OA20831A - Methods for treating filoviridae virus infections. - Google Patents

Abstract

Provided are compounds, methods, and pharmaceutical compositions for treating Filoviridae virus infections by administering ribosides, riboside phosphates and prodrugs thereof, of Formula IV:

Description

METHODS FOR TREATING FILOVIRIDAE VIRUS INFECTIONS

FIELD OF THE INVENTION

[000 J ] The invention relates generally to methods and compounds for treating Filoviridae virus infections, particularly methods and nucleosides for treating Ebola virus, Marburg virus and Cueva virus.

BACKGROUND OF THE INVENTION (0002] Filoviruses (e.g., Ebola virus (EBOV) and Marburg virus (MARV)) are among the most léthal and destructive viruses. They cause severe, often fatal viral hémorrhagie fevers in humans and nonhuman primates (e.g., monkeys, gorillas, and chimpanzees). Filoviruses are of particular concem as possible biological weapons since they hâve the potential for aérosol dissémination and weaponization.

[0003] The incubation period for Filovirus infection ranges from 2 to 21 days. The onset of îllness is abrupt and is characterized by high fever, headaches, joint and muscle aches, sore throat, fatigue, diarrhea, vomiting, and stomach pain. A rash, red eyes, hiccups and internai and extemal bleeding may be seen in some patients. Within one week of becoming infected with the virus, most patients expérience chest pains and multiple organ failure, go into shock, and die. Some patients also expérience blindness and extensive bleeding before dyîng.

[0004] Filoviridae are a family of RNA viruses. Two members of the Filoviridae family hâve been identified: EBOV and MARV. Two key pathogénie types of the Filoviridae family hâve been identified: Ebolavirus and MARV. There is one identified variant of MARV and five identified species of ebolavirus: Zaïre (i.e. Ebola virus, EBOV), Sudan, Tai Forest, Bundibugyo, and Reston. The exact origin, locations, and natural habitat of Filoviridae are unknown. However, on the basis of available evidence and the nature of similar viruses, it is postulated that Filoviridae are zoonotic (i.e., animal-borne) and are normally maintained in an animal host that is native to the African continent.

[0005] For more than 30 years, ebolaviruses hâve been associated with periodic épisodes of hémorrhagie fever in Central Africa that produce severe disease in infected patients. Mortality rates in outbreaks hâve ranged from 50% for the Sudan species of ebolavirus (SEBOV) to up to 90% for the Zaïre species of ebolavirus (EBOV, ZEBOV) (Sanchez etal., Filoviridae: Marburg and Ebola Viruses, in Fields Virology (eds. Knipe, D.M. & Howley, P.M.) 1409-1448 (Lippincott Williams & Wilkins, Philadelphia)). An outbreak late in 2007 caused by an apparently new species of ebolavirus in Uganda resulted in a fatality rate of about 25% (Towner et al., PLoS Pathog., 4:el000212 (2008)). ZEBOV has also decimated populations of wild apes in this sanie région of Africa (Walsh étal., Nature, 422:611-614 (2003)).

[0006] Prévention and treatment of filovirus infections, includîng ebolaviruses (i.e. EBOV) présents many challenges. In fact, there are no vaccines or post exposure treatment modalities available for preventing or managing EBOV infections. Patients instead receive supportive therapy. Le., electrolyte and fluid balancing, oxygen, blood pressure maintenance, and treatment for any secondary infections.

[0007] Thus, there is a need for compositions and methods for treating EBOV infections. The présent invention addresses these and other needs.

SUMMARY OF THE INVENTION

[0008] Provîded, is a method for treating a Filoviridae infection in a human in need thereof comprising administering a therapeutically effective amount of a compound of Formula IV:

OH ÔH

Formula IV or a pharmaceutically acceptable sait, hydrate or ester, thereof;

wherein,

R⁷ is selected from the group consisting of

a) H, -0(=0)^, -C(=O)ORⁿ, -C(=0)NRⁿRⁱ², -C(=O)SRⁿ, -S(O)₂Rⁿ, -S(O)(ORⁿ), -SiOMOR¹¹! or-SOzNR^R¹²;

b)

HO—P--$/ §

HO ^? ,

c) a group selected from:

R^c is selected from the group of phenyl, 1-naphthyl, 2maphthyl,

R^d is selected from the group of H or CH3;

R^el and R^e2 are each independently selected from the group of H, (Ci-Cfi)alkyl or benzyl;

R^f is selected from the group of from H, (Ci-Cs)alkyl, benzyl, (C3Cô)cycloalkyl, and -CH2-(C3-Cé)cycloalkyl;

R^g is selected from selected from the group of (Ci-Cs)alkyl, O-(Ci-Cs)alkyl, benzyl, -O-benzyl, CH₂-(C₃-C6)cycloalkyl, -O-CH₂-(C₃-C₆)cycloalkyl, and CF₃; and n' is an integer selected from the group of 1, 2, 3, and 4; and

d) a group of the formula:

Q

Z² J wherein:

Q is selected from the group of O, S, NR, ⁺N(O)(R), N(0R), N(O)(0R), or N-NR₂;

Z¹ and Z², when taken together, are -Q^QR^XQ¹-; wherein each Q¹ is independently selected from the group of O, S, or NR; and each R^y is independently selected from the group of H, F, Cl, Br, I, OH, R, -C(=Q²)R, -C(=Q²)OR, C(=Q²)N(R)2, -N(R)2, -⁺N(R)3, -SR, -S(O)R, S(O)2R, -S(O)(OR), -S(O)₂(OR), -OC(=Q¹)R, OC(=Q²)OR, -OC(=Q²)(N(R)2), -SC( --Q^;)R SC(=Q²)OR, -SC(=Q²)(N(R)2), -N(R)C(=Q²)R, N(R)C(=Q²)0R, -N(R)C(=Q³)N(R)_2j -so₂nr₂, -CN, -N3, -NO2, -OR, or Z³; or when taken together, two R^y on the same carbon atom form a carbocyclic ring of 3 to 7 carbon atoms, each Q² is independently, O, S, NR, ⁺N(0)(R), N(0R), N(O)(OR), or N-NR₂; or

Z¹ and Z² are each, independently, a group of the Formula la;

Formula la wherein:

each Q³ is independently selected from the group of a bond, O, CR2, NR, ⁺N(0)(R), N(OR), ⁴N(O)(OR), N-NR2, S, S-S, S(O), or S(O)2;

M2 ïs an integer selected from the group of 0, 1 or 2, each R^x is independently R^y or the formula:

wherein:

each Mla, Mlc, and Mld is an integer independently selected from the group of 0 or I;

M12c is an integer selected from the group of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

Z³ is Z⁴ or Z⁵;

Z⁴ is R, -C(Q²)R^y, -C(Q²)Z⁵, -SOJV, or -SO₂Z⁵;

and

Z⁵ is a carbocycle or a heterocycle wherein Z⁵ is independently substituted with 0 to 3 R^y groups;

each R¹¹ or R¹² is independently H, (Ci-Ce)alkyl, (C2-C8)alkenyl, (C2-Cg)alkynyl, (C4-C8)carbocyclylalkyl, (Cô-C^oioptionally substituted aryl, optionally substituted heteroaryl, -C(=O)(Ci-C8)alkyl, -S(O)n(Ci-Cs)alkyl or (C6-C2o)aryl(Ci~C8)alkyI, or R^1! and R¹² taken together with a nitrogen to which they are both attached form a 3 to 7 membered heterocyclic ring wherein any one carbon atom of said heterocyclic ring can optionally be replaced with -O-, -S- or -NR^a-;

each R^a is independently selected from the group of H, (Ci-C8)alkyl, (Cz-C^aikenyl, (C₂-Cs)alkynyl, (C6-C2o)aryl(Ci-Cs)alkyl, (C4-Cs)carbocyclylalkyl, -C(^O)R, C(=O)OR, -C(O)NR₂, -C(=O)SR, -S(O)R, -S(O)₂R, -S(O)(OR), -S(O)₂(OR), or -SO₂NR₂, wherein each R is independently selected from the group of H, (Ci-Ce) alkyl, (Ci-Cg) substituted alkyl, (Cî-Csjaikenyl, (C₂-Ce) substituted alkenyl, (C₂-Cs) alkynyl, (C₂-C₈) substituted alkynyl, (C<5-C₂o)aryl, (Cs-C₂o)substituted aryl, (C₂-C₂o)heterocyclyl, (C₂-C₂o)substîtuted heterocyclyl, (C6-C₂o)aryl(Ci-Cs)alkyl or substituted (Cû-C₂o)aryl(Ci-Cs)alkyl;

each n is an integer independently selected from the group of 0, 1, or 2; and wherein each (Ci-Cg)alkyl, (C₂-Cg)alkenyl, (C₂-Cs)alkynyl or (C6-C₂o)aryl(Ci“Cs)alkyl of each R¹¹ or R¹² is, independently, optionally substituted with one or more substituents selected from the group of halo, hydroxy, CN, Ns, N(R^a)₂ or OR^a; and wherein one or more of the non-terminal carbon atoms of each said (CiC«)alkyl may be optionally replaced with -O-, -S- or -NR^a-,

[0009] In another embodiment, the présent invention provides a compound that is

or a pharmaceutically acceptable sait, hydrate, or ester thereof.

DETA1LED DESCRIPTION OF THE INVENTION

I. DEFINITIONS

[0010] Unless stated otherwise, the foilowîng tenus and phrases as used herein are intended to hâve the foilowîng meanings:

[0011] When trade names are used herein, applîcants intend to independently include the trade name product and the active pharmaceutical ingredient(s) of the trade name product.

[0012] As used herein, a compound of the invention or a compound of Formula IV means a compound of Formula IV or a pharmaceutically acceptable sait, thereof Similarly, with respect to isolatable intermédiares, the phrase a compound of Formula (number) means a compound ofthat formula and pharmaceutically acceptable salis, thereof.

[0013] “Alkyl” is hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms. For example, an alkyl group can hâve 1 to 20 carbon atoms (Le, C1-C20 alkyl), 1 to 8 carbon atoms (Le., Ci-Cs alkyl), or 1 to 6 carbon atoms (Le., Ci-Cô alkyl). Examples of suîtable alkyl groups include, but are not limited to, methyl (Me, -CH₃), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH₃)₂), 1-butyl (n-Bu, n-butyi, CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -CiCHjh), 1 -pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH₃)CH₂CH2CH₃), 3-pentyl (-CH(CH₂CH₃)2), 2-methyl2-butyl (-C(CH3)₂CH₂CH3), 3-methyl-2-butyl (-CH(CH₃)CH(CH3)₂), 3-methyl-1-butyl (-CH2CH2CH(CH₃)2), 2-methyl-1-butyl (-CH₂CH(CH₃)CH2CH3), 1-hexyl (-CH2CH₂CH₂CH2CH2CH₃), 2-hexyl (-CH(CH3)CH2CH₂CH₂CH3), 3-hexyl (CH(CH2CH₃)(CH₂CH2CH3))₁ 2-methyl-2-pentyl (-C(CH3)₂CH₂CH₂CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH₂CH₃), 4-methyl-2-pentyl (-CH(CH₃)CH2CH(CH₃)2), 3-methyl-3-pentyl (-C(CH₃)(CH₂CH3)2), 2-methyl-3-pentyl (-CH(CH₂CH₃)CH(CH₃)₂)₅ 2,3-dimethyl-2-butyï (-C(CH₃)2CH(CH₃)2), 3,3-dimethyl-2-butyl (-CH(CH₃)C(CH₃)₃, and octyi (-(C^^CHa).

