for about 2 hours. Additional p-nitrophenol (50 mg) and triethylamine (736 μl, 5.3 mmol) were added and the reaction mixture was stirred for about 1 hour .

The reaction mixture was then concentrated under reduced pressure, diluted with EtOAc, and washed twice with 5% aqueous citric acid and then with saturated aqueous sodium chloride. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude material was purified on a silica gel column (0-15% EtOAc in hexane) to give compound H. 1H NMR (400 MHz, CDCl ₃ ) δ 8.17 (m, 2H), 7.38-7.13 (m, 10H), 7.13-7.02 (m, 2H), 4.95 (m, 1H), 4.31 (m, 1H), 3.69 (m, 1H), 3.02 (dd, J =6.1, 1.8 Hz, 2H), 1.21-1.08 (m, 6H). ³¹ P NMR (162 MHz, CDCl ₃ ) δ -2.96, -2.98. MS m/z=485.0 [M+H].

Preparation of (2S)-isopropyl-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)- 5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate (compound 25).

Compound 1 (58 mg, 0.2 mmol) and compound H (116 mg, 0.24 mmol) were mixed and 2 ml of anhydrous DMF was added. The reaction mixture was stirred under nitrogen at room temperature. 1 M tBuMgCl in THF (300 µl, 0.3 mmol) was added dropwise over 3 min and then the reaction mixture was stirred for about 16 h. The reaction mixture was diluted with EtOAc and washed with 5% aqueous citric acid, saturated aqueous bicarbonate sodium and then saturated aqueous sodium chloride solution. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified on a silica gel column (0-5% MeOH in DCM) to give compound 25. 1H NMR (400 MHz, CD3OD) δ 7.84 (m, 1H), 7.27-7.08 (m, 8H ), 7.08-6.97(m, 2H), 6.88(m, 2H), 4.91-4.84(m, 1H), 4.74(m, 1H), 4.26( m, 1H), 4.19-4.04 (m, 2H), 4.04-3.91 (m, 2H), 2.97 (m, 1H), 2.82 (m, 1H), 1 .14 (m, 3H), 1.06 (m, 3H). ³¹ P NMR (162 MHz, CD3OD) δ 3.63, 3.25. MS m/z=637.0 [M+H].

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Example 29 (S)-methyl-2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine -7-yl)-5-cyano3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphopyl)amino)propanoate (26).

Preparation of (S)-methyl-2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7 -yl)-5-cyano3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate is described below.

Compound 1 (100 mg, 0.34 mmol) was dissolved in THF (2 ml) and cooled in an ice bath. Then 1 M t-BuMgCl (0.52 ml, 0.77 mmol) was slowly added dropwise. The resulting mixture was stirred for about 30 minutes at room temperature. Compound I (prepared according to WO 2012142085, 219 mg, 0.52 mmol) in THF (2 ml) was then added over 5 minutes and the resulting mixture was stirred for about 24 hours at room temperature. The reaction mixture was then diluted with EtOAc, cooled in an ice bath, washed with aqueous NaHCO ₃ (2 ml), washed with brine, dried over sodium sulfate and concentrated under vacuum. The resulting mixture was purified by silica gel column chromatography (0 to 20% MeOH in DCM) and preparative HPLC (10 to 80% acetonitrile in water) to give compound 26. 1H NMR (400 MHz, CD3OD) δ 7.86 ( s, 1H), 7.29 (dd, J=8.6, 7.2 Hz, 2H), 7.21-7.09 (m, 3H), 6.94-6.81 (m, 2H) , 4.79 (d, J=5.4 Hz, 1H), 4.38 (ddc, J=10.8, 5.3, 2.7 Hz, 2H), 4.33-4.23 (m , 1H), 4.18 (t, J=5.5 Hz, 1H), 3.86 (dc, J=9.9, 7.1 Hz, 1H), 3.62 (s, 3H), 1 .27 (dd, J=7.2, 1.1 Hz, 3H). MS m/z = 533 (M+1)+.

Example 30 (S)-neopentyl-2-(((S)-(((2R,3S,4R,5R)-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7 -yl)-5cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphopyl)amino)propanoate (27).

Preparation of (S)-neopentyl-2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triαzine-7 -yl)-5cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate is described below.

Compound 1 (100 mg, 0.34 mmol) was dissolved in THF (2 ml) and cooled in an ice bath. Then 1 M t-BuMgCl (0.52 ml, 0.77 mmol) was slowly added dropwise. The resulting mixture was stirred for about 30 minutes at room temperature. Compound J (prepared according to WO2012075140, 248 mg, 0.52 mmol) was then added over about 5 min and the resulting mixture was stirred for about 24 h at room temperature, diluted with EtOAc, cooled in an ice bath, treated with aqueous NaHCO ₃ (2 ml), washed with brine, dried over sodium sulfate and concentrated in vacuo. The resulting mixture was purified by silica gel column chromatography (0 to 20% MeOH in DCM) and preparative HPLC (10 to 80% acetonitrile in water) to give compound 27. 1H NMR (400 MHz, CD3OD) δ 7.86 ( s, 1H), 7.36-7.24 (m, 2H), 7.23-7.10 (m, 3H), 6.96-6.85 (m, 2H), 4.78 (d, J=5.4 Hz, 1H), 4.38 (tdd, J=10.0, 4.9, 2.5 Hz, 2H), 4.32-4.24 (m, 1H), 4.17 (t, J=5.6 Hz, 1H), 3.91 (dc, J=9.8, 7.1 Hz, 1H), 3.81 (d, J=10.5 Hz, 1H), 3 .69 (d, J=10.5 Hz, 1H), 1.31 (dd, J=7.2, 1.1 Hz, 3H), 0.89 (s, 9H). MC m/z=589 (M+1)+.

Example 31 (2S)-cyclopentyl-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl )-5cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphopyl)amino)propanoate (28).

Preparation of (2S)-cyclopentyl-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)- 5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate is described below.

Compound 1 (100 mg, 0.34 mmol) was dissolved in THF (2 ml) and cooled in an ice bath. Then 1 M t-BuMgCl (0.52 ml, 0.77 mmol) was slowly added dropwise. The resulting mixture was stirred for about 30 minutes at room temperature. Compound K (prepared according to WO2012075140, 247 mg, 0.52 mmol) in THF (2 ml) was then added over about 5 min and the resulting mixture was stirred for about 24 h at room temperature, diluted with EtOAc, cooled in an ice bath, worked up aq. NaHCO ₃ (2 ml), washed with brine, dried over sodium sulfate and concentrated in vacuo. The resulting mixture was purified by silica gel column chromatography (0 to 20% MeOH in DCM) and preparative HPLC (10 to 80% acetonitrile in water) to give Example 28. 1H NMR (400 MHz, CD3OD) δ 7.85 ( s, 1H), 7.33-7.22 (m, 2H), 7.14 (tdd, J=7.6, 2.1, 1.1 Hz, 3H), 6.95-6.87 ( m, 2H), 5.13-5.00 (m, 1H), 4.78 (d, J=5.4 Hz, 1H), 4.48-4.35 (m, 2H), 4.30 (ddd, J=10.6, 5.7, 3.6 Hz, 1H), 4.19 (t, J=5.4 Hz, 1H), 3.78 (dc, J=9.2, 7 .1 Hz, 1H), 1.81 (dtd, J=12.5, 5.9, 2.4 Hz, 2H), 1.74-1.49 (m, 6H), 1.21 (dd, J=7.1, 1.2 Hz, 3H). MS m/z = 587 (M+1) ⁺ .

Example 32 (2S)-cyclohexyl-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl )-5cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphopyl)amino)propanoate (29).

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_{Diisopropylethylamine} (0.075 ml, 0.43 mmol) for about 5 minutes at about 0°C. The resulting mixture was stirred at about 50° C. for about 1 hour. The reaction mixture was then cooled in an ice bath, treated with 1 M citric acid solution (0.5 ml) and purified directly by preparative HPLC (0 to 70% ACN in water ) to give compound 29. 1H NMR (400 MHz, CD3OD) δ 7.84 (s, 1H), 7.32-7.23 (m, 2H), 7.18-7.10 (m, 3H), 6.93-6.87 (m, 2H), 4.78 (d, J=5.4 Hz, 1H), 4.67 (td, J=8.7, 4.2 Hz, 1H), 4 .48-4.35 (m, 2H), 4.30 (ddd, J=10.8, 5.7, 3.7 Hz, 1H), 4.20 (t, J=5.4 Hz, 1H ), 3.88-3.71 (m, 1H), 1.83-1.63 (m, 4H), 1.58-1.46 (m, 1H), 1.46-1.24 (m , 5H), 1.24 (s, 3H). ³¹ P NMR (162 MHz, CD ₃ OD) δ 3.75. MS m/z=601 (M+1)+.

Example 33 Ethyl 2-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano -3,4dihydroxytetrahydrofuran-2-yl)methoxy)((phenoxy)phosphoryl)amino)-2-methylpropanoate (30).

Preparation of ethyl 2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano- 3,4dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-2-methylpropanoate is described below.

Preparation of ethyl 2-((tert-butoxycarbonyl)amino)-2-methylpropanoate.