[0014] “Alkoxy” means a group having the formula -O-alkyl, in which an alkyl group, as defined above, is attached to the parent molécule via an oxygen atom. The alkyl portion of an alkoxy group can hâve 1 to 20 carbon atoms (Le., C1-C20 alkoxy), I to 12 carbon atoms(/.e., CiC12 alkoxy), or 1 to 6 carbon atoms(/.e., Ci-Cc alkoxy). Examples of suitable alkoxy groups include, but are not limited to, methoxy (-O-CH3 or -OMe), ethoxy (-OCH₂CH₃ or -OEt), tbutoxy (-O-C(CH₃)₃ or-OtBu) and the like.

[0015] “Halo alkyl” is an alkyl group, as defmed above, in which one or more hydrogen atoms of the alkyl group is replaced with a halogen atom. The alkyl portion of a haloalkyl group can hâve 1 to 20 carbon atoms (i.e., C1-C20 haloalkyl), 1 to 12 carbon atoms(7.e., C1-C12 haloalkyl), or 1 to 6 carbon atoms(r.e., Ci-Cô alkyl). Examples of suitable haloalkyl groups include, but are not limited to, -CF₃, -CHF2, -CFH2, -CH₃CF₃, and the like.

[0016] “Alkenyl” is a hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms with ai least one site of unsaturation, Le. a carbon-carbon, sp² double bond. For example, an alkenyl group can hâve 2 to 20 carbon atoms (i.e., C2-C20 alkenyl), 2 to 8 carbon atoms (Le., C2-C8 alkenyl), or 2 to 6 carbon atoms (Le., C₂-Cô alkenyl). Examples of suitable alkenyl groups include, but are not limited to, ethylene or vinyl (-CH=CH₂), allyl (-CH2CH=CH₂), cyclopentenyl (-C5H7), and 5-hexenyI (-CH2CH₂CH2CH₂CH=CH2).

[0017] “Alkynyl” is a hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms with at least one site of unsaturation, i.e. a carbon-carbon, sp triple bond. For example, an alkynyl group can hâve 2 to 20 carbon atoms (i.e., C2-C20 alkynyl), 2 to 8 carbon atoms (i.e., C2Cs alkyne,), or 2 to 6 carbon atoms (Le., C2-C6 alkynyl). Examples of suitable alkynyl groups include, but are not limited to, acetylenic (-C^CH), propargyl (-CHaC^CH), and the like.

[0018] “Alkylene” refers to a saturated, branched or straîght chain or cyclic hydrocarbon radical having two monovalent radical cent ers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. For example, an alkylene group can hâve 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. Typical alkylene radicals include, but are not limited to, methylene (-CH₂-), 1,1-ethyl (-CH(CH₃)-), 1,2-ethyl (-CH2CH2-), ί,Ι-propyl (-CH(CH₂CH₃)-), 1,2-propyï (-CH₂CH(CH₃)-), 1,3-propyl (-CH₂CH₂CH₂-), 1,4-butyl (-CH2CH2CH2CH2-), and the like.

[0019] “Alkenylene” refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical having two monovalent radical centers derived by the removal of two hydrogen atoms ÎTom the same or two different carbon atoms of a parent alkene. For example, and alkenylene group can hâve 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. Typical alkenylene radicals include, but are not limited to, 1,2-ethylene (-CH=CH-).

[0020] “Alkynylene” refers to an unsaturated, branched or straight chain or cyclic hydrocarbon radical having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkyne. For example, an alkynylene group can hâve 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. Typical alkynylene radicals include, but are not limited to, acetylene (-C=C-), propargyl (-CH₂C=C-), and4-pentynyl (-CH₂CH₂CH₂C=C-).

[0021] “Amino” refers generally to a nitrogen radical which can be considered a dérivative of ammonia, having the formula -N(X)₂, where each “X” is independently H, substituted or unsubstituted alkyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, etc. The hybridization of the nitrogen is approximately sp³. Nonlimiting types of amino include ~NH₂, -N(alkyl)₂, -NH(alkyl), -N(carbocyclyl)₂, -NH( carbocyclyl), N(heterocyclyl)₂, -NH(heterocyclyl), -N(aryl)₂, -NH(aryl), -N(alkyl)(aryl), N(alkyl)( heterocyclyl), -N(carbocyclyl)(heterocyclyl), -N(aryl)(heteroaryl), N(alkyl)(heteroaryl), etc. The term “alkylamino” refers to an amino group substituted with at least one alkyl group. Nonlimiting examples of amino groups include -NH₂, -NH(CH₃), N(CH₃)₂, -NH(CH₂CH₃), - N(CH₂CH₃)₂, -NH(phenyl), -N(phenyl)₂, -NH(benzyl), -N(benzyl)₂, etc. Substituted alkylamino refers generally to alkylamino groups, as defined above, in which at least one substituted alkyl, as defined herein, is attached to the amino nitrogen atom. Nonlimiting examples of substituted alkylamino includes -NH(alkylene-C(O)-OH), -NH(alkyleneC(O)-O-alkyl), -N(alkyIene-C(O)-OH)₂, -N(alkylene-C(O)-O-alkyl)₂, etc.

[0022] “Aryl” means an aromatic hydrocarbon radical derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring System. For example, an aryl group can hâve 6 to 20 carbon atoms, 6 to 14 carbon atoms, or 6 to 10 carbon atoms. Typical aryl groups include, but are not limited to, radical s derived from benzene (e.g., phenyl), substituted benzene, naphthalene, anthracene, biphenyl, and the like.

[0023] “Arylalkyl” refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp³ carbon atom, is replaced with an aryl radical. Typical arylalkyl groups include, but are not limited to, benzyi, 2-phenyIethan-l-yl, naphthylmethyl, 2-naphthylethan-l-yl, naphthobenzyl, 2-naphthophenylethan-l-yl and the like. The arylalkyl group can comprise 7 to 20 carbon atoms, e.g., the alkyl moiety is 1 to 6 carbon atoms and the aryl moiety is 6 to 14 carbon atoms.

[0024] “Arylalkenyl” refers to an acyclic alkenyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp³ carbon atom, but also an sp² carbon atom, is replaced with an aryl radical. The aryl portion of the arylalkenyl can include, for example, any of the aryl groups disclosed herein, and the alkenyl portion of the arylalkenyl can include, for example, any of the alkenyl groups disclosed herein. The arylalkenyl group can comprise 8 to 20 carbon atoms, e.g., the alkenyl moiety is 2 to 6 carbon atoms and the aryl moiety is 6 to 14 carbon atoms.

[0025] “Arylalkynyl” refers to an acyclic alkynyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp³ carbon atom, but also an sp carbon atom, is replaced with an aryl radical. The aryl portion of the arylalkynyl can include, for example, any of the aryl groups disclosed herein, and the alkynyl portion of the arylalkynyl can include, for example, any of the alkynyl groups disclosed herein. The arylalkynyl group can comprise 8 to 20 carbon atoms, e.g., the alkynyl moiety is 2 to 6 carbon atoms and the aryl moiety is 6 to 14 carbon atoms.

[0026] The terni “substituted” in reference to alkyl, alkylene, aryl, arylalkyl, alkoxy, heterocyclyl, heteroaryl, carbocyclyl, etc. , for example, “substituted alkyl”, “substituted alkylene”, “substituted aryl”, “substituted arylalkyl”, “substituted heterocyclyl”, and “substituted carbocyclyl” means alkyl, alkylene, aryl, arylalkyl, heterocyclyl, carbocyclyl respectively, in which one or more hydrogen atoms are each independently replaced with a nonhydrogen substituent. Typical substituents include, but are not limited to, -X, -R^b, -O, =O, -OR^b, -SR¹’, -S, -NR^b2, -N⁺R\ NR^!; -CX3, -CN, -OCN, -SCN, -N=O0, -NCS, -NO, -NO₂, =N₂, -N₃, -NHC(=0)R^b, -OC(=O)R^b, -NHC(-0)NR^b3, -S(=O)2-, -S(=O)2OH, -S(=O)2R^b, -OS(=O)2OR^b, -S(=O)2NR^b2, -S(=O)R^b, -OP(=O)(OR^b)2, -P(=O)(OR^b)2, -P(=O)(O )₂,

-P(=O)(OHh, -P(O)(OR^b)(O), -C(=O)R^b, -C(=O)X, -C(S)R^b, -C(O)OR^b, -C(O)O; -C(S)OR^b, -C(O)SR^b, -C(S)SR^b, -C(O)NR^b2, -C(S)NR^b2, -C(=NR^b)NR^b2, where each X is independently a halogen: F, Cl, Br, or I; and each R^b is independently H, alkyl, aryl, arylalkyl, a heterocycle, or a protecting group or prodrug moîety. Alkylene, alkenylene, and alkynylene groups may also be similarly substituted. Unless otherwise indicated, when the terni substituted is used in conjunction with groups such as arylalkyl, which hâve two or more moieties capable of substitution, the substituents can be attached to the aryl moiety, the alkyl moiety, or both.

[0027] The term “prodrug” as used herein refers to any compound that when administered to a biological System generates the drug substance, i.e., active ingrédient, as a resuit of spontaneous Chemical reaction(s), enzyme catalyzed Chemical reaction(s), photolysis, and/or metabolîc Chemical reaction(s). A prodrug is thus a covalently modified analog or latent form of a therapeutically active compound.

[0028] One skilled in the art will recognize that substituents and other moieties of the compounds of Formula IV should be selected in order to provide a compound which is sufficiently stable to provide a pharmaceutically usefùl compound which can be formulated înto an acceptably stable pharmaceutical composition. Compounds of Formula IV which hâve such stability are contemplated as falling within the scope of the présent invention.

[0029] “Heteroalkyl” refers to an alkyl group where one or more carbon atoms hâve been replaced with a heteroatom, such as, O, N, or S. For example, if the carbon atom of the alkyl group which is attached to the parent molécule is replaced with a heteroatom (e.g., O, N, or S) the resulting heteroalkyl groups are, respectively, an alkoxy group (e.g., -OCH3, etc.), an amine (e.g., -NHCH3, -N(CH3)2, etc.), or a thioalkyl group (e.g., -SCH3). If a non-terminal carbon atom of the alkyl group which is not attached to the parent molécule is replaced with a heteroatom (e.g., O, N, or S) the resulting heteroalkyl groups are, respectively, an alkyl ether (e.g., -CH2CH2-O-CH3, etc.), an alkyl amine (e.g., -CH2NHCH3, -CHîN(CH3)2, etc.), or a thioalkyl ether (e.g.rCFb-S-CHb). If a terminal carbon atom of the alkyl group is replaced with a heteroatom (e.g., O, N, or S), the resulting heteroalkyl groups are, respectively, a hydroxyalkyl group (e.g., -CH2CH2-OH), an aminoalkyl group (e.g., -CH2NH2), or an alkyl thiol group (e.g., -CH2CH2-SH). A heteroalkyl group can hâve, for example, 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. A Ci-Cô heteroalkyl group means a heteroalkyl group having 1 to 6 carbon atoms.

[0030] “Heterocycle” or “heterocyclyl” as used hereîn includes by way of example and not limitation those heterocycles described in Paquette, Léo A.; Principles of Modem Heterocyclic Chemistry (W.A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; The Chemistry of Heterocyclic Compounds. A Sériés of Monographs” (John Wiley & Sons, New York, 1950 to présent), in particular Volumes 13, 14, 16, 19, and 28, and .7. Am. Chem. Soc. (1960) 82:5566. In one spécifie embodiment ofthe invention “heterocycle” includes a “carbocycle” as defined herein, wherein one or more {e.g. 1, 2, 3, or 4) carbon atoms hâve been replaced with a heteroatom {e.g. O, N, or S). The terms “heterocycle” or “heterocyclyl” includes saturated rings, partially unsaturated rings, and aromatic rings (i.e., heteroaromatic rings). Substituted heterocyclyls include, for example, heterocyclic rings substituted with any of the substituents disclosed herein including carbonyl groups. A non-lîmiting example of a carbonyl substituted heterocyclyl is:

10031] Examples of heterocycles include by way of example and not limitation pyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl, tetrahydrothiophenyl, sulfur oxidized tetrahydrothîophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyi, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolînyl, azocinyl, triazinyl, 6H-l,2,5-thiadiazinyl, 2H,6H-l,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, IH-indazoly, purinyl, 4Hquinolizinyl, phthalazinyl, naphthyrîdinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, β-carboiinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, fiirazanyl, phenoxazinyl, isochromanyl, chromanyl, îmidazolidinyl, îmîdazolinyl, pyrazolîdinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, isatinoyl, and bis-tetrahydrofuranyl:

[0032] By way of example and not limitation, carbon bonded heterocycles are bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole, position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of an aziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6, 7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of an isoquinolîne. Still more typically, carbon bonded heterocycles include 2-pyridyi, 3-pyridyl, 4-pyrîdyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazînyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5thiazolyl.

[0033] By way of example and not limitation, nitrogen bonded heterocycles are bonded at position I of an aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroIine, 3-pyrroIine, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoïine, piperidine, piperazine, indole, indoline, IH-indazole, position 2 ofa isoindoie, or isoindoline, position 4 of a morpboline, and position 9 of a carbazole, or β-carboline. Still more typically, nitrogen bonded heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1pyrazolyl, and 1-piperidinyf

[0034] “Heterocyclylalkyl” refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp³ carbon atom, is replaced with a heterocyclyl radical (i.e., a heterocyclyl-alkylene- moiety). Typical heterocyclyl alkyl groups include, but are not limited to heterocyclyl-CHz-, 2-(heterocyclyl)ethan-l-yl, and the like, wherein the “heterocyclyl” portion includes any of the heterocyclyl groups described above, including those described in Principles of Modem Heterocyclic Chemistry. One skilled in the art will also understand that the heterocyclyl group can be attached to the alkyl portion of the heterocyclyl alkyl by means of a carbon-carbon bond or a carbon-heteroatom bond, with the proviso that the resulting group is chemicaliy stable. The heterocyclyl alkyl group comprises 3 to 20 carbon atoms, e.g., the alkyl portion of the arylalkyl group is 1 to 6 carbon atoms and the heterocyclyl moiety is 2 to 14 carbon atoms. Examples of heterocyclylalkyls include by way of example and not limitation 5-membered su!fur, oxygen, and/or nitrogen containing heterocycles such as thiazolylmethyl, 2-thiazolylethan-l-yl, imidazolylmethyl, oxazolylmethyl, thiadiazoiylmethyl, etc., 6-membered sulfur, oxygen, and/or nitrogen containing heterocycles such as piperidinylmethyl, piperazinylmethyl, morpholinylmethyl, pyridinylmethyl, pyridizylmethyl, pyrimidylmethyl, pyrazinylmethyl, etc.

[0035] “Heterocyclylalkenyl” refers to an acyclic alkenyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp³ carbon atom, but also a sp² carbon atom, îs replaced with a heterocyclyl radical (j.e., a heterocyclyl-alkenylene- moiety). The heterocyclyl portion of the heterocyclyl alkenyl group includes any of the heterocyclyl groups described hereîn, including those described in Principles of Modem Heterocyclic Chemistry, and the alkenyl portion of the heterocyclyl alkenyl group includes any of the alkenyl groups disclosed herein. One skilled in the art will also understand that the heterocyclyl group can be attached to the alkenyl portion of the heterocyclyl alkenyl by means of a carbon-carbon bond or a carbon-heteroatom bond, with the proviso that the resulting group is chemically stable. The heterocyclyl alkenyl group comprises 4 to 20 carbon atoms, e.g., the alkenyl portion of the heterocyclyl alkenyl group is 2 to 6 carbon atoms and the heterocyclyl moiety is 2 to 14 carbon atoms.

[0036] “Heterocyclylalkynyl” refers to an acyclic alkynyl radical in which one ofthe hydrogen atoms bonded to a carbon atom, typically a terminal or sp³ carbon atom, but also an sp carbon atom, is replaced with a heterocyclyl radical (i.e., a heterocyclyl-alkynylene- moiety). The heterocyclyl portion of the heterocyclyl alkynyl group includes any of the heterocyclyl groups described herein, including those described in Principles of Modem Heterocyclic Chemistry, and the alkynyl portion ofthe heterocyclyl alkynyl group includes any ofthe alkynyl groups disclosed herein. One skilled in the art will also understand that the heterocyclyl group can be attached to the alkynyl portion of the heterocyclyl alkynyl by means of a carbon-carbon bond or a carbon-heteroatom bond, with the proviso that the resulting group is chemically stable. The heterocyclyl alkynyl group comprises 4 to 20 carbon atoms, e.g., the alkynyl portion of the heterocyclyl alkynyl group is 2 to 6 carbon atoms and the heterocyclyl moiety is 2 to 14 carbon atoms.

[0037] “Heteroaryl” refers to an aromatic heterocyclyl having at least one heteroatom in the ring. Non-limiting examples of suitable heteroatoms which can be included in the aromatic ring include oxygen, sulfur, and nitrogen. Non-limiting examples of heteroaryl rings include ail of those aromatic rings listed inthe définition of “heterocyclyl”, including pyridmyf pyrrolyl, oxazolyl, indolyl, isoindolyl, purinyl, furanyi, thienyl, benzofuranyl, benzothiophenyl, carbazolyl, imidazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, quinolyl, isoquinolyl, pyridazyl, pyrimidyl, pyrazyl, etc.

[0038] “Carbocycle” or “carbocyclyl” refers to a saturated (i.e., cycioalkyl), partially unsaturated (e.g., cycloalkenyl, cycloalkadienyl, etc.) or aromatic ring having 3 to 7 carbon atoms as a monocycle, 7 to 12 carbon atoms as a bicycle, and up to about 20 carbon atoms as a polycycle. Monocyclic carbocycles hâve 3 to 7 ring atoms, still more typically 5 or 6 ring atoms. Bicyclic carbocycles hâve 7 to 12 ring atoms, e.g., arranged as a bicyclo [4,5], [5,5], [5,6] or [6,6] System, or 9 or 10 ring atoms arranged as a bicyclo [5,6] or [6,6] system, or spiroftised rings. Non-limiting examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, I-cyclopent-1-enyl, l-cyclopent-2-enyl, l-cyclopent-3-enyl, cyclohexyl, 1cyclohex-l-enyl, l-cyclohex-2-enyl, l-cyclohex-3-enyl, and phenyl. Non-limiting examples of bicyclo carbocycles includes naphthyl, tetrahydronaphthalene, and decaline.

[0039] “Carbocyclylalkyl” refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom is replaced with a carbocyclyl radical as described herein. Typical, but non-limiting, examples of carbocyclylalkyl groups include cyclopropylmethyl, cyclopropylethyl, cyclobutyl methyl, cyclopentyl methyl and cycloh exylmethyl.

[0040] “Arylheteroalkyl” refers to a heteroalkyl as defined herein, in which a hydrogen atom (which may be attached either to a carbon atom or a heteroatom) has been replaced with an aryl group as defined herein. The aryl groups may be bonded to a carbon atom of the heteroalkyl group, or to a heteroatom of the heteroalkyl group, provided that the resulting arylheteroalkyl group provides a chemically stable moiety. For example, an arylheteroalkyl group can hâve the general formulae -alkylene-O-aryl, -alkylene-O-alkylene-aryl, -alkylene-NH-aryl,

-alkylene-NH-alkylene-aryl, -alkylene-S-aryl, -alkylene-S-alkylene-aryl, etc. In addition, any of the alkylene moieties in the general formulae above can be further substituted with any of the substituents defined or exemplified herein.

[0041] “Heteroaryl alkyl” refers to an alkyl group, as defined herein, in which a hydrogen atom has been replaced with a heteroaryl group as defined herein. Non-limiting examples of heteroaryl alkyl include -CHs-pyridinyl, -CHz-pyrrolyl, -CHi-oxazolyl, -CHs-indolyl, -CHi-isoindolyl, -CHa-purinyl, -CHi-fùranyl, -CHa-thienyl, -CH2-benzofuranyl,

-CH2-benzothiophenyl, -CKb-carbazolyl, -CFh-imidazolyl, -CHz-thiazolyl, -CHs-isoxazolyl, -Cth-pyrazolyl, -CH2-isothiazolyl, -CFh-quinolyl, -CFh-isoquinolyl, -CFh-pyridazyl, -CHî-pyrimidyt -CFb-pyrazyl, -CH(CHy)-pyridinyl, -CH(CH3)-pyrrolyl, -CH(CH₃)-oxazoly1, -CH(CH₃)-indoIyI, -CH(CH3)-isoindolyl, -CH(CH3)-purinyl, -CH(CH3)-fùranyl, -CH(CH3)-thienyl, -CH(CH3)-benzofuranyl, -CH(CH3)-benzothiophenyl, -CH(CH3)-carbazolyl, -CH(CH₃)-imidazolyl, -CH(CH₃)-thiazolyl, -CH(CH₃)-isoxazolyl, -CH(CH₃)-pyrazolyl, -CH(CH₃)-isothiazolyl, -CH(CH₃)-quinolyl, -CH(CH₃)-isoquinolyl, -CH(CH₃)-pyridazyJ, -CH(CH3)-pyrimidyl, -CH(CH3)-pyrazyl, etc.