Triphenylphosphine (6.18 g, 25.00 mmol) was added to THF (30 ml). DIAD (4.92 mL, 25.00 mmol) was then added and stirred at room temperature for 10 min. Dissolve 2-((tert-butoxycarbonyl)amino)-2-methylpropanoic acid (5.08 g, 25.00 mmol) in THF (20 ml) and add to the reaction mixture, followed by the addition of ethanol (2.19 ml, 37.49 mmol ). The reaction mixture was allowed to stir at room temperature for about 1 hour. The solvents were removed under reduced pressure and the crude material was dissolved in Et ₂ O:hexane 1:1 (120 ml). Solid triphenylphosphine oxide was filtered off and the solvent was removed under reduced pressure. The crude material was taken up in minimal CH ₂ Cl ₂ and purified by silica gel chromatography eluting with 0-50% EtOAc/Hex to give ethyl 2-((tert-butoxycarbonyl)amino)2-methylpropanoate. ¹ H NMR (400 MHz, chloroform-d) δ 4.18 (quart, J=7.1 Hz, 2H), 1.49 (s, 6H), 1.43 (s, 9H), 1.27 ( t, J=7.1 Hz, 3H).

Preparation of ethyl 2-amino-2-methylpropanoate hydrochloride.

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Ethyl 2-((tert-butoxycarbonyl)amino)-2-methylpropanoate (2.71 g, 11.72 mmol) was added to CH ₂ Cl ₂ (25 ml), 4N was added slowly. HCl in dioxane (25 mmol) and stirred at room temperature. After 1 h, it was determined by TLC that the reaction was complete. The solvents were removed under reduced pressure and the crude material was co-evaporated twice with Et ₂ O and then placed under high vacuum to give ethyl 2-amino-2-methylpropanoate hydrochloride. 1H NMR (400 MHz,

DMSO-d ₆ ) δ: 8.70 (s, 3H), 4.18 (quart, J=7.1 Hz, 2H), 1.46 (s, 6H), 1.21 (t, J=7 .1 Hz, 3H).

Preparation of ethyl 2-methyl-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (compound N).

Phenyl dichlorophosphate (0.97 ml, 6.50 mmol) and ethyl 2-amino-2-methylpropanoate hydrochloride (1.09 g, 6.50 mmol) were added to CH ₂ Cl ₂ (50 ml). The reaction mixture was cooled to about 0° C. and TEA (1.75 ml, 12.45 mmol) was added slowly. The cold bath was removed and the reaction mixture was allowed to stir at room temperature. After about 2 hours, it was determined by ³¹ P NMR that the addition of the amino acid was complete. P-nitrophenol (0.860 g, 6.17 mmol) was added followed by TEA (0.87 g, 7.69 mmol). The reaction mixture was allowed to stir at room temperature. After about 2 hours, it was determined by LC/MS that the reaction was complete. The reaction mixture was diluted with Et ₂ O and the TEA-HCl salts were filtered off. The crude material was concentrated and purified by silica gel chromatography (0-50% EtOAc/Hex) to give compound N. 1H NMR (400

MHz, DMSO-d ₆ ) 8.37-8.21 (m, 2H), 7.55-7.44 (m, 2H), 7.43-7.33 (m, 2H), 7.30 -7.09 (m, 3H), 6.57 (d, J=10.1 Hz, 1H), 3.99 (quart, J=7.1 Hz, 2H), 1.39 (s, 6H) , 1.08 (t, J=7.1 Hz, 3H). ³¹ R NMR (162 MHz, DMSO-d ₆ ) δ 2.87. LC/MS: t _R = 1.65 min, MS m/z = 408.97 [M+1].; LC system: Thermo Accela 1250 for 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 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.

Preparation of ethyl 2-(((((2R,3 S,4R,5R)-5 -(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano -3,4dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-2-methylpropanoate (compound 30).

Compound 1 (66 mg, 0.23 mmol) was added to NMP (2.0 ml). The mixture was cooled to about 0° C. and tBuMgCl (1.0 M in THF, 0.34 ml, 0.34 mmol) was added slowly. The reaction mixture was allowed to stir at about 0°C for about 30 min, and then a solution of compound N (139 mg, 0.34 mmol) in THF (1.0 ml) was added. The cold bath was removed and the reaction mixture was placed in an oil bath preheated to about 50°C. After about 2 hours the reaction mixture was cooled to room temperature and quenched with acetic acid and methanol. The crude material was concentrated and purified by reverse phase HPLC without modifier to give compound 30. 1H NMR (400 MHz, DMSO-d ₆ ) δ 7.89 (m, 3H), 7.31 (quart, J=8.1 Hz, 2H), 7.22-7.05 (m, 3H), 6.87 (d, J=4.5, 1H), 6.80 (d, J=4.5 Hz, 1H), 6 .27 (d, J=11.7, 1H), 5.81 (d, J=9.7, 1H), 5.35 (d, J=5.6 Hz, 1H), 4.64 (dt , J=9.0, 5.6 Hz, 1H), 4.24(m, 2H), 4.11(m, 1H), 4.04-3.90(m, 3H), 1.39- 1.23 (m, 6H), 1.10 (t, J=7.1, 3H). ³¹ R NMR (162 MHz, DMSO-d ₆ ) δ 2.45, 2.41. LC/MS: t _R =1.03 min, MS m/z=561.03 [M+1]; LC system: Thermo Accela 1250 for 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 min - 2.80 min 100% ACN, 2.8 min - 2.85 min 100% - 2%

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ACN, 2.85 min - 3.0 min 2% ACN at 1.8 ml/min.

Example 34 Isopropyl-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5- cyano3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-2-methylpropanoate (31).

Preparation of isopropyl 2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano3, 4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-2-methylpropanoate is described below.

Preparation of isopropyl 2-((tert-butoxycarbonyl)amino)-2-methylpropanoate.

O I PPh ₃ , DIAD, THF O I

BoSIM ^ oN *% OH ---------► ΒοαΗΝ ^ΛθΛ

Triphenylphosphine (6.17 g, 25.00 mmol) was added to THF (30 ml). DIAD (4.92 mL, 25.00 mmol) was then added and stirred at room temperature for about 10 minutes. 2-((tert-butoxycarbonyl)amino)-2-methylpropanoic acid (5.07 g, 25.00 mmol) was taken up in THF (20 ml) and added to the reaction mixture followed by the addition of isopropanol (1.91 ml, 25.00 mmol ). The reaction mixture was allowed to stir at room temperature for about 1 hour. The solvents were removed under reduced pressure and the crude material was dissolved in Et ₂ O:hexane 1:1 (120 ml). Solid triphenylphosphine oxide was filtered off and the solvent was removed under reduced pressure. The resulting crude material was taken up in minimal CH ₂ Cl ₂ and purified by silica gel chromatography (0-50% EtOAc/Hex) to give isopropyl 2-((tert-butoxycarbonyl)amino)-2-methylpropanoate. 1H NMR (400 MHz, chloroform-d) δ 5.03 (n, J=6.2 Hz, 1H), 1.48 (s, 6H), 1.40 (d, J=6.2 Hz, 9H ), 1.24 (d, J=6.3 Hz, 6H).

Preparation of isopropyl-2-amino-2-methylpropanoate hydrochloride.

Isopropyl 2-((tert-butoxycarbonyl)amino)-2-methylpropanoate (4.09 g, 16.67 mmol) was added to CH2Cl2 (50 mL), 4N was added slowly. HCl in dioxane (50 mmol) and stirred at room temperature. After about 1 hour, it was determined by TLC that the reaction was complete. The solvents were removed under reduced pressure and the crude material was co-evaporated twice with Et ₂ O and then placed under high vacuum to give isopropyl-2-amino-2-methylpropanoate hydrochloride. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.61 (s, 3H), 4.96 (n, J=6.2 Hz, 1H), 1.44 (s, 6H), 1.22 (d, J=6.2 Hz, 6H).

Preparation of isopropyl 2-methyl-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (compound O).

Phenyl dichlorophosphate (0.83 ml, 5.58 mmol) and isopropyl 2-amino-2-methylpropanoate hydrochloride (1.01 g, 5.58 mmol) were added to CH ₂ Cl ₂ (50 ml). The reaction mixture was cooled to 0° C. and TEA (1.61 ml, 11.45 mmol) was added slowly. The cold bath was removed and the reaction mixture was allowed to stir at room temperature. After about 2 hours, it was determined by ³¹ P NMR that the addition of the amino acid was complete. P-nitrophenol (0.74 g, 5.30 mmol) was added followed by TEA (0.81, 5.84 mmol). The reaction mixture was allowed to stir at room temperature. After about 2 hours, it was determined by LC/MS that the reaction was complete. The reaction mixture was diluted with Et ₂ O and the TEA-HCl salts were filtered off.

The crude material was concentrated and purified by silica gel chromatography (050% EtOAc/Hex) to give compound O. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.42-8.19 (m, 2H), 7.55 -7.43

- 47 039561 (m, 2H), 7.39 (dd, J=8.6, 7.2 Hz, 2H), 7.30-7.12 (m, 3H), 6.53 (d, J= 10.1 Hz, 1H), 4.82 (hept, J=6.3 Hz, 1H), 1.38 (s, 6H), 1.09 (d, J=6.3, 6H). ³¹ P NMR (162 MHz, DMSO-d ₆ ) δ -2.84. LC/MS: t _R =1.73 min, MS m/z=422.92 [M+1]; LC system: Thermo Accela 1250 for 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 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.

Preparation of isopropyl 2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]Ίriazin-7-yl)-5-cyano- 3,4dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-2-methylpropanoate (compound 31).

Compound 1 (66 mg, 0.23 mmol) was added to NMP (2.0 ml). The mixture was cooled to about 0° C. and tBuMgCl (1.0 M in THF, 0.57 mL, 0.57 mmol) was added slowly. The reaction mixture was allowed to stir at about 0°C for about 30 min, and then a solution of compound O (143 mg, 0.34 mmol) in THF (1.0 ml) was added. The cold bath was removed and the reaction mixture was placed in an oil bath preheated to about 50°C. After about 2 hours the reaction mixture was cooled to room temperature and quenched with acetic acid and methanol. The crude material was concentrated and purified by reverse phase HPLC without modifier to give compound 31. 1H NMR (400 MHz, DMSO-d ₆ ) δ 7.88 (m, 3H), 7.30 (td, J=8.5 , 7.0 Hz, 2H), 7.20-7.04 (m, 3H), 6.87 (d, J=4.5, 1H), 6.80 (d, J=4.5 Hz, 1H), 6.27 (d, 6.1 Hz, 1H), 5.75 (t, J=9.1 Hz, 1H), 5.34 (d, J=5.7 Hz, 1H), 4 .81 (n, J=6.3 Hz, 1H), 4.71-4.50 (m, 1H), 4.23 (m, 2H), 4.11 (m, 1H), 4.03- 3.83 (m, 1H), 1.37-1.23 (m, 6H), 1.18-1.04 (m, 6H). ³¹ R NMR (162 MHz, DMSO) δ 2.47, 2.43. LC/MS: t _R = 1.08 min, MS m/z = 575.06 [M+1]; LC system: Thermo Accela 1250 for 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 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 35 (S)-2-ethylbutyl-2-(((S)- (((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4 ]triazin-7-yl)-5cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphopyl)amino)propanoate (32).

Preparation of (S)-2-ethylbutyl-2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine -7-yl)-5cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate is described below.

Preparation of (3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)dihydrofuran-2(3H)-one.

(3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol (15.0 g) was combined with MTBE (60.0 ml), KBr (424, 5 mg), an aqueous solution of K ₂ HPO ₄ (2.5 M, 14.3 ml) and TEMPO (56 mg). This mixture was cooled to about 1°C. An aqueous solution of sodium hypochlorite (7.9 wt.%) was slowly introduced in portions until complete consumption of the starting material, noted using the starch/iodide test. The layers were separated and the aqueous layer was subjected to MTBE extraction. The combined organic phase was dried over MgSO ₄ and concentrated under reduced pressure to give the title product as a solid.

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

- 48 039561

N-iodosuccinimide (17 01 g; 75.6 mmol) while maintaining the temperature of the contents at about 0°C. After completion of the reaction (after about 3 hours at about 0°C), the reaction mixture was transferred into 1 M aqueous sodium hydroxide solution (11 g NaOH and 276 ml water) while maintaining the temperature of the contents at about 20-30°C. The resulting suspension was shaken at about 22° C. for 1.5 hours and then filtered. The solids were washed with water (50 ml) and dried at about 50° C. under vacuum to give 4-amino-7-iodopyrrolo[2,1-f][1,2,4]triazine as a solid. 1H NMR (400 MHz, DMSO-d ₆ ) δ 7.90 (s, 1H), 7.78 (br s, 2H), 6.98 (d, J=4.4 Hz, 1H), 6, 82 (d, J=4.4 Hz, 1H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 155.7, 149.1, 118.8, 118.1, 104.4, 71.9. MS m/z=260.97 [M+H].

Preparation of (3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy )methyl)tetrahydrofuran-2-ol using (4-amino-7-iodopyrrolo[2.1-1][1,2,4]triazine).

Iodine-substituted base (iodobase) 2 (81 g) and THF (1.6 l) were added to the reactor under nitrogen atmosphere. The resulting solution was cooled to about 5° C. and TMSCl (68 g) was added. PhMgCl (345 ml, 1.8 M in THF) was then added slowly while maintaining the internal temperature at approximately <5°C. The reaction mixture was stirred at about 0°C for 30 min and then cooled to about -15°C. Slowly made iPrMgCl-LiCl (311 ml, 1.1 M in THF) while maintaining the internal temperature below about -12°C. After about 10 min stirring at about -15°C, the reaction mixture was cooled to about -20°C and made a solution of lactone 1 (130 g) in THF (400 ml). The reaction mixture was then shaken at about -20° C. for about 1 hour and quenched with AcOH (57 ml). The reaction mixture was heated to about 0°C and the pH was adjusted to 7-8 with an aqueous solution of NaHCO ₃ (5 wt.%, 1300 ml). Then the reaction mixture was diluted with EtOAc (1300 ml) and the organic and aqueous layers were separated. The organic layer was washed with 1N. HCl (1300 ml), aqueous NaHCO ₃ solution (5 wt.%, 1300 ml) and brine (1300 ml), and then dried over anhydrous Na ₂ SO ₄ and concentrated to dryness. Purification by silica gel column chromatography using a MeOH/EtOAc gradient afforded the title product.

Preparation of ((2S)-2-ethylbutyl-2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate) (mixture of Sp and Rp).

L-alanine 2-ethylbutyl ester hydrochloride (5.0 g, 23.84 mmol) was combined with methylene chloride (40 ml), cooled to about -78° C. and phenyl dichlorophosphate (3.65 ml, 23.84 mmol) was added. Triethylamine (6.6 ml, 47.68 mmol) was added over about 60 minutes at about -78°C and the resulting mixture was stirred at ambient temperature for 3 hours. The reaction mixture was cooled to about 0°C and pentafluorophenol (4 .4 g, 23.84 mmol). Triethylamine (3.3 mL, 23.84 mmol) was added over about 60 minutes. The resulting mixture was stirred for about 3 hours at ambient temperature and concentrated under reduced pressure. The residue was dissolved in EtOAc, washed several times with aqueous sodium carbonate solution and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of EtOAc and hexane (0 to 30%). Fractions containing product were concentrated under reduced pressure to give (2S)-2-ethylbutyl-2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate as a solid. 1H NMR (400 MHz, chloroform-d) δ 7.41-7.32 (m, 4H), 7.30-7.17 (m, 6H), 4.24-4.16 (m, 1H), 4.13-4.03(m, 4H), 4.01-3.89(m, 1H), 1.59-1.42(m, 8H), 1.40-1.31(m, 8H ), 0.88 (t,

- 49 039561

J=7.5 Hz, 12H). ³¹ R NMR (162 MHz, chloroform-d) δ -1.52. ¹⁹ F NMR (377 MHz, chloroform-d) δ -153.63, 153.93 (m), -160.05 (td, J=21.9, 3.6 Hz), -162.65 (qd, J=22.4, 20.5, 4.5 Hz). MS m/z=496 [M+H].

Preparation of ((2S)-2-ethylbutyl-2-(((n-perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate).

i) PhOP(O)CI ₂ , E1 ₃ MDCM n)4-nitrophenol, Et ₃ N, DCM

1 ₂ "HC1"

0Et ₃ N, iPAc/n-heptane

F F

L-alanine 2-ethylbutyl ester hydrochloride (40.10 g, 0.191 mmol) was dissolved in dichloromethane (533 g) and the solution was cooled with stirring to about -15° C. under N ₂ (g). Phenyl dichlorophosphate (40.32 g, 0.191 mol) was added followed by the slow addition of triethylamine (41.58 g, 0.411 mmol) and the reaction mixture was stirred at about -15° C. for about 1.5 hours. Pentafluorophenol (35.14 g , 0.191 mol) and then triethylamine (19.23 g, 0.190 mol) and the reaction mixture was stirred for about 2 hours. The reaction mixture was heated to about 0° C. and 0.5 M HCl (279.19 g) was added. The mixture was heated to about 22°C and the organic layer was separated and washed with 5% aqueous KHCO3 (281 g) and then with water (281 g). An aliquot of the organic layer (453.10 g out of a 604.30 g solution) was concentrated to a volume of about 120 ml, isopropyl acetate (157 g) was added and the solution was concentrated to dryness. The residue was dissolved in isopropyl acetate (158 g). The resulting solution was concentrated to a volume of about 120 ml and the temperature was brought to about 45°C. n-heptane (165 g) was added and the mixture was cooled to 22°C over about 1 hour. n-heptane (167 g) was added and the mixture was cooled to about 0°C. Triethylamine (2.90 g, 0.0287 mol) was added and the resulting mixture was stirred at 0°C for about 17 hours. The mixture was filtered, the solids were washed with n-heptane (145 g) and the solids were dried under vacuum at about 40° C for about 15 hours to give 2-ethylbutyl-((S)-(pentafluorophenoxy)(phenoxy)phosphoryl)-L-alaninate.