[0042] The term “optionally substituted” in référencé to a particular moiety of the compound of Formula IV (e.g., an optionally substituted aryl group) refers to a moiety wherein ail substituents are hydrogen or wherein one or more of the hydrogens of the moiety may be replaced by substituents such as those listed under the définition of “substituted”.

[0043] The term “optionally replaced” in référencé to a particular moiety of the compound of Formula IV (e.g., the carbon atoms of said (Ci-Cs)alkyl may be optionally replaced by -O-, -S-, or -NR^a-) means that one or more of the methylene groups of the (Ci-Cs)alkyl may be replaced by 0, 1, 2, or more of the groups specified (e.g., -O-, -S-, or -NR^a-).

[0044] The term “non-terminal carbon atom(s)” in référencé to an alkyl, alkenyl, alkynyl, alkylene, alkenylene, or alkynylene moiety refers to the carbon atoms in the moiety that intervene between the first carbon atom of the moiety and the last carbon atom in the moiety. Therefore, by way of example and not limitation, in the alkyl moiety -CH2(C*)H2(C*)H2CH3 or alkylene moiety -CH2(C’)H2(C*)H2CH2- the C* atoms would be considered to be the nonterminal carbon atoms.

[0045] Certain Q and Q¹ alternatives are nitrogen oxides such as ⁺N(O)(R) or ⁺N(O)(OR). These nitrogen oxides, as shown here attached to a carbon atom, can also be represented by charge separated groups such as

0“ O ^Ror OR, respectively, and are intended to be équivalent to the aforementioned représentations for the purposes of describing this invention.

[0046] Linker or “link” means a Chemical moiety comprisîng a covalent bond or a chain of atoms. Linkers include repeating units of alkyloxy (e.g. polyethyleneoxy, PEG, polymethyleneoxy) and alkylamino (e.g. polyethyleneamino, Jeffamine™); and diacid ester and amides including succinate, succinamide, diglycolate, malonate, and caproamide.

[0047] The terms such as “oxygen-linked”, “nitrogen-linked”, “carbon-linked”, “sulfurlinked”, or “phosphorous-linked” mean that if a bond between two moieties can be formed by using more than one type of atom in a moiety, then the bond formed between the moieties is through the atom specified. For example, a nitrogen-linked amino acid would be bonded through a nitrogen atom of the amino acid rather than through an oxygen or carbon atom of the amino acid.

[0048] In some embodiments of the compounds of Formula IV, one or more of Z¹ or Z² are independently a radical of a nitrogen-linked naturally occurring ct-amino acid ester. Examples of naturally occurring amino acids include isoleucine, leucîne, lysine, méthionine, phenylalanine, threonine, tryptophan, valine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, selenocysteine, serine, tyrosine, arginine, histîdine, ornithine and taurine. The esters of these amino acids comprise any of those described for the substituent R, particularly those in which Ris optîonally substituted (Ci-Cs)alkyl.

[0049] The term “purine” or “pyrimidine” base comprises, but is not limited to, adenine, N⁶alkylpurines, N⁶~acylpurines (wherein acyl is C(O)(alkyl, aryl, alkylaryl, or arylalkyl), N⁶benzylpurine, N^û-halopurine, N⁶-vinylpurine, N⁶-acetylenic purîne, N⁶-acyl purine, N⁶hydroxyalkyl purine, N⁶-allylaminopurine, N^-thioallyl purine, N²-alkylpurines, N²-alkyl-6thiopurines, thymine, cytosine, 5-fluorocytosine, 5-methylcytosine, 6-azapyrimidine, including 6-azacytosine, 2- and/or4-mercaptopyrmidine, uracil, 5-halouracil, including 5-fluorouracil, C⁵alkylpyrimidînes, C⁵-benzylpyrimidines, C⁵-halopyrimidines, C⁵-vinylpyrimidine, C³-acetylenîc pyrimidine, C⁵-acyl pyrimidine, C⁵-hydroxyalkyl purine, C³-amidopyrimidîne, C⁵cyanopyrimidine, C?-5-iodopyrimidine, C⁶-iodo-pyrimidine, Cf-Br-vinyl pyrimidine, C⁶-Brvinyl pyrimidine, C⁵-nitropyrimidîne, CF-amino-pyrimidine, N²-alkylpurines, N²-alkyl-6thiopurines, 5-azacytidinyl, 5-azauracilyl, triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, and pyrazolopyrimidinyl. Purine bases include, but are not limited to, guanine, adenine, hypoxanthine, 2,6-diaminopurine, and 6-chloro purine. The purine and pyrimidine bases are linked to the ribose sugar, or analog thereof, through a nitrogen atom of the base. Functional oxygen and nitrogen groups on the base can be protected as necessary or desired. Suitable protecting groups are well known to those skilled in the art, and include trimethylsilyl, dimethyihexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl groups, and acyl groups such as acetyl and propionyl, methanesulfonyl, and p-toluenesulfonyl.

[0050J Unless otherwise specified, the carbon atoms of the compounds of Formula IV are intended to hâve a valence of four. In some Chemical structure représentations where carbon atoms do not hâve a sufficient number of variables attached to produce a valence of four, the remaining carbon substituents needed to provide a valence of four should be assumed to be hydrogen. For example,

has the same meaning as

[0051] “Protecting group” refers to a moiety of a compound that masks or alters the properties of a functional group or the properties of the compound as a whole. The Chemical substructure of a protecting group varies widely. One function of a protecting group is to serve as an intermediate in the synthesis of the parental drug substance. Chemical protecting groups and strategies for protection/deprotectîon are well known in the art. See: Protective Groups in Organic Chemîstry, Theodora W. Greene (John Wiley & Sons, Inc., New York, 1991. See also

Protective Groups in Organic Chemistry, Peter G. M. Wuts and Theodora W. Greene, 4th Ed., 2006. Protecting groups are often utilized to mask the reactivity of certain functional groups, to assist in the efficiency of desired Chemical reactions, e.g. making and breaking Chemical bonds in an ordered and planned fashion. Protection of functional groups of a compound alters other physical properties besides the reactivity of the protected functional group, such as the polarîty, lipophilîcity (hydrophobîcîty), and other properties which can be measured by common analytical tools. Chemically protected intermediates may themselves be bîologically active or inactive. “Hydroxy protecting groups” refers to those protecting groups useful for protecting hydroxy groups (-OH).

[0052] Protected compounds may also exhibit altered, and in some cases, optimized properties in vitro and in vivo, such as passage through cellular membranes and résistance to enzymatic dégradation or séquestration. In this rôle, protected compounds with intended therapeutic effects may be referred to as prodrugs. Another fùnction of a protecting group is to convert the parental drug into a prodrug, whereby the parental drug is released upon conversion of the prodrug in vivo. Because active prodrugs may be absorbed more effectively than the parental drug, prodrugs may possess greater potency in vivo than the parental drug. Protecting groups are removed either in vitro, in the instance of Chemical intermediates, or in vivo, in the case of prodrugs. With Chemical intermediates, it is not particularly important that the resulting products after deprotection, e.g. alcohols, be physiologically acceptable, although in general it is more désirable if the products are pharmacologically innocuous.

[0053] The term chiral refers to molécules which hâve the property of nonsuperîmposability of the mirror image partner, while the term achiral refers to molécules which are superimposable on their mirror image partner.

[0054] The term stereoisomers refers to compounds which hâve identical Chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

[0055] Diastereomer refers to a stereoisomer with two or more centers of chirality and whose molécules are not mirror images of one another. Diastereomers hâve different physical properties, e.g. melting points, boiling points, spectral properties, reactivities and biological properties. For example, the compounds of Formula IV may hâve a chiral phosphorus atom when R⁷ is

and Z¹ and Z² are different. When at least one of either Z¹ or Z² aiso has a chiral center, for example with Z¹ or Z² is a nitrogen-linked, chiral, naturally occurring α-amino acid ester, then the compound of Formula IV will exists as diastereomers because there are two centers of chirality in the molécule. AU such diastereomers and their uses described hereîn are encompassed by the instant invention. Mixtures of diastereomers may be separate under high resolution analytical procedures such as electrophoresis, crystallization and/or chromatography. Diastereomers may hâve different physical attribut es such as, but not limited to, solubility, Chemical stabilities and crystallinity and may also hâve different biological properties such as, but not limited to, enzymatic stability, absorption and metabolic stability.

[0056] Enantiomers refer to two stereoisomers of a compound which are nonsuperimposable mirror images of one another.

[0057] The modifier about used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (e.g., includes the degree of error associated with measurement of the particular quantity).

[0058] The term “treatîng”, as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term “treatment”, as used hereîn, refers to the act of treatîng, as “treatîng” is defined immediately above.

[0059] The term “therapeutically effective amount”, as used herein, is the amount of compound of Formula IV présent in a composition described herein that is needed to provide a desired level of drug in the sécrétions and tissues of the airways and lungs, or alternatively, in the bloodstream of a subject to be treated to give an anticipated physiological response or desired biological effect when such a composition is administered by the chosen route of administration. The précisé amount will dépend upon numerous factors, for example the particular compound of Formula IV, the spécifie activity of the composition, the delivery device employed, the physical characteristics of the composition, its intended use, as well as patient considérations such as severity ofthe disease State, patient coopération, etc., and can readily be determîned by one skilled in the art based upon the information provided herein.

[0060] The term “normal saline” means a water solution containing 0.9% (w/v) NaCl.

10061] The term “hypertonie saline” means a water solution containing greaterthan 0.9% (w/v) NaCl. For example, 3% hypertonie saline would contain 3% (w/v) NaCl.

[0062] “Forming a reaction mixture” refers to the process of bringing into contact at least two distinct species such that they mix together and can react. It should be appreciated, however, the resulting reaction product can be produced direct ly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture.

[0063] “Coupling agent” refers to an agent capable of coupling two disparate compounds. Coupling agents can be catalytic or stoichiometric. For example, the coupling agents can be a lithium based coupling agent or a magnésium based coupling agent such as a Grignard reagent. Exemplary coupling agents include, but are not limited to, n-BuLi, MgCh, iPrMgCl, tBuMgCl, PhMgCl or combinations thereof.

[0064] “Silane” refers to a Silicon containing group having the formula S1R4, where each R group can be alkyl, alkenyl, cycloalkyl, phenyl, or other Silicon containing groups. When the silane is linked to another compound, the silane is referred to as a “silyl” and has the formula -S1R3.

[0065] “Halo-silane” refers to a silane having at least one halogen group linked to the Silicon atom. Représentative halo-silanes hâve the formula Halo-SiRa, where each R group can be alkyl, alkenyl, cycloalkyl, phenyl, or other Silicon containing groups. Spécifie halo-silanes include Cl-Si(CH3)₃, and Cl-SiiCH^CFhCHzSitCHs^-Cl.

[0066] “Non-nucleophilic base” refers to an électron donor, a Lewis base, such as nitrogen bases including triethylamine, diisopropylethyl amine, Ν,Ν-diethylaniline, pyridine, 2,6-lutidine, 2,4,6-collidine, 4-dimethylaminopyridine, and quinuclidine.