Preparation of 2-ethylbutyl-((S)-(4-nitrophenoxy)(phenoxy)phosphoryl)-L-alaninate.

i) PhOP(O)CI _? , Et ₃ N, iPAc''M-nitposenol. Et ₃ N.iPAc

1g*HC1 shDBU, iPAc/n-heptane

no ₂

OPh

A suspension of L-alanine 2-ethylbutyl ester hydrochloride (20.08 g, 95.8 mmol) and isopropyl acetate (174 g) was cooled with stirring to about -20°C. Phenyl dichlorophosphate (20.37 g, 96.5 mmol) was added followed by the slow addition of triethylamine (20.97 g, 207.2 mmol) and the resulting mixture was stirred at about -20° C. for about 1 hour. 4-nitrophenol ( 13.23 g, 95.1 mmol) followed by slow addition of triethylamine (10.01 g, 98.8 mmol) and the reaction mixture was stirred for about 1.5 h. 5 M HCl (140 g). The organic layer was separated and washed with 5% Na ₂ CO ₃ (2x100 g) and 10% NaCl (2x100 g). The organic layer was then concentrated to a volume of about 80 ml and isopropyl acetate (4 g) was added followed by n-heptane (110 g). Product seeds (0.100 g) were added followed by a second portion of n-heptane (110 g) and the mixture was cooled to about 0°C. 1,8 Diazabicycloundec-7-ene (1.49 g, 9.79 mmol) was added and the resulting mixture was stirred at about 0° C. for about 21 hours. The resulting solids were filtered and washed first with n-heptane (61 g) and then H ₂ O (2x100 g). The solids were stirred with H ₂ O (200 g) for about 1.5 h, filtered and washed with H ₂ O (3x100 g) and then n-heptane (61 g). The resulting solids were dried under vacuum at about 40° C. for about 19 hours to give 2-ethylbutyl-((S)-(4nitrophenoxy)(phenoxy)phosphoryl)-L-alaninate.

Preparation of the title compound (mixture of Sp and Rp).

F but

iBuMgCl, DMF

Nucleoside (29 mg, 0.1 mmol), phosphonamide (60 mg, 0.12 mmol) and N,N-dimethylformamide (2 ml) were combined at ambient temperature. tert-Butyl magnesium chloride (1M in THF, 0.15 ml) was added slowly. After about 1 hour, the reaction mixture was diluted with ethyl acetate, washed with aqueous citric acid (5 wt%), saturated aqueous NaHCO ₃ and brine. The organic phase was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of methanol and CH2Cl2 (0 to 5%). Fractions containing product were concentrated under reduced pressure to give the title product.

Preparation of (3aR,4R,6R,6aR)-4-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-6-(hydroxymethyl)-2,2dimethyltetrahydrofuro[3, 4-d][1,3]dioxole-4-carbonitrile.

To a mixture of (2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2-carbonitrile (5.8 g, 0.02 mol), 2,2-dimethoxypropane (11.59 ml, 0.09 mol) and acetone (145 ml) at ambient temperature was added sulfuric acid (18 M, 1.44 ml). The mixture was heated to about 45°C. After about 30 minutes, the mixture was cooled to ambient temperature and sodium bicarbonate (5.8 g) and water (5.8 ml) were added. After 15 minutes the mixture was concentrated under reduced pressure. The residue was taken up in ethyl acetate (150 ml) and water (50 ml). The aqueous layer was subjected to extraction with ethyl acetate (2x50 ml). The combined organic phase was dried over sodium sulfate and concentrated under reduced pressure to give crude (2R,3R,4S,5R)-2-(4aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)- 3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile. ¹ H NMR (400 MHz, CD3OD) δ 7.84 (s, 1H), 6.93 (d, J=4.6 Hz, 1H), 6.89 (d, J=4.6 Hz, 1H) , 5.40 (d, J=6.7 Hz, 1H), 5.00 (dd, J=6.7, 3.3 Hz, 1H), 4.48-4.40 (m, 1H), 3.81-3.72 (m, 2H), 1.71 (s, 3H), 1.40 (s, 3H). MS m/z=332.23 [M+1].

Preparation of TsOH salt (3aR,4R,6R,6aR)-4-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-6-(hydroxymethyl)-2,2- dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-carbonitrile.

To a mixture of (2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2-carbonitrile (5.0 g, 17.2 mmol, 1.0 eq.), 2,2-dimethoxypropane (10.5 ml, 86 mmol, 5.0 eq.) and acetone (25 ml) at ambient temperature, ptolylsulfonic acid (3.59 g, 1.1 eq.) was added. The resulting mixture was stirred at ambient temperature. After about 30 minutes, isopropyl acetate (25 ml) was added over about one hour. The resulting suspension was filtered and washed with heptane:isopropyl acetate 2:1 (25 ml). The product was dried under vacuum at about 40°C.

Preparation of (3aR,4R,6R,6aR)-4-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-6-(hydroxymethyl)-2,2dimethyltetrahydrofuro[3, 4-d][1,3]dioxole-4-carbonitrile.

To a mixture of (2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2-carbonitrile (5 g, 17.2 mmol, 1.0 eq.), 2,2-dimethoxypropane (10.5 ml, 86 mmol, 5.0 eq.) and acetone (25 ml) at ambient temperature n-tolylsulfonic acid (3.59 g, 1.1 eq.) was added to the medium. The resulting mixture was stirred at ambient temperature. After 30 minutes, isopropyl acetate (25 ml) was added over 1 hour. The resulting suspension was filtered and washed

- 51 039561 with a mixture of heptane:isopropyl acetate 2:1 (25 ml). The product was dried under vacuum at 40°C. The isolated solid was added to the reactor and 5% K ₂ CO ₃ solution (50 ml) and ethyl acetate (50 ml) were added. The layers were separated and the aqueous layer was washed with ethyl acetate (25 ml). The combined organic layers were washed with water (25 ml) and then concentrated to about 25 ml. The reactor was filled with isopropyl acetate (25 ml) and concentrated to about 25 ml. The reactor was refilled with isopropyl acetate (25 ml) and concentrated to 25 ml. The resulting solution was seeded to obtain a thick suspension. Heptane (25 ml) was added to this suspension over 1 hour. The resulting suspension was filtered and washed with heptane:isopropyl acetate 2:1 (25 ml). The product was dried under vacuum at 40°C. () (2R,3R,4S,5R)-2(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran -2 carbonitrile. 1H NMR (400 MHz, CD3OD) δ 7.84 (s, 1H), 6.93 (d, J=4.6 Hz, 1H), 6.89 (d, J=4.6 Hz, 1H), 5.40 (d, J=6.7 Hz, 1H), 5.00 (dd, J=6.7, 3.3 Hz, 1H), 4.48-4.40 (m, 1H), 3 .81-3.72 (m, 2H), 1.71 (s, 3H), 1.40 (s, 3H). MS m/z = 332.23 [M+1].

Preparation of (2S)-2-ethylbutyl-2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl )-5cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate.

Acetonitrile (100 mL) was combined with (2S)-2-ethylbutyl-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (9.6g, 21.31mmol), alcohol as substrate (6 .6 g, 0.02 mol), magnesium chloride (1.9 g, 19.91 mmol) at ambient temperature. The mixture was shaken for about 15 minutes and N,N-diisopropylethylamine (8.67 mL, 49.78 mmol) was added. After about 4 h, the reaction mixture was diluted with ethyl acetate (100 ml), cooled to about 0°C and combined with an aqueous solution of citric acid (5 wt.%, 100 ml). The organic phase was washed with aqueous citric acid (5 wt%, 100 ml) and saturated aqueous ammonium chloride (40 ml), aqueous potassium carbonate (10 wt%, 2x100 ml) and saturated aqueous brine (100 ml). The organic phase was dried over sodium sulfate and concentrated under reduced pressure to give the crude product. 1H NMR (400 MHz, CD3OD) δ 7.86 (s, 1H), 7.31-7.22 (m, 2H), 7.17-7.09 (m, 3H), 6.93-6, 84 (m, 2H), 5.34 (d, J=6.7 Hz, 1H), 4.98 (dd, J=6.6, 3.5 Hz, 1H), 4.59-4.50 (m, 1H), 4.36-4.22 (m, 2H), 4.02 (dd, J=10.9, 5.7 Hz, 1H), 3.91 (dd, J=10.9 , 5.7 Hz, 1H), 3.83 (dc, J=9.7, 7.1 Hz, 1H), 1.70 (s, 3H), 1.50-1.41 (m, 1H) , 1.39 (s, 3H), 1.36-1.21 (m, 7H), 0.86 (t, J=7.4 Hz, 6H). MS m/z=643.21 [M+1].

Preparation of (S)-2-ethylbutyl-2-(((S)-(((2R,3 S,4R,5R)-5-(4-amino-pyrrolo[2,1-f] [1,2,4] triazin-7-yl)-5cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (compound 32).

The crude acetonide (12.85 g) was combined with tetrahydrofuran (50 ml) and concentrated under reduced pressure. The residue was dissolved in tetrahydrofuran (100 ml), cooled to about 0° C. and concentrated HCl (20 ml) was added slowly. The mixture was allowed to warm up to ambient temperature. After consumption of the starting acetonide, determined by HPLC analysis, water (100 ml) was added, followed by saturated aqueous sodium bicarbonate (200 ml). The mixture was subjected to extraction with ethyl acetate (100 ml), the organic phase was washed with saturated aqueous brine (50 ml), dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of methanol and ethyl acetate (0 to 20%). Fractions containing product were concentrated under reduced pressure to give the title product.

Preparation of (S)-2-ethylbutyl-2-(((S)-(((2R,3 S,4R,5R)-5-(4-amino-pyrrolo[2,1-f] [1,2,4] triazin-7-yl)-5cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (compound 32).

- 52 039561

Into a vial containing (S)-2-ethylbutyl-2-(((S)-(((3aR,4R,6R,6aR)-6-(4-aminopyrrolo[2,1f][1,2,4] triazin-7-yl)-6-cyano-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (30 mg, 0, 05 mmol), 80% aqueous formic acid solution (1.5 ml) was added. After 18 hours at about 20°C, complete conversion was confirmed by HPLC and LC/MS. MS (m/z)=603 (M+1)+.

Preparation of (S)-2-ethylbutyl-2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine -7-yl)-5cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (compound 32) using direct coupling.

To a mixture of (2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2-carbonitrile (0.5 g, 2 mmol), (S)-2-ethylbutyl-2-(((S)-(4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (0.9g, 2 mmol) and MgCl ₂ (0.2 g, 2 mmol) were added N,N-dimethylacetamide (10 ml). The resulting mixture was heated to about 30°C with constant stirring. Then N,N-diisopropylethylamine (0.7 ml, 4 mmol) was added slowly and the reaction mixture was stirred for about 6 h. Water (H2O) (10 ml) was added followed by 2-MeTHF (10 ml) and the organic and water phase. The aqueous layer was then back extracted with 2-MeTHF (10 ml). The organic layer was combined and washed with 10 wt.% citric acid solution (10 ml) and then with 10 wt.% solution of K ₂ CO ₃ (10 ml) and H 2 O (10 ml). Before separating the layers, a small amount of brine was added to dissolve the emulsions in the wash water. The organic layer was evaporated to dryness to give 0.65 g of a foam. Then added iPrOAc (2.6 ml) and the mixture was heated to about 40°C to ensure dissolution. The solution was cooled to about 20°C and the resulting mixture was stirred for about 3 days. Solids were isolated by filtration and the filter cake was washed with a small amount of iPrOAc. The solids were dried to give (S)-2-ethylbutyl-2(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2, 4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate.

To a mixture of (2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2-carbonitrile (0.2 g, 0.7 mmol), (S)-2-ethylbutyl-2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (0.3g, 0.7 mmol) and MgCl ₂ (0.1 g, 1 mmol) were added N,Ndimethylacetamide (4 ml). The resulting mixture was heated to about 30°C with constant stirring. Then N,N-diisopropylethylamine (0.3 ml, 2 mmol) was added slowly and the reaction mixture was stirred for 5 hours. The conversion to the product was confirmed by HPLC analysis.

Preparation of (3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-bis((tert-butyldimethylsilyl)oxy)- 5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-ol.

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A solution of 7-iodopyrrolo[2,1-1][1,2,4]triazine-4-amine (13.9 g, 53.5 mmol) in THF (280 ml) was obtained. The solution was cooled to about 0° C. and TMSCl (13.6 ml, 107 mmol) was added. The reaction mixture was stirred for about 20 minutes and then PhMgCl (2M in THF; 53.5 ml, 56.8 mmol) was added while maintaining the internal temperature below about 5°C. The reaction mixture was shaken at about 0°C for about 30 minutes and then cooled to about -20°C. Then added iPrMgClLiCl (1.3 M in THF, 43.1 ml, 56 mmol) while maintaining the internal temperature below about 15°C. The reaction mixture was shaken for about 30 minutes at about -20°C.

In a separate flask, a solution of (3R,4R,5R)-3,4-bis((tert-butyldimethylsilyl)oxy)-5-(((m-tert-butyldimethylsilyl)oxy)methyl)dihydrofuran-2(3H)-one (25.0 g, 50.9 mmol, 0.83 eq.) in LaCl ₃ -2LiCl (0.6 M in THF, 85 ml, 50.9 mmol). Then, this solution was transferred to the solution containing the Grignard reagent while maintaining the internal temperature below -20°C. The resulting reaction mixture was shaken at about -20°C for about 4 hours.

The reaction was quenched with 1 M HCl (140 ml) and the mixture was warmed to ambient temperature. EtOAc (140 ml) was added and the organic and aqueous phases were separated. The aqueous layer was subjected to extraction with EtOAc (200 ml). The combined EtOAc layers were subjected to extraction successively with saturated aqueous NaHCO ₃ (2x200 ml), water (200 ml) and brine (200 ml). The organic layer was concentrated and then purified by silica gel chromatography (30% EtOAc/hexane) to give (3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine -7-yl)-3,4-bis((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-ol. 1H NMR (300 MHz, CDCl ₃ ) δ 8.15-7.88 (m, 1H), 7.51 (d, J=4.8 Hz, 0.5H), 7.02-6.92 (m , 0.5H), 6.65-6.57 (m, 1H), 5.665.24 (m, 3H), 4.49-3.50 (m, 4H), 0.97-0.78 (26H ), 0.65 (s, 1.5H), 0.19-0.00 (m, 15.5H), -0.22 (s, 1H), -0.55 (s, 1H). MS m/z=626 (M+H).

Preparation of (2R,3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-bis((tert-butyldimethylsilyl)oxy )-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile.

A solution of (3R,4R,5R)-2-(4-αminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-bis((tert-butyldimethylsilyl)oxy)- 5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-ol (1.50 g, 2.40 mmol) in CH ₂ Cl ₂ (15 ml) was cooled to about -40°C. Trifluoroacetic acid (0.555 ml, 7.20 mmol) was added while maintaining the temperature below -20°C. In a separate flask, trimethylsilyl trifluoromethanesulfonate (2.60 ml, 14.4 mmol) was added to 5 ml CH ₂ Cl ₂ (5 ml) at about 15°C followed by trimethylsilyl cyanide (1.92 ml, 14.4 mmol) and the solution was cooled down to about -30°C. The cooled solution was added to a solution of (3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-bis((tert-butyldimethylsilyl)oxy )-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-ol while maintaining the temperature below -25°C. The reaction mixture was stirred for 15 min at about 30°C. The reaction was quenched with triethylamine (3.34 ml, 24.0 mmol) and the mixture was heated to about 0°C. Water (50 ml) was added while maintaining the temperature below about 20°C. When the addition was complete, the mixture was stirred for 15 minutes at room temperature. The layers were separated and the organic layer was washed successively with KOH (20 ml), water (20 ml) and brine (20 ml). The organic layer was dried over Na ₂ SO ₄ , concentrated and then purified by silica gel chromatography (30% EtOAc/hexane) to give the title product as a 3.8:1 mixture of diastereomers. The mixture was further purified by preparative HPLC (0 to 95% ACN in water) to give the title product as a single diastereomer. 1H NMR (400 MHz, DMSO-d ₆ ) δ 8.147.92 (m, 2H), 7.89 (s, 1H), 6.95 (d, J=4.8 Hz, 1H), 6.88 ( d, J=4.4 Hz, 1H), 5.27 (d, J=4.6 Hz, 1H), 5.10 (dd, J=7.7, 4.6 Hz, 1H), 4, 31 (dd, J=4.7, 1.4Hz, 1H), 4.12 (ddd, J=5.9, 4.1, 1.4Hz, 1H), 3.80-3.69 ( m, 1H), 3.56 (td, J=7.8, 3.9 Hz, 1H), 0.93 (s, 9H), 0.75 (s, 9H), 0.11 (s, 3H ), 0.09 (s, 3H), -0.15 (s, 3H), -0.62 (s, 3H). MS m/z=520 (M+H).

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Preparation of (S)-2-ethylbutyl-2-(((S)-(((2R,3R,4R,5R)-5-(4-aminopyrrolo[2.1-f][1,2,4]Ίpiazine-7 -yl)3,4-bis((tert-butyldimethylsilyl)oxy)-5-cyanothetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate.

To the mixture hydroxy)-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile (16mg, 0.03mmol), (S)-2-ethylbutyl-2-(((S)(4-nitrophenoxy)(phenoxy)phosphoryl)amino) propanoate (17 mg, 0.04 mmol) and MgCl ₂ (4 mg, 0.05 mmol) were added THF (0.3 ml). The resulting mixture was heated to about 50°C with constant stirring. K,K-diisopropylethylamine (0.013 ml, 0.08 mmol) was then added and the reaction mixture was stirred for 21 h. Conversion to product was confirmed by UHPLC and LC/MS analysis. MS m/z=831 (M+H).