[0067] “Leaving group” refers to groups that maintain the bonding électron pair during heterolytic bond cleavage. For example, a leavîng group is readily displaced during a nucleophilic displacement reaction. Suitable leaving groups include, but are not limited to, chlorîde, bromide, mesylate, tosylate, triilate, 4-nitrobenzenesulfonate, 4-chlorobenzenesulfonate, 4-nitrophenoxy, pentafluorophenoxy, etc. One of skill in the art will recognize other leaving groups useful in the présent invention.

[0068] “Deprotection agent” refers to any agent capable of removing a protecting group. The deprotection agent will dépend on the type of protecting group used. Représentative deprotection agents are known in the art and can be found in Proteclive Groups in Organic Chemistry, Peter G. M. Wuts and Theodora W. Greene, 4th Ed., 2006.

Π. COMPOUNDS OF THE PRESENT INVENTION

[0069] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying description, structures and formulas. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover ail alternatives, modifications, and équivalents, which may be included within the scope of the présent invention.

[0070] Provided, is a method fortreating & Filoviridae infection in a human in need thereof comprising administering a therapeutically effective amount of a compound of Formula I:

R⁸

or a pharmaceutically acceptable sait, hydrate or ester, thereof; wherein:

each R¹ is H or halogen;

each R², R³, R⁴ or R⁵ is independently H, 0R^a, N(R^a)2, Ns, CN, NO2, S(O)_nR^a, halogen, (Ci-Cs)alkyl, (C4-Cs)carbocyclylalkyl, (Ci-Cs)substituÎed alkyl, (C2-Cs)alkenyl, (C2”Cs)substituted alkenyl, (C2-Cs)alkynyl or (C2~Cs)substituted alkynyl;

wherein each R^a is independently H, (Ci-Cs)alkyl, (C2-Cs)alkenyl, (C2-Cs)alkynyl, aryl(Ci-Cs)alkyl, (C4-C8)carbocyclylalkyl, -C(=0)R, -C(=O)OR, C(=0)NR₂, -C( O)SR. -S(O)R, -S(O)₂R, -S(O)(OR), -S(O)₂(OR), or -SO2NR2;

each R is independently H, (Ci-Cs) alkyl, (Ci-Cs) substituted alkyl, (C2Cs)alkenyl, (C2-Cg) substituted alkenyl, (Cz-Cs) alkynyl, (C2-Cs) substituted alkynyl, C6-C20 aryl, C6-C20 substituted aryl, C2-C20 heterocyclyl, C2-C20 substituted heterocyclyl, arylalkyl or substituted arylalkyl;

or any two R², R³, R⁴ or R⁵ on adjacent carbon atoms when taken together are -O(CO)Oor when taken together with the ring carbon atoms to which they are attached form a double bond;

R⁶ is OR^a, N(R^a)2, N3, CN, NOî, S(O)nR^a, -CC^jR¹¹, -C(=O)ORⁿ, -C(=0)NR^HR¹², C(=O)SR^H, -S(O)Rⁿ, -S(O)2R^h, -S(O)(ORⁿ), -S(O)2(OR* *), -SO2NRⁿR¹², halogen, (Ci-Cs)alkyl, (C^Cgjcarbocyclylalkyl, (Ci-Cg)substituted alkyl, (C2-Cs)alkenyl, (C2-Cs)substituted alkenyl, (C2-Cs)alkynyl, (Cz-Cgjsubstituted alkynyl, or aryl(Ci-Cg)alkyl;

wherein each R¹¹ or R¹² is independently H, (Ci-Cs)alkyl, (C2-Cs)alkenyl, (C2-Cs)alkynyl, (C4-Cs)carbocyclyialkyl, optionally substituted aryl, optionally substituted heteroaryl, -C(=O)(Ci-Cg)alkyl, -S(O)_n(Ci-Cs)alkyl or aryl(CiCg)alkyl; or R¹¹ and R¹² taken together with a nitrogen to which they are both attached form a 3 to 7 membered heterocyclic ring wherein any one carbon atom of said heterocyclic ring can optionally be replaced with -O-, -S- or -NR^a-;

each n is independently 0, 1, or 2;

R⁷ is selected from a group consisting of

a) H, -C^OjR¹¹, -C(=O)OR^h, -C(=O)NR^1]R¹², -C^OjSR¹¹, -S(O)Rⁿ, -S(O)2Rⁿ, S(O)(OR^1]), -S(O)2(ORⁿ), or -SO₂NR^1,R¹², wherein each (Ci-Cs)alkyl, (C2-Cs)alkenyl, (C2-Cs)alkynyl or aryl(Ci-C8)alkyl of each R¹¹ or R¹² is, independently, optionally substituted with one or more halo, hydroxy, CN, N3, N(R^a)₂or OR^a; and wherein one or more ofthe non-terminal carbon atoms ofeach said (Ci-Cs)alkyl may be optionally replaced with -O-, -S- or—NR^a-, and il ξ

HO—P--Ç / 5

HO

II HO—P.

/^XO

HO

c) a group selected from:

and wherein:

R^c is selected from phenyl, 1-naphthyl, 2-naphthyl,

R^d is H or CH3;

R^S1 and R^e2 are each independently H, Ci-Ce alkyl or benzyl;

R^f is selected from H, Ci-Cs alkyl, benzyl, C3-C6 cycloalkyl, and -CH2-C3-C6 cycloalkyl;

R^g is selected from Ci-Cg alkyl, -O-Ci-Cs alkyl, benzyl, -O-benzyl, -CH2-C3-C6 cycloalkyl, -O-CH2-C3-C6 cycloalkyl, and CF3; and n’ is selected from 1, 2, 3, and 4; and

d) a group of the formula:

Z^a wherein

Q is O, S, NR, ⁺N(0)(R), N(0R), ⁺N(0)(0R), or N-NR2;

Z¹ and Z², when taken together, are -Q'fCfR^i^Q¹-;

wherein each Q¹ is independently O, S, or NR; and each R^y is independently H, F, Cl, Br, I, OH, R, -C(=Q²)R, -C(=Q²)OR, C(=Q²)N(R)2, -N(R)2, -W, -SR, -S(O)R, -S(0)2R, -S(O)(OR), S(O)2(OR), -OC^Q^R, -OC(=Q²)OR, -OC(=Q²)(N(R)2)_s -SC(=Q²)R, SC(=Q²)OR, -SC(=Q²)(N(R)2), -N(R)C(=Q²)R, -N(R)C(=Q²)OR, N(R)C(=Q²)N(R)2, -SO2NR2, -CN, -N₃, -NO₂, -OR, or Z³; or when taken together, two R^y on the same carbon atom form a carbocyclic ring of 3 to 7 carbon atoms;

each Q² is independently, O, S, NR, “N(0)(R), N(0R), N(O)(OR), orN-NR₂;or Z¹ and Z² are each, independently, a group of the Formula la:

Formula la wherein:

each Q³ is independently a bond, O, CR2, NR, ^hN(0)(R), N(0R), ⁺N(0)(0R), N-NR2, S, S-S, S(O), or S(0)₂;

M2 is 0, 1 or 2;

each R* is independently R^y or the formula:

whereîn:

each Mla, Mlc, and Mld is independently 0 or I, M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

Z³ isZ⁴ or Z⁵;

Z⁴ is R, -C(Q²)R^y, -C(Q²)Z⁵, -SO2R^y, or -SO2Z⁵; and

Z⁵ is a carbocycle or a heterocycle whereîn Z⁵ is independently substituted with 0 to 3 R^y groups;

each R^s is halogen, NRⁿR¹², N(Rⁿ)0Rⁿ, NR^R^R¹², N3, NO, NO2, CHO, CN, CH(=NRⁿ), -CH=NNHRⁿ, -CH^OR¹¹), -CH(OR^h)2, -C(=0)NRⁿR¹², -C(=S)NR^lIR¹², -C(=0)0Rⁿ, (Ci-Cg)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C4“C₈)carbocyclylalkyl, optionally substituted aryl, optionally substituted heteroaryl, -C(=O)(Ci-Cg)aIkyl, -S(O)_n(C]-C₈)alkyl, aryl(Ci-C₈)alkyl, OR’¹ or SR¹¹;

each R⁹ or R^w is independently H, halogen, NR”R¹², N^JOR¹¹, NR’WR¹², N3, NO, NO2, CHO, CN, -CH(=NRⁿ), -CH=NHNRⁿ, -CH=N(0R^h), -CH(0R¹¹)2, -C(=O)NRⁿR¹², -C(=S)NRⁿR¹², -C(=O)ORⁿ, R¹¹, OR¹¹ or SRⁿ; and whereîn each (Ci-Cg)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl or aryl(Ci-C₈)alkyl of each R², R³, R⁵, or R⁶ is, independently, optionally substituted with one or more halo, hydroxy, CN, N3, N(R^a)2 or OR^a; and whereîn one or more of the non-terminal carbon atoms of each said (Cj-C8)alkyl may be optionally replaced with -O-, -Sor -NR^a-,

[0071] In another embodiment, provîded is a compound of Formula IV:

Formula IV or a pharmaceutically acceptable sait, hydrate or ester, thereof;

wherein R⁷ is as defined above for Formula I.

[0072] Provided, is a method for treating a Filoviridae infection in a hutnan in need thereof comprising administering a therapeutically effective amount of a compound of Formula IV:

Formula IV or a pharmaceutically acceptable sait or ester, thereof; wherein:

R⁷ is selected from the group of

a) H, O)R^;1, -C(=O)OR^H, -C(=O)NR¹¹R¹², -C(=O)SR^U, -S(O)R^H, -S(O)2R^1!, S(O)(ORⁿ), -S(O)2(OR^h), or-SO₂NR^hR¹² , wherein each R¹¹ or R¹² is independently H, (Ci-Cs)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C4Cg)carbocyclyIalkyl, optionally substituted aryl, optionally substituted heteroaryl, -C(=O)(Ci-C₈)alkyl, -S(O)_n(Ci-C₈)alkyl or aryI(Ci-C₈)alkyI; or R¹¹ and R¹² taken together with a nitrogen to which they are both attached form a 3 to 7 membered heterocyclic ring wherein any one carbon atom of said heterocyclic ring can optionally be replaced with -O-, -S- or-NR^a-;

each R^a is independently H, (Ci-Cs)alkyl, (C₂-Cg)alkenyl, (C2-Cg)alkynyl, aryl(CiC₈)a1kyl, (C4-C₈)carbocyclylalkyl, -C(=0)R, -C(=O)OR, -C(=O)NR₂, -C(=0)SR, -S(O)R, -S(O)₂R, -S(O)(OR), -S(0)₂(0R), or-SO₂NR₂;

wherein each R is independently H, (Ci-C₈) alkyl, (Ci-Cg) substituted alkyl, (C₂C₈)alkenyl, (C₂-Cs) substituted alkenyl, (Cs-Cg) alkynyl, (C₂-C₈) substituted alkynyl, O-Cso aryl, C&-C₂o substituted aryl, C₂-C₂o heterocyclyl, C₂-C₂o substituted heterocyclyl, arylalkyl or substituted arylalkyl; and wherein each (Ci-C₈)alkyl, (C₂-Cs)alkenyl, (C₂-Cg)alkynyl or aryI(Ci-Cs)alkyl of each R¹¹ or R¹² is, independently, optionally substituted with one or more halo, hydroxy, CN, N2, N(R^a)2or OR^a; and wherein one or more of the non-terminal carbon atoms of each said (Ci-Cs)alkyl may be optionally replaced with -O-, -Sor -NR^a-, and

b)

O //

HO—P--$/ j

HO ,

c) a group selected from:

wherein:

R^c is selected from phenyl, l-naphthyl, 2-naphthyl,

R^; is H or CH₃;

R^e is H or Ci-Ce alkyl;

R¹ is selected from H, Ci-Cg alkyl, benzyl, C₃-Cs cycloalkyl, and -CH2-C3-C6 cycloalkyl;

R^s is selected from Ci-Ce alkyl, -O-Ci-Cs alkyl, benzyl, -O-benzyl, -CH2-C3-C6 cycloalkyl, -O“CH₂-C₃-C6 cycloalkyl, and CF₃; and n’ is selected from l, 2, 3, and 4; and

d) a group of the formula:

wherein

Q is O, S, NR, ^HN(0)(R), N(0R), ⁺N(O)(OR), or N-NR·;

Z¹ and Z², when taken together, are -Q'ÇCfR^lsQ¹-, wherein each Q¹ is independently O, S, or NR; and each R^v is independently H, F, Cl, Br, I, OH, R, -C(=Q²)R, -C(=Q²)OR, C(=Q²)N(R)2, -N(R)2, -WX -SR, -S(O)R, -S(O)2R, -S(OXOR), S(O)2(OR), -OC(=Q‘)R, -OC(=Q²)OR, -OC(=Q²)(N(R)2), -SC(=Q²)R, SC( Q²)OR: -SC(=Q²)(N(R)2), -N(R)C(=Q²)R, -N(R)C(=Q²)OR, N(R)C(=Q²)N(R)₂, -SO₂NR₂, -CN, -N₃, -NO2, -OR, or Z³, or when taken together, two R^y on the same carbon atom form a carbocyclic ring of 3 to 7 carbon atoms;

each Q² is independently, O, S, NR, ⁺N(0)(R), N(0R), ⁺N(O)(OR), orN-NR₂;or Z¹ and Z² are each, independently, a group of the Formula la:

Formula la wherein:

each Q³ is independently a bond, O, CR2, NR, ⁺N(O)(R), N(0R), ⁺N(O)(OR), N~NR2> S, S-S, S(O), or S(O)₂;

M2 is 0, 1 or 2;

each R^x is independently R^y or the formula:

wherein:

each Mla, Mlc, and Mld is independently 0 or 1;

M12c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

Z³ is Z⁴ or Z⁵;

Z⁴ is R, -C(Q²)R^y, -C(Q²)Z⁵, -SO2R^y, or -SO2Z⁵; and

Z⁵ is a carbocycle or a heterocycle wherein Z⁵ is independently substituted with 0 to 3 R^y groups.

[0073] Provided, is a method for treatîng a Filoviridae infection in a human in need thereof comprising administering a therapeutically effective amount of a compound of Formula IV:

Formula IV or a pharmaceutically acceptable sait, hydrate or ester, thereof; wherein.

R⁷ is selected from the group consisting of

a) H, -C(=O)R^H, -C(=0)0R^h, -C(=0)NR^llR¹², -C(=O)SR^U, -S(O)Rⁿ, -S(O)2R^h, -S(O)(OR^h), -S(O)2(OR^u), or -SO^’R¹²;

b)

O il HO—P--5 / £

HO

c) a group selected from:

R^c is selected from the group of phenyl, 1-naphthyl, 2-naphthyl,

R^d is selected from the group of H or CH3,

R^el and R^e2 are each independently selected from the group of H, (Ci-Ce)alkyl or benzyl;

R^f is selected from the group of from H, (Ci-Cs)alkyl, benzyl, (C3Cô)cycloalkyl, and -CH2-(C₃-C6)cycloalkyl;

R^g is selected from selected from the group of (Ci-Cs)alkyl, 0-(Ci-C8)alkyl, benzyl, -O-benzyl, CH₂-(C₃-C₆)cycloalkyl, -O-CH₂-(C₃-C₆)cycloalkyl, and CF₃, and n' is an integer selected from the group of 1, 2, 3, and 4; and d) a group of the formula:

Q

Z² wherein:

Q is selected from the group of O, S, NR, N(0)(R), N(0R), ⁺N(O)(OR), or N-NR2;

Z¹ and Z², when taken together, are -Q^l(C(R^y)2)₃Q’-;

wherein each Q¹ is independently selected from the group of O, S, or NR; and each R^y is independently selected from the group of H, F, Cl, Br, I, OH, R, -C(=Q²)R, -C(=Q²)OR, C(=Q³)N(R)2, -N(R)2, -⁺N(R)3s -SR, -S(O)R, S(O)₂R, -S(O)(OR), -S(0)2(0R), -OC(=Q‘)R, OC(=Q²)OR, -OC(=Q²XN(R)2), -SC(=Q²)R, SC(=Q²)OR, -SC(=Q²XN(R)2), -N(R)C(=Q²)R, 33

N(RKX-Q^?)OR, -N(R)C(=Q²)N(R)₂, -SO₂NR₂, -CN, -N3, -NO₂, -OR, or Z³; or when taken together, two R^y on the same carbon atom form a carbocyclic ring of 3 to 7 carbon atoms;

each Q² is independently, O, S, NR, 'N(0)(R), N(0R), ^hN(0)(0R), or N-NR₂; or

Z¹ and Z² are each, independently, a group of the Formula la:

wherein:

each Q³ is independently selected front the group of a bond, O, CR2, NR, ⁺N(0)(R), N(0R), ⁺N(0)(0R), N-NR2, S, S-S, S(O), or S(O)₂;

M2 is an integer selected front the group of 0, 1 or 2; each R^x is independently R^y or the formula:

wherein:

each Mla, Mlc, and Mld is an integer independently selected front the group of 0 or 1;

M12c is an integer selected front the group of 0, I, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

Z³ is Z⁴ or Z⁵;

Z⁴ is R, -C(Q²)R^y, -C(Q²)Z⁵, -SO2R^y, or -SO2Z³;

and

Z⁵ is a carbocycle or a heterocycle wherein Z⁵ is independently substituted with 0 to 3 R^y group s;

each R¹¹ or R¹² is independently H, (Ci-Cs)alkyl, (C2-Cs)alkenyl, (Cî-Cs)alkynyl, (C4-Cs)carbocyclylalkyl, (C6-C2o)optionally substituted aryl, optionally substituted heteroaryl, -C(=O)(Ci-Cs)alkyl, -S(O)n(Ci-Cs)alkyI or (C6-C2o)aryl(Ci-C8)alkyî, or R¹¹ and R¹² taken together with a nitrogen to which they are both attached fbrm a 3 to 7 membered heterocyclic ring wherein any one carbon atom of said heterocyclic ring can optionally be replaced with -O-, -S- or -NR^a-, each R^a is independently selected from the group of H, (Ci-Csjalkyl, (C2-Cs)alkenyl, (C2-Cs)a1kynyl, (C6-C₂o)aryl(Ci-C₈)alkyl, (C4-C8)carbocyclyl alkyl, -C(=0)R, C(- O)OR, -C(=O)NR₂, -C(=O)SR, -S(O)R, -S(O)2R, -S(OXOR). -S(O)2(OR), or -SO2NR2, wherein each R is independently selected from the group of H, (Ci-Cs) alkyl, (Ci-Cs) substituted alkyl, (C2-Cg)alkenyl, (C₂-Cs) substituted alkenyl, (Cs-Ce) alkynyl, (C2-C8) substituted alkynyl, (C₆-C₂ü)aryl, (C6-C₂o)substituted aryl, (C2-C₂o)heterocyclyl, (C₂-C2o)substituted heterocyclyl, (C6-C2o)aryl(Ci-C₈)alkyl or substituted (Câ-C2o)aryl(Ci-Cs)alkyl;

each n is an integer independently selected from the group of 0, 1, or 2; and wherein each (Ci-Cs)alkyl, (C₂-Cg)alkenyl, (C₂-Cs)alkynyl or (C6-C₂o)aryl(Ci-Cs)alkyi of each R¹¹ or R¹² is, independently, optionally substituted with one or more substituents selected from the group of halo, hydroxy, CN, Ns, N(R^a)₂ or OR^a; and wherein one or more of the non-terminal carbon atoms of each said (CiCs)alkyl may be optionally replaced with -O-, -S- or NR^a[0074] In another embodiment of a compound of Formula IV, R⁷ can be H. In another embodiment of a compound of Formula IV, R⁷ is selected from the group of a), b), or c) as defined for Formula IV.

[0075] In some embodiments, R^el and R^e2 can each independently be selected from the group of H, Ci-Cû alkyl or benzyl. In some embodiments, R^el can be H, Ci-Cô alkyl or benzyl, and R^e2 can be H or Ci-Cô alkyl. In some embodiments, R^el and R^e2 can each independently be H or Ci-Ce alkyl. In some embodiments, R^eI and R^e2 can each independently be H or benzyl. In some embodiments, R^el can be H, methyl or benzyl, and R^e2 can be H or methyl. In some embodiments, R^el can be H or methyl, and R^e2 can be H or methyl. In some embodiments, R^e) can be methyl, and R^e2 can be H or methyl. In some embodiments, R^el can be H or benzyl, and R^e2 can be H or methyl.

[0076] In another embodiment of a compound of Formula IV, R⁷ is

wherein R^f is selected from the group of H, Ci-Cs alkyl, benzyl, Cï-Cs cycloalkyl, and -CH2-C3Cc cycloalkyl. In another embodiment of a compound of Formula IV, R^f is Ci-Cs alkyl.

[0077] In another embodiment of a compound of Formula IV, R⁷ is

wherein

R^f is selected from the group of H, Ci-Cs alkyl, benzyl, Cî-Cô cycloalkyl, and -CH2-C3Cs cycloalkyl; and

R⁸ is selected from the group of Ci-Cg alkyl, -O-Ci-Cs alkyl, benzyl, -O-benzyl, -CH₂C₃-Cô cycloalkyl, -O-CH2-C3-C6 cycloalkyl, and CF₃.

[0078] In another embodiment of a compound of Formula IV, R⁷ is

wherein R^f is selected from the group of H, Ci-Cs alkyl, benzyl, C₃-Cô cycloalkyl, and CH2-C3Ce cycloalkyl. In another embodiment of a compound of Formula IV, R^f is Ci-Cs alkyl. In another embodiment of a compound of Formula IV, R^f is Ci-Cr, alkyl.