A solution of (2R,3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]Ίpiazin-7-yl)-3,4-bis((Ίret-butyldimethylsilyl)oxy )-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile (16 mg, 0.03 mmol) in THF (0.3 ml) was cooled to -10°C. tBuMgCl (0.07 mL, 0.07 mmol) was added dropwise followed by a solution of (S)-2-ethylbutyl-2(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (22 mg, 0 .04 mmol) in THF (0.15 ml). The reaction mixture was heated to 5° C. and stirred for 16 hours. The reaction was quenched with MeOH, concentrated and then purified by silica gel chromatography (EtOAc/hexane) to give the title product. 1H NMR (400 MHz, CDCl ₃ ) δ 7.97 (s, 1H), 7.38-7.29 (m, 2H), 7.25-7.21 (m, 2H), 7.21-7 .13 (m, 1H), 7.11 (d, J=4.6 Hz, 1H), 6.65 (d, J=4.6 Hz, 1H), 5.88 (br s, 2H) , 5.35 (d, J=4.4 Hz, 1H), 4.49-4.41 (m, 1H), 4.41-4.35 (m, 1H), 4.32-4.26 (m, 1H), 4.24 (dd, J=4.5, 1.7 Hz, 1H), 4.10-3.99 (m, 2H), 3.96 (dd, J=10.9 , 5.7 Hz, 1H), 3.80-3.72 (m, 1H), 1.48 (g, J=6.2 Hz, 1H), 1.39-1.28 (m, 7H) , 0.96 (s, 9H), 0.85 (t, J=7.5 Hz, 6H), 0.80 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H), -0.13 (s, 3H), -0.56 (s, 3H). ³¹ P NMR (162 MHz, CDC^) δ 2.74 (s). MS m/z=831 (M+H).

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

Crude solution of (S)-2-ethylbutyl-2-(((S)-(((2R,3R,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4] triazin-7-yl)-3,4-bis((tert-butyldimethylsilyl)oxy)-5-cyanothetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate was cooled to about 0°C and concentrated HCl was added slowly (0.05 ml, 0.62 mmol). The reaction mixture was stirred for about 72 hours at about 20°C. The conversion to product was confirmed by UPLC and LC/MS analysis. MS m/z=603 (M+H).

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Solution of (S)-2-ethylbutyl-2-(((S)-(((2R,3R,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine -7-yl)-3,4bis((tert-butyldimethylsilyl)oxy)-5-cyanothetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate in fluoride or acid can provide deprotection to form a solution (S )-2-ethylbutyl-2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7- yl)-5-cyano-3,4dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate.

Representative fluorides include, but are not limited to, TBAF, KF, pyridinium hydrofluoride, triethylammonium hydrofluoride, hydrogen fluoride, hydrochloric acid, toluenesulfonic acid, or any other suitable fluoride source. Exemplary acids include, but are not limited to, those listed in Greene, TW; Wuts, PGM Protective Groups In Organic Synthesis, 4th ^Ed ., John Wiley & Sons: New York, 2006.

Example 35-a. ((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran- 2-yl)methoxy)oxidophosphoryl)alaninate (compound 35).

2-ethylbutyl-((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5- cyano-3,4dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (130 mg, 0.216 mmol) was dissolved in a mixture of acetonitrile (6 ml) and water (2 ml). Aqueous sodium hydroxide solution (2N, 0.5 ml) was added dropwise over 5 minutes at room temperature and the reaction mixture was stirred. After 2 hours the resulting mixture was concentrated under reduced pressure and the residue was purified by HPLC on a C18 column eluting with water to give the desired product as the bis sodium salt. Ή NMR (400 MHz, D ₂ o) δ 7.79 (s, 1H), 6.86 (d, J=4.7 Hz, 1H), 6.80 (d, J=4.7 Hz, 1H ), 4.86 (d, J=5.4 Hz, 1H), 4.40-4.34 (m, 1H), 4.30 (dd, J=5.3, 3.0 Hz, 1H) , 3.75 (cdd, J=11.6, 4.5, 3.1 Hz, 2H), 3.20 (dc, J=8.6, 7.1 Hz, 1H), 0.86 (d , J=7.0 Hz, 3H). ³¹ P NMR (162 MHz, D2O) δ 7.30. LC/MS m/z 442.95 [M+H]. HPLC (gradient 2-98% MeCN-H ₂ O with 0.1% TFA as modifier over 8.5 min, 1.5 ml/min, column: Phenomenex Kinetex C18, 2.6 µm 100 A, 4, 6x100 mm) t _R =2.694 min.

A. Antiviral activity.

Another aspect of the invention relates to methods for inhibiting viral infections, comprising the step of treating a sample or subject that is expected to require such inhibition with a composition according to the present invention.

In the context of the present invention, samples suspected of containing a virus include natural or artificial materials such as living organisms; tissues 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; samples of food, water or air; samples of bioproducts, such as extracts of cells, in particular recombinant cells that synthesize the desired glycoprotein; etc. Typically, the sample is expected to have an organism that induces a viral infection, often a pathogenic organism such as a tumor virus. Samples may be contained in any medium, including water and mixtures of organic solvents and water. Samples include living organisms such as humans and artificial materials such as cell cultures.

If desired, the antiviral activity of a compound of the present invention after application of the composition can be observed by any method, including direct and indirect methods for detecting such activity. The present invention includes quantitative, qualitative and semi-quantitative methods for determining such activity. 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 of the present invention can be measured using standard screening protocols that are known in the art. For example, the antiviral activity of a compound can be measured using the following general protocols.

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Virus cell line Tablet Format Number cells Multiplicity of infection (PFU per cell) Anise incubators (days) Definition Values EBOV (Zaire) Hela 384 4000 0.5 2 HCS EU50 EBOV (Zaire) HFF-1 2 HCS EBOV-GFP Huh-7 96 10000 0.1 4 GFP EBOV-GFP HMVEC-TERT GFP EBOV-LUC Huh-7 LUC MARV-GFP Huh-7 GFP NiV Hela CPE NiV-GFP HMVEC-TERT GFP NiV-LUC HMVEC-TERT LUC

EBOV: Ebola virus, Zaire species EBOV-GFP: Ebola reporter virus expressing green fluorescent protein EBOV-LUC: Ebola reporter virus expressing luciferase MARV-GFP: Marburg virus expressing green fluorescent protein NiV: Nipah virus NiV-GFP: Nipah reporter virus expressing green fluorescent protein NiV-LUC: Nipah reporter virus expressing luciferase HCS: Simultaneous multiparameter imaging (Ebola virus GP immunostaining) GFP: Green fluorescent protein LUC: Luciferase CPE Cytopathic effects as determined by the CellTiterGlo (CTG) reagent Hela: Hela epithelial cell (cervical carcinomas) HFF-1: Human foreskin fibroblast Huh-7: Hepatocyte HVMEC-TERT: Human vascular endothelial cells immortalized with telomerase catalytic protein

Example 36 Ebola virus antiviral activity assay and cytotoxicity assay.

The antiviral activity of compound 1 and compound 9 was determined against Ebola virus (EBOV), Marburg virus (MARV) (Table 2) and Nipah virus (MV) (Table 3) using fully replicating reporter viruses expressing luciferase or green fluorescent protein (GFP) (Uebelhoer, L.S., 2014. AVR; Hoenen, T., 2013. AVR). Also, the antiviral activity of compound 1 and compound 9 was determined against Ebola virus (EBOV), Marburg virus (MARV) (Table 2a), using fully replicating reporter viruses expressing luciferase or green fluorescent protein (GFP) (Uebelhoer, L.S., 2014. AVR Hoenen, T. 2013. AVR). All studies were conducted under biosafety level 4 (BSL-4) conditions at the Centers for Disease Control and Prevention (CDC). Ebola virus antiviral activity assays were performed in primary human endothelial microvascular cells immortalized with telomerase catalytic protein (HMVEC-TERT) and Huh-7 cells (Shao, R., 2004, BBRC). Antiviral activity against the Nipah virus was determined in HMVEC-TERT and Hela cells.

Antiviral activity assays were performed in 96-well plates. Received eighty concentrations of the compound by 3-fold serial dilution in the medium and 100 μl per well of each diluted solution in triplicate was transferred to the plates, which were previously sown in a monolayer of cells. The plates were transferred to BSL-4 conditions and an appropriately diluted virus solution prepared in cell culture medium and for which the virus titer was preliminarily determined was added to the plates loaded with cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as controls for 0% and 100% virus inhibition, respectively. After infection, assay plates were incubated for 3-4 days in a cell culture incubator. After incubation, virus replication was determined using an Envision plate reader by direct fluorescence of GFP-expressing reporter viruses or luciferase-expressing reporter viruses after appropriate addition of lu substrate.

- 57 039561 ciferase. For virus yield analysis, media from infected cells were removed and a portion was used to quantify viral RNA by quantitative reverse transcription polymerase chain reaction (RT-qPCR). The remaining media were serially diluted and the amount of infectious virus was quantified using the diluted media to infect fresh cell monolayers, thus determining the tissue culture infectious dose that caused 50% cytopathic effects (TCID50) using the Cell TiterGlo reagent (Promega, Madison, WI) . To analyze the virus-induced cytopathic effect (CPE), the viability of infected cells was determined using the Cell TiterGlo reagent.

Percent inhibition was calculated for each test concentration relative to controls corresponding to 0% and 100% inhibition, and the EC ₅₀ value for each compound was determined by non-linear regression as the effective compound concentration that inhibited viral replication by 50%.

Example 37 EBOV-GFP, HMVEC-TERT cells.