[0079] In another embodiment of a compound of Formula IV, R⁷ is:

O

wherein R^g is selected from the group of Ci-Cs alkyl, -O-Ci-Cg alkyl, benzyl, -O-benzyl, -CH₂C₃-Cû cycloalkyl, -O-CH₂-C₃-C₆ cycloalkyl, and CF₃ In another embodiment of a compound of Formula IV, R^f is Ci-Cs alkyl. In another embodiment of a compound of Formula IV, R^f is CiCe alkyl.

[0080] In another embodiment of a compound of Formula IV, R⁷ is selected from the group of

[0081] In another embodiment of a compound of Formula IV, R⁷ is

O // £

HO—P--S/ 5

HO '

[0082] In another embodîment, provided is a compound of Formula IV that is:

or a pharmaceutically acceptable sait or ester thereof.

[0083] In another embodiments, provided is a compound of Formula IV that is:

or a pharmaceutically acceptable sait or ester thereof.

[0084] In another embodiments, provided is a compound of Formula IV that is:

or a pharmaceutîcaily acceptable sait, hydrate, or ester thereof.

[0085] In another embodîment, provided is a compound of Formula IV that is

or a pharmaceutically acceptable sait or ester thereof.

[0086] In another embodiment, the présent invention provides a compound that is

or a pharmaceutically acceptable sait or ester thereof.

[0087] In another embodiment, the présent invention provides a compound that is

or a pharmaceutically acceptable sait, hydrate, or ester thereof.

[0088] In another embodiment, the présent invention provides a compound that is

or a pharmaceutically acceptable sait or ester thereof.

[0089] In another embodiment, the présent invention provides a compound that is

or a pharmaceutically acceptable sait or ester thereof

[0090] In another embodiment, the présent invention provides a compound that is

or a pharmaceutically acceptable sait or ester thereof.

[0091] Names of compounds of the présent disclosure are provided usmg ACD/Name software for naming Chemical compounds (Advanced Chemistry Development, Inc., Toronto, Canada). Other compounds or radicals may be named with common names or systematic or non-systematic names. The naming and numbering of the compounds of the disclosure is illustrated with a représentative compound of Formula IV:

which is named (2S)-2-ethylbutyl 2-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[l,2-f|[l,2,4]triazin-7y 1 )- 5 -cyano-3,4-dihy droxytetrahydrofuran-2-yl)methoxy )(phenoxy )phosphoryl amino) propanoate. Other compounds of the présent invention include:

which is named (S)-2-ethylbutyl 2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,l-f][î,2,4] triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl) amino)propanoate, and

which is named (S)-2-ethylbutyl 2-(((R)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,l-f][l,2,4] triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl) amino)propanoate.

(S)-2-ethylbutyl 2-(((R)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,l-f][l,2,4]triazin-7-yl)-5-cyano3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate may also be illustrated as

[0092] Any reference to the compounds of the invention described herein also includes a reference to a physiologically acceptable sait thereof. Examples of physiologically acceptable salts of the compounds of the invention include salts derived from an appropriate base, such as an alkali métal or an alkaline earth (for example, Na⁺, Li⁺, K⁺’ Ca⁺2 and Mg⁺^), ammonium and NR4” (wherein R is defined herein). Physiologically acceptable salis of a nitrogen atom or an amino group include (a) acid addition salts formed with inorganic acids, for example, hydrochioric acid, hydrobromic acid, sulfurîc 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, cîtric acid, malic acid, ascorbic acid, benzoic acid, isethionic acid, lactobionic acid, tannic acid, palmitîc acid, alginîc acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, malonic acid, sulfosalîcylic acid, glycolic acid, 2-hydroxy-3-naphthoate, pamoate, salicylic acid, stearîc acid, phthalic acid, mandelic acid, lactic acid, ethanesulfonic acid, lysine, arginine, glutamic acid, glycine, serine, threonine, alanine, isoleucine, leucine and the like, and (c) salts formed from elemental anions for example, chlorîne, bromine, and iodine. Physiologically acceptable salts of a compound of a hydroxy group include the anion of saîd compound in combination with a suitable cation such as Na⁺ and NRZ.

[0093] A compound of Formula IV and its pharmaceutically acceptable salts may exist as different polymorphs or pseudopolymorphs. As used herein, crystalline polymorphism means the abîlity of a crystalline compound to exist in different crystal structures. The crystalline polymorphism may resuit from différences in crystal packing (packing polymorphism) or différences in packing between different conformers of the same molécule (conformât!onal polymorphism). As used herein, crystalline pseudopolymorphism means the ability of a hydrate or solvaté of a compound to exist in different crystal structures. The pseudopolymorphs of the instant invention may exist due to différences in crystal packing (packing pseudopolymorphism) or due to différences in packing between different conformers of the same molécule (conformational pseudopolymorphism). The instant invention comprises ail polymorphs and pseudopolymorphs of the compounds of Formula IV and their pharmaceutîcaily acceptable salts.

[0094] A compound of Formula IV and its pharmaceutîcaily acceptable salis may also exist as an amorphous solid. As used herein, an amorphous solid is a solid in which there is no longrange order ofthe positions of the atoms in the solid. This définition applies as well when the crystal size is two nanometers or less. Additives, including solvents, may be used to create the amorphous forms of the instant invention. The instant invention comprises ail amorphous forms of the compounds of Formula IV and their pharmaceutîcaily acceptable salts.

[0095] For therapeutic use, salts of active ingrédients of the compounds of the invention will be physiologically acceptable, i.e. they will be salts derîved from a physiologically acceptable acid or base. However, salts of acids or bases which are not physiologically acceptable may also find use, for example, in the préparation or purification of a physiologically acceptable compound. Ail salts, whether or not derived form a physiologically acceptable acid or base, are within the scope of the présent invention.

[0096] Finally, it is to be understood that the compositions herein comprise compounds of the invention in their un-ionized, as well as zwitterionic form, and combinations with stoichiometric amounts of water as in hydrates.

[0097] It is to be noted that ail enantîomers, diastereomers, and racemic mixtures, tautomers, polymorphs, pseudopolymorphs of compounds within the scope of Formula IV and pharmaceutîcaily acceptable salts thereof are embraced by the présent invention. Ail mixtures of such enantîomers and diastereomers are within the scope of the présent invention.

[0098] The compounds of the invention, exemplified by Formula IV may hâve chiral centers, e.g. chiral carbon or phosphorus atoms. The compounds of the invention thus include racemic mixtures of ail stereoisomers, including enantîomers, diastereomers, and atropisomers. In addition, the compounds of the invention include enriched or resolved optical isomers at any or ail asymmetric, chiral atoms. In other words, the chiral centers apparent from the depictions are provided as the chiral isomers or racemic mixtures. Both racemic and diastereomeric mixtures, as well as the individual optical isomers isolated or synthesized, substantially fiee of their enantiomeric or diastereomeric partners, are ail within the scope of the invention. The racemic mixtures are separated into their individual, substantially optically pure isomers through wellknown techniques such as, for example, the séparation of diastereomeric salts formed with optically active adjuncts, e.g., acids or bases followed by conversion back to the optically active substances. In most instances, the desired optical isomer is synthesized by means of stereospecific réactions, beginning with the appropriate stereoisomer of the desired starting material.

[0099] Stereochemical définitions and conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wîlen, S., Stereochemistry ofOrganic Compounds (1994) John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active forms, i.e., they hâve the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the préfixés D and L or R and S are used to dénoté the absolute configuration of the molécule about its chiral center(s). The préfixés d and 1, D and L, or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with S, (-), or 1 meanîng that the compound is levorotatory while a compound prefixed with R, (+), or d is dextrorotatory. For a given Chemical structure, these stereoisomers are identical except that they are mirror images of one another. A spécifie stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a Chemical reaction or process. The terms racemic mixture and racemate refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.

[0100] The compounds of the invention can also exist as tautomeric isomers in certain cases. Although only one delocalized résonance structure may be depicted, ail such forms are contemplated within the scope of the invention. For example, ene-amine tautomers can exist for purine, pyrimidine, imidazole, guanidine, amidine, and tetrazole Systems and ail their possible tautomeric forms are within the scope of the invention.

10101 ] Any formula or structure given herein, including Formula IV compounds, is also intended to represent unlabeled forms as well as isotopically labeled forms of the compounds. Isotopically labeled compounds hâve structures depicted by the formulas given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into compounds of the disclosure include isotopes ofhydrogen, carbon, nîtrogen, oxygen, phosphorous, fluorine and chlorine, such as, but not limited to ²H (deuterium, D), ³H (tritium), ^HC, ¹³C, ¹⁴C, ’⁵N, ¹⁸F, ³¹P, ³²P, ³⁵S, ³⁶C1 and ⁱ²⁵I. Varions isotopically labeled compounds of the présent disclosure, for example those into which radioactive isotopes such as ³H, ^!3C and ¹⁴C are incorporated. Such isotopically labeled compounds may be useful in metabolic studies, reaction kinetic studies, détection or imaging techniques, such as positron émission tomography (PET) or single-photon émission computed tomography (SPECT) including drug or substrate tissue distribution assays or in radioactive treatment of patients.

[0102] The disclosure also included compounds of Formula IV in which from 1 to n hydrogens attached to a carbon atom is/are replaced by deuterium, in which n is the number of hydrogens in the molécule. Such compounds exhibit increased résistance to métabolisai and are thus useful for increasing the half-life of any compound of Formula IV when administered to a mammal, particularly a human. See, for example, Foster, “Deuterium Isotope Effects in Studies of Drug Métabolisai”, Trends Pharmacol. Sci. 5(12):524-527 (1984). Such compounds are synthesized by means well known in the art, for example by employing starting materials in which one or more hydrogens hâve been replaced by deuterium.

[0103] Deuterium labeled or substituted therapeutîc compounds of the disclosure may hâve improved DMPK (drug metabolism and pharmacokinetics) properties, relating to distribution, metabolism and excrétion (ADME). Substitution with heavier isotopes such as deuterium may afford certain therapeutîc advantages resulting from greater metabolic stability, for example increased in vivo half-life, reduced dosage requirements and/or an improvement in therapeutîc index. An ^1SF labeled compound may be useful for PET or SPECT studies. Isotopically labeled compounds of this disclosure and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and préparations described beiow by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent. It is understood that deuterium in this context is regarded as a substituent in the compound of Formula IV.

[0104] The concentration of such a heavier isotope, specifically deuterium, may be defmed by an isotopic enrichment factor. In the compounds of this disclosure any atom not specifically designated as a particular isotope is meant to represent any stable isotope of that atom. Unless otherwise stated, when a position is designated specîfically as H or hydrogen, the position is understood to hâve hydrogen at îts natural abundance isotopic composition. Accordîngly, in the compounds of this disclosure any atom specifically designated as a deuterium (D) is meant to represent deuterium.

[0105] Whenever a compound described herein is substituted with more than one of the same designated group, e.g., R or R¹, then it will be understood that the groups may be the same or different, î.e., each group is independently selected. Wavy lines, , indicate the site of covalent bond attachments to the adjoining sub structures, groups, moieties, or atoms.