HMVEC-TERT cells were seeded in 96-well plates. Eight to ten concentrations of the compound were obtained by 3-fold serial dilution and 100 μl per well of each diluted solution in triplicate was transferred to plates, on which HMVEC-TERT cells were previously seeded in a monolayer. The plates were transferred to BSL-4 conditions, and an appropriately diluted solution of EBOVGFP virus, which was prepared in cell culture medium and for which the virus titer was previously determined, was added to the plates loaded with cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as controls for 0% and 100% virus inhibition, respectively. After infection, assay plates were incubated for 3-4 days in a cell culture incubator. After incubation, virus replication was determined using an Envision plate reader by direct fluorescence to determine GFP expression from the reporter virus. Percent inhibition was calculated for each test concentration relative to controls corresponding to 0% and 100% inhibition, and the EC50 value for each compound was determined by non-linear regression as the effective compound concentration that inhibited virus replication by 50%.

Example 38 EBOV-GFP, Huh-7 cells.

Huh-7 cells were seeded in 96-well plates. Eight to ten concentrations of the compound were obtained by 3-fold serial dilution, and 100 μl per well of each solution was transferred in triplicate to plates on which Huh-7 cells were previously seeded as a monolayer. The plates were transferred to BSL-4 conditions and an appropriately diluted solution of EBOV-GFP virus, which was prepared in cell culture medium and for which the virus titer was preliminarily determined, was added to the plates loaded with cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as controls for 0% and 100% virus inhibition, respectively. After infection, assay plates were incubated for 3-4 days in a cell culture incubator. After incubation, virus replication was determined using an Envision plate reader by direct fluorescence to determine GFP expression from the reporter virus. Percent inhibition was calculated for each test concentration relative to controls corresponding to 0% and 100% inhibition, and the EC ₅₀ value for each compound was determined by non-linear regression as the effective compound concentration that inhibited virus replication by 50%.

Example 39 EBOV-Luc, Huh-7 cells.

Huh-7 cells were seeded in 96-well plates. Eight to ten concentrations of the compound were obtained by 3-fold serial dilution, and 100 μl per well of each solution was transferred in triplicate to plates, on which cells were previously seeded in a monolayer. The plates were transferred to BSL-4 conditions and an appropriately diluted solution of EBOV-Luc virus, which was prepared in cell culture medium and for which the virus titer was preliminarily determined, was added to the plates loaded with cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as controls for 0% and 100% virus inhibition, respectively. After infection, assay plates were incubated for 3-4 days in a cell culture incubator. After incubation, virus replication was determined using an Envision plate reader after appropriate addition of luciferase substrate. Percent inhibition was calculated for each test concentration relative to controls corresponding to 0% and 100% inhibition, and the EC ₅₀ value for each compound was determined by non-linear regression as the effective compound concentration that inhibited virus replication by 50%.

Example 40 MARV-GFP, Huh-7 cells.

Huh-7 cells were seeded in 96-well plates. Eight to ten concentrations of the compound were obtained by 3-fold serial dilution, and 100 μl per well of each solution was transferred in triplicate to plates on which Huh-7 cells were previously seeded as a monolayer. Tablets pe

- 58 039561 was transferred to BSL-4 conditions and an appropriately diluted solution of MARVGFP virus, which was prepared in cell culture medium and for which the virus titer was previously determined, was added. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as controls for 0% and 100% virus inhibition, respectively. After infection, assay plates were incubated for 3-4 days in a cell culture incubator. After incubation, virus replication was determined using an Envision plate reader by direct fluorescence to determine GFP expression from the reporter virus. Percent inhibition was calculated for each test concentration relative to controls corresponding to 0% and 100% inhibition, and the EC ₅₀ value for each compound was determined by non-linear regression as the effective compound concentration that inhibited virus replication by 50%.

Example 41 Ebola virus, Huh-7 cells (RNA).

Huh-7 cells were seeded in 96-well plates. Eight to ten concentrations of the compound were obtained by 3-fold serial dilution, and 100 μl per well of each diluted solution was transferred in triplicate to plates on which Huh-7 cells were previously seeded in a monolayer. The plates were transferred to BSL-4 conditions and an appropriately diluted solution of EBOV virus, which was prepared in cell culture medium and for which the virus titer was previously determined, was added to the plates loaded with cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as controls for 0% and 100% virus inhibition, respectively. After infection, assay plates were incubated for 3-4 days in a cell culture incubator. After incubation, the medium was removed from the infected cells and a portion was used to quantify viral RNA by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Percent inhibition was calculated for each test concentration relative to controls corresponding to 0% and 100% inhibition, and the EC ₅₀ value for each compound was determined by non-linear regression as the effective compound concentration that inhibited virus replication by 50%.

Example 42 Ebola virus, Huh-7 cells (yield).

Huh-7 cells were seeded in 96-well plates. Eight to ten concentrations of the compound were obtained by 3-fold serial dilution, and 100 μl per well of each diluted solution was transferred in triplicate to plates on which Huh-7 cells were previously seeded in a monolayer. The plates were transferred to BSL-4 conditions and an appropriately diluted solution of EBOV virus, which was prepared in cell culture medium and for which the virus titer was previously determined, was added to the plates loaded with cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as controls for 0% and 100% virus inhibition, respectively. After infection, assay plates were incubated for 3-4 days in a cell culture incubator. After incubation, the medium from infected cells was removed and diluted to 10-fold serial dilutions. The amount of infectious virus was determined using diluted infection media for fresh cell monolayers and thus the tissue culture infection dose that caused 50% cytopathic effects (TCID50) was determined using the Cell TiterGlo reagent (Promega, Madison, WI). Percent inhibition was calculated for each test concentration relative to controls corresponding to 0% and 100% inhibition, and the EC50 value for each compound was determined by non-linear regression as the effective compound concentration that inhibited virus replication by 50%.

Example 43 Ebola virus, HeLa cells.

The antiviral activity of the selected compounds was determined against Ebola virus (EBOV), Zaire species, under biosafety level 4 (BSL-4) conditions at the US Army Medical Research Institute for Infections Disease (USAMRIID) ). Hela cells were seeded in 384-well plates at 5000 cells per well. The antiviral activity of each compound was determined in quadruplicate. Eight to ten concentrations of the compound were added in 3-fold serial dilutions directly to cell cultures using the digital dispenser HP300 2 hours before infection. The plates were transferred to BSL-4 conditions and an appropriately diluted virus solution prepared in cell culture medium and for which the virus titer was preliminarily determined was added to the plates loaded with cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as controls for 0% and 100% virus inhibition, respectively. After infection, assay plates were incubated for 2 days in a cell culture incubator. After incubation, cells were fixed in formalin solution and virus replication was determined by quantifying Ebola virus glycoproteins after immunostaining and simultaneous multi-parameter imaging using a Perkin confocal microscopy instrument.

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Elmer Opera. Percent inhibition was calculated for each test concentration relative to controls corresponding to 0% and 100% inhibition, and the EC ₅₀ value for each compound was determined by non-linear regression as the effective compound concentration that inhibited virus replication by 50%.

Example 44 Ebola Virus Macrophage Cultures

The antiviral activity of the selected compounds was determined against Ebola virus (EBOV), Zaire species, under biosafety level 4 (BSL-4) conditions at the US Army Medical Research Institute for Infections Disease (USAMRIID) ). Macrophage cultures were isolated from fresh human peripheral blood mononuclear cells (PBMCs) and differentiated in the presence of 5 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 50 μM β-mercaptoethanol. Media were changed every 2 days and cells adhering to the tissue culture plate were removed after 7 days with 0.5M EDTA in 1x phosphate buffered saline (PBS), concentrated by centrifugation at 200xg for 10 min and placed in 384-well tablets for research in the amount of 40,000 cells per well. The antiviral activity of each compound was determined in quadruplicate. Eight to ten concentrations of the compound were added in 3-fold serial dilutions directly to cell cultures using the digital dispenser HP300 2 hours before infection. The plates were transferred to BSL-4 conditions and an appropriately diluted virus solution prepared in cell culture medium and for which the virus titer was preliminarily determined was added to the plates loaded with cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as controls for 0% and 100% virus inhibition, respectively. After infection, assay plates were incubated for 2 days in a cell culture incubator. After incubation, cells were fixed in formalin solution and virus replication was determined by quantifying Ebola virus glycoproteins after immunostaining and simultaneous multiparameter imaging using a Perkin Elmer Opera confocal microscope. Percent inhibition was calculated for each test concentration relative to controls corresponding to 0% and 100% inhibition, and the EC50 value for each compound was determined by non-linear regression as the effective compound concentration that inhibited virus replication by 50%.

Example 45 Nipah-GFP, HMVEC-TERT cells.

HMVEC-TERT cells were seeded in 96-well plates. Eight to ten concentrations of the compound were obtained by 3-fold serial dilution, and 100 μl per well of each solution was transferred in triplicate to plates on which HMVEC-TERT cells were previously seeded in a monolayer. The plates were transferred to BSL-4 conditions and an appropriately diluted solution of NiV-GFP virus, which was prepared in cell culture medium and for which the virus titer was preliminarily determined, was added to the plates loaded with cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as controls for 0% and 100% virus inhibition, respectively. After infection, assay plates were incubated for 3-4 days in a cell culture incubator. After incubation, virus replication was determined in an Envision plate reader by direct fluorescence to determine GFP expression from the reporter virus. Percent inhibition was calculated for each test concentration relative to controls corresponding to 0% and 100% inhibition, and the EC ₅₀ value for each compound was determined by non-linear regression as the effective compound concentration that inhibited virus replication by 50%.

Example 46 NiV-Luc, HMVEC-TERT.

HMVEC-TERT cells were seeded in 96-well plates. Received eight to ten concentrations of the compound by 3-fold serial dilution and 100 μl per well of each diluted solution in triplicate was transferred to the plates, which were previously seeded in a monolayer of cells. The plates were transferred to BSL-4 conditions and appropriately diluted Niv-Luc virus solution prepared in cell culture medium and pre-determined virus titer was added to the plates loaded with cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as controls for 0% and 100% virus inhibition, respectively. After infection, assay plates were incubated for 3-4 days in a cell culture incubator. After incubation, virus replication was determined using an Envision plate reader after appropriate addition of luciferase substrate. Percent inhibition was calculated for each test concentration relative to controls corresponding to 0% and 100% inhibition, and the EC50 value for each compound was determined by non-linear regression as the effective compound concentration that inhibited virus replication by 50%.

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Example 47 NiV, Hela (yield).

Hela cells were seeded in 96-well plates. Eight to ten concentrations of the compound were obtained by 3-fold serial dilution and 100 μl per well of each diluted solution in triplicate were transferred to plates, on which Hela cells were previously seeded in a monolayer. The plates were transferred to BSL-4 conditions and appropriately diluted Niv virus solution prepared in cell culture medium and for which the virus titer was previously determined was added to the plates loaded with cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as controls for 0% and 100% virus inhibition, respectively. After infection, assay plates were incubated for 4 days in a cell culture incubator. After incubation, the medium from infected cells was removed and diluted in 10-fold serial dilutions. The amount of infectious virus was determined using diluted infection media for fresh cell monolayers and thus the tissue culture infection dose that caused 50% cytopathic effects (TCID50) was determined using the Cell TiterGlo reagent (Promega, Madison, WI). Percent inhibition was calculated for each test concentration relative to controls corresponding to 0% and 100% inhibition, and the EC ₅₀ value for each compound was determined by non-linear regression as the effective compound concentration that inhibited virus replication by 50%.

Example 48 Niv, Hela (RNA).

Huh-7 cells were seeded in 96-well plates. Eight to ten concentrations of the compound were obtained by 3-fold serial dilution and 100 μl per well of each diluted solution in triplicate were transferred to plates, on which Hela cells were previously seeded in a monolayer. The plates were transferred to BSL-4 conditions and appropriately diluted Niv virus solution prepared in cell culture medium and pre-determined virus titer was added to the plates loaded with cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as controls for 0% and 100% virus inhibition, respectively. After infection, assay plates were incubated for 3-4 days in a cell culture incubator. After incubation, the medium was removed from the infected cells and a portion was used to quantify viral RNA by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Percent inhibition was calculated for each test concentration relative to controls corresponding to 0% and 100% inhibition, and the EC ₅₀ value for each compound was determined by non-linear regression as the effective compound concentration that inhibited virus replication by 50%.

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Table 2. Antiviral activity assays against Ebola virus and Marburg virus

EU ₅₀ (nM) Analysis Reporter virus RNA Output Antigen Expression (Simultaneous Multiparameter Imaging) Virus EBOV-GFP EVOV Luc MARVGFP Ebola virus cell line HMVECTERT Huh-7 Huh-7 Huh-7 Huh-7 Hela Macrophage Compound 1 771 1492 3126 1726 BUT BUT >20000 >20000 Compound 9 121 90 BUT BUT one 1029 290 501 (K)-diastereomer Compound 9 62 70 but but but BUT (8)-diastereomer of Compound 9 (Compound 32) 40 81 but but but but Compound 10 Compound 15 630 271 but but but but Compound 21 905 270 Compound 22 but BUT but but but but Compound 23 458 1650 243.350 Compound 24 Compound 25 Compound 26 283 970, 1180 1180 Compound 27 82 182 Compound 28 102 975 120 Compound 29 Compound 30 Compound 31 11061 >20000 1230

EBOV-GFP: Ebola virus expressing reporter gene

GFP EBOV-Luc: Ebola virus expressing the luciferase reporter gene

MARV-GFP: Marburg virus expressing reporter gene

GFP Ebola virus: Ebola virus strain 2014

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Table 2-a. Analysis of antiviral activity against Ebola virus and Marburg virus

EU ₅₀ (nM) Analysis Reporter virus RNA Output Antigen expression (simultaneous multiparameter imaging) Virus EBOV-GFP EVOV Luc MARVGFP ebola cell line HMVECTERT Huh-7 Huh-7 Huh-7 Huh-7 Hela Macrophage Compound 1 771 1492 3126 1726 BUT but >20000 >20000 Compound 9 121 90 BUT BUT one 1029 290.270 501, 70 (K)-diastereomer Compound 9 62 70 but but but but 210 112 (8)-diastereomer of Compound 9 (Compound 32) 40 81 but but but but 100 87 Compound 10 3200 Compound 15 630 271 but but but but 520 501 Compound 21 905, 473 270 Compound 22 but BUT but but but but 11570 Compound 23 458 1650, 1845 243.350, 297 Compound 24 785 Compound 25 6720 Compound 26 283 970, 1180, 1103 1180, 1290 Compound 27 82 182 Compound 28 102 975.682 120 Compound 29 275 Compound 30 11061 >20000 1230 Compound 31 >20000, >10000

EBOV-GFP: Ebola virus expressing reporter gene

GFP EBOV-Luc: Ebola virus expressing the luciferase reporter gene

MARV-GFP: Marburg virus expressing reporter gene

GFP Ebola virus: Ebola virus strain 2014

Table 3. Analysis of antiviral activity against Nipah virus and Hendra virus

EU ₅₀ (nM) Analysis Reporter virus Cytopathic effect (CPE) Output Virus NiV GFP NiV Luc NiV cell line HMVEC-TERT Hela Compound 1 13420 3500 1484 1000 Compound 9 60 thirty H/O H/O

NiV GFP: Nipah virus expressing reporter gene

GFP NiV-Luc: Nipah virus expressing the luciferase reporter gene

NiV: Nipah Virus

All publications, patents and patent documents mentioned above are incorporated herein by reference as if they were incorporated by reference individually.

The present invention has been described with reference to various specific and preferred embodiments and techniques. However, one skilled in the art will recognize that many variations and modifications can be made without departing from the spirit and scope of the present invention.

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Claims (10)

CLAIM 1. A compound selected from the group consisting of - 64 039561 or a pharmaceutically acceptable salt thereof. 2. A pharmaceutical composition for the treatment or prevention of Filoviridae infections, containing a therapeutically effective amount of a compound according to claim 1 or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers or excipients. 3. Pharmaceutical composition for the treatment or prevention of Filoviridae infections, containing a therapeutically effective amount of the compound according to claim 1 or a pharmaceutically acceptable salt thereof and at least one other therapeutic agent selected from the group consisting of ribavirin, palivizumab, motavizumab, RSV-IGIV ( RespiGam®), motavizumab-YTE (MEDI-557), 3-(8)-(1-(2fluorophenyl)-3-(2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][ 1,4]diazepin-3-yl)-ureas (A-60444), 2-[[2hydroxy-5-[(E)-(5-methyltetrazol-1-yl)iminomethyl]phenyl]-(4-hydroxyphenyl) methyl]-4-[(E)-(5-methyltetrazol-1-yl)iminomethyl]phenol (MDT-637), 1-cyclopropyl-3-[[1-(4-hydroxybutyl)-1Hbenzimidazol-2-yl monohydrate ]methyl]-1,3-dihydroimidazo[4,5-c]pyridin-2-one bis-hydrochloride (BMS-433771), amiodarone, dronedarone, verapamil, convalescent plasma from Ebola virus survivors (ECP), TKM-100201, VCX4430 ((28.38.4B,5B)-2-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5-(hydroxymethyl)pyrrolidin-3,4-diol), favipiravir (also known as like T-705 or Avigan), T-705 monophosphate, T-705 diphosphate, T-705 triphosphate, FGI-106 (1-X,7-X-bis[3-(dimethylamino)propyl]-3,9dimethylquinoline[8 ,7-b]quinolone-1,7-diamine), JK-05, TKM-Ebola, ZMapp, recombinant nematode anticoagulant protein c2 (rNAPc2), VRC-EBOADC076-00-VP, OS-2966, MVA-BN filo, brincidofovir, Vaxart adenovirus vector 5 Ebola vaccines, Ad26-ZEBOV, FiloVax vaccine (FiloVax), GOVX-E301, GOVX-E302, Ebola entry inhibitors (NPC1 inhibitors) and rVSV-EBOV, and mixtures thereof. 4. The pharmaceutical composition of claim 3, wherein said at least one other therapeutic agent is ZMapp. 5. The use of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 2 to 4, for the treatment of a Filoviridae viral infection in a human. 6. Use according to claim 5, wherein the Filoviridae viral infection is an Ebola virus infection. 7. Use according to claim 5, wherein the Filoviridae viral infection is a Marburg virus infection. 8. The use of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 2 to 4, for the manufacture of a medicament for the treatment of a human Filoviridae viral infection. 9. Use according to claim 8, wherein the Filoviridae viral infection is an Ebola virus infection. 10. Use according to claim 8, wherein the Filoviridae viral infection is a Marburg virus infection.