[0106] Selected substituents comprising the compounds of Formula IV are présent to a recursive degree. In this context, “recursive substituent” means that a substituent may récité another instance of itself. Because of the recursive nature of such substituents, theoretically, a large number of compounds may be présent in any given embodiment. For example, R^N comprises a R^y substituent. R^y can be R. R can be Z³. Z³ can be Z⁴ and Z⁴ can be R or comprise substituents comprising R^y. Altematively, Z³ can be Z⁵ which can comprise substituents comprising R^y One of ordinary ski 11 in the art of médicinal chemistry understands that the total number of such substituents is reasonably limited by the desired properties ofthe compound intended. Such properties include, by way of example and not limitation, physîcal properties such as molecular weight, solubility or log P, application properties such as activity against the intended target, and practical properties such as ease of synthesis.

[0107] By way of example and not limitation, Z³ and R^y are recursive substituents in certain embodiments. Typically, each recursive substituent can independently occur 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0, tîmes in a given embodiment. More typically, each recursive substituent can independently occur 12 or fewer times in a given embodiment. Even more typically, each recursive substituent can independently occur 3 or fewer times in a given embodiment. For example, Z³ will occur 0 to 8 times, R^y will occur 0 to 6 times in a given embodiment. Even more typically, Z³ will occur 0 to 6 times and R^y will occur 0 to 4 times in a given embodiment.

[0108] Recursîve substituents are an intended aspect of the invention. One of ordînary ski 11 in the art of médicinal chemistry understands the versatilîty of such substituents. To the degree that recursîve substituents are présent in an embodiment of the invention, the total number will be determined as set forth above.

[0109] The compounds of the présent invention can be prepared by methods known to one of skill in the art. For example, the compounds of the présent invention can be prepared according to the methods described in U.S. Patent No. 8,008,264 and U.S. Application Publication No. US 2012/0027752.

A. Métabolites of the Compounds of the Invention

[0110] Also falling within the scope ofthis invention are the in vivo metabolic products ofthe compounds described herein, to the extent such products are novel and unobvious over the prior art. Such products may resuit for example from the oxidation, réduction, hydrolysis, amidation, estérification and the like of the administered compound, primarîly due to enzymatic processes. Accordingly, the invention includes novel and unobvious compounds produced by a process comprising contacting a compound of this invention with a mammal for a period of tîme sufficîent to yield a metabolic product thereof. Such products typically are identified by preparîng a radiolabelled (e.g. or 3H) compound of the invention, administering it parenterally in a détectable dose (e.g. greaterthan about 0.5 mg/kg) to an animal such as rat, mouse, guinea pig, monkey, or to man, allowing sufficient time for metabolism to occur (typically about 30 seconds to 30 hours) and isolating its conversion products from the urine, blood or other biological samples. These products are easily isolated since they are labeled (others are isolated by the use of antibodies capable of binding epitopes surviving in the métabolite). The métabolite structures are determined in conventional fashion, e.g. by MS or NMR analysis. In general, analysis of métabolites is done in the same way as conventional drug metabolism studîes well-known to those skill ed in the art. The conversion products, so long as they are not otherwise found in vivo, are usefol in diagnostic assays for therapeutic dosing of the compounds of the invention even if they possess no anti Filoviridae activity of their own.

[OUI] Recipes and methods for determining stability of compounds in surrogate gastrointestinal sécrétions are known. Compounds are defîned herein as stable in the gastrointestinal tract where less than about 50 mole percent ofthe protected groups are deprotected in surrogate intestinal or gastric juice upon incubation for 1 hour at 37 °C. Simply because the compounds are stable to the gastrointestinal tract does not mean that they cannot be hydrolyzed m vivo. The prodrugs of the invention typically wîl 1 be stable in the digestive System but may be substantially hydrolyzed to the parental drug in the digestive lumen, liver or other metabolic organ, or within cells in general.

IIL PHARMACEUTICAL FORMULATIONS

[0112] The compounds of this invention are formulated with conventional carriers and excipients, which will be selected in accord with ordinary practice. Tablets will contain excipients, glidants, fillers, binders and the like. Aqueous formulations are prepared in stérile form, and when intended for delivery by other than oral administration generally will be isotonie. Ail formulations will optionally contain excipients such as those set forth in the Handbook of Pharmaceuticaï Excipients (1986). Excipients include ascorbic acid and other antioxidants, chelatîng agents such as EDTA, carbohydrates such as dextran, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and the like. The pH ofthe formulations ranges from about 3 to about 11, but is ordinarily about 7 to 10. In some embodiments, the pH of the formulations ranges from about 2 to about 5, but is ordinarily about 3 to about 4. In some embodiments, the pH of the formulations ranges from about 2 to about 10, but is ordinarily about 3.5 to about 8.5.

[0113] While it is possible for the active ingrédients to be admînistered alone it may be préférable to présent them as pharmaceuticaï formulations. The formulations, both for veterînary and for human use, of the invention comprise at least one active ingrédient, as above defined, together with one or more acceptable carriers therefor and optionally other therapeutîc ingrédients, particularly those additional therapeutîc ingrédients as discussed herein. The carrier(s) must be acceptable in the sense of being compatible with the other ingrédients of the formulation and physiologically innocuous to the récipient thereof.

[0114] The formulations include those suitable for the foregoing administration routes. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations generally are found in Remington's Pharmaceuticaï Sciences (Mack Publîshing Co., Easton, PA). Such methods include the step of bringing into association the active ingrédient with the carrier which constitutes one or more accessory ingrédients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingrédient with liquid carriers or fînely divided solid carriers or both, and then, if necessary, shaping the product.

[0115] Formulations of the présent invention suitable for oral administration may be presented as discrète units such as capsules, cachets or tablets each containîng a predetermined amount of the active ingrédient; as a powder or granules, as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid émulsion or a water-in-oil liquid émulsion. The active ingrédient may also be administered as a bol us, electuary or paste.

[0116] A tablet is made by compression or molding, optionally with one or more accessory ingrédients. Compressed tablets may be prepared by compressing in a suitable machine the active ingrédient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservatîve, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingrédient moistened with an inert liquid diluent. The tablets may optionally be coated or scored and optionally are formulated so as to provide slow or controlled release of the active ingrédient therefrom.

[0117] For infections of the eye or other external tissues e.g. mouth and skin, the formulations are preferably applied as a topical ointment or cream containîng the active ingredient(s) in an amount of, for example, 0.075 to 20% w/w (including active ingredient(s) in a range between 0.1% and 20% in incréments of 0.1% w/w such as 0.6% w/w, 0.7% w/w, etc.), preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w. When formulated in an ointment, the active ingrédients may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingrédients may be formulated in a cream with an oil-in-water cream base.

[0118] If desired, the aqueous phase of the cream base may include, for example, at least 30% w/w of a polyhydric alcohol, i.e. an alcohol having two or more hydroxyl groups such as propylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400) and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or pénétration of the active ingrédient through the skin or other affected areas. Examples of such dermal pénétration enhancers include dimethyl sulphoxide and related analogs.

[0119] The oily phase of the émulsions ofthis invention may be constituted from known ingrédients in a known manner. While the phase may comprise merely an emulsifier (otherwise known as an emulgent), it desîrabiy comprises a mixture of at least one emulsifier with a fat or an oii or with both a fat and an oil. Preferably, a hydrophiiîc emulsifier is încluded together with a lipophilie emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax, and the wax together with the oil and fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.

[0120] Emulgents and émulsion stabilizers suitable for use in the formulation of the invention include Tween® 60, Span® 80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodium lauryl sulfate. Further emulgents and émulsion stabilizers suitable for use in the formulation of the invention include Tween® 80.

[0121] The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties. The cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as dî-îsoadipate, isocetyl stéarate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stéarate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination dependîng on the properties required. Alternative!y, high melting point lipids such as white soft paraffm and/or liquîd parafïin or other minerai oils are used.

[0122] Pharmaceutical formulations according to the présent invention comprise a combination according to the invention together with one or more pharmaceutically acceptable carriers or excipients and optionally other therapeutic agents. Pharmaceutical formulations containing the active ingrédient may be in any form suitable for the intended method of administration. When used for oral use for example, tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, émulsions, hard or soft capsules, syrups or élixirs may be prepared. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents including sweetening agents, flavoring agents, coloring agents and preserving agents, in orderto provide a palatable préparation. Tablets containing the active ingrédient in admixture with non-toxic pharmaceutically acceptable excipient which are suitable for manufacture of tablets are acceptable. These excipients may be, for example, inert diluent s, su ch as calcium or sodium carbonate, lactose, calcium or sodium phosphate; granulating and disintegrating agents, such as maize starch, or alginic acid; binding agents, such as starch, gelât in or acacia; and lubricating agents, such as magnésium stéarate, stearic acid or talc. Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.

[0123] Formulations for oral use may be also présented as hard gelatin capsules where the active ingrédient is mixed with an inert solid diluent, for example calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingrédient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.

[0124] Aqueous suspensions of the invention contain the active materîals in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcelluose, sodium algînate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally-occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stéarate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbîtan monooleate). Further non-limiting examples of suspending agents include Captisol® (sulfobutyl ether betacyclodextrin, SBE-p-CD). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose or saccharin.

[0125] Oil suspensions may be formulated by suspending the active ingrédient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a minerai oil such as liquid paraffin. The oral suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palatable oral préparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

[0126] Dispersible powders and granules of the invention suitable for préparation of an aqueous suspension by the addition of water provide the active ingrédient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those disclosed above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be présent.

[0127] The pharmaceuticai compositions of the invention may also be in the form of oil-inwater émulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, a minerai oil, such as liquid paraffin, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally-occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The émulsion may also contain sweetening and flavoring agents. Syrups and élixirs may be formulated with sweetening agents, such as glycerol, sorbîtol or sucrose. Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent.

[0128] The pharmaceuticai compositions of the invention may be in the form of a stérile injectable préparation, such as a stérile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art usîng those suitable dispersing or wetting agents and suspending agents which hâve been mentioned above. The stérile injectable préparation may also be a stérile injectable solution or suspension in a non-toxîc parenterally acceptable diluent or solvent, such as a solution in 1,3-butane-diol or prepared as a lyophilized powder. The stérile injectable préparation may also be a stérile injectable solution or suspension in a parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonie sodium chloride solution. In addition, stérile fixed oiis may conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may likewîse be used in the préparation of injectables. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution isotonie sodium chloride solution, hypertonie sodium chloride solution, and hypotonie sodium chloride solution.

[0129] The amount of active ingrédient that may be combined with the carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a time-release formulation intended for oral administration to humans may contain approximately 1 to 1000 mg of active material compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95% of the total compositions (weight:weight). The pharmaceutical composition can be prepared to provide easily measurable amounts for administration. For example, an aqueous solution intended for intravenous infusion may contain from about 3 to 500 pg ofthe active ingrédient per millîliter of solution in order that infusion of a suitable volume at a rate of about 30 mL/hr can occur.

[0130] Formulations suitable for topical administration to the eye also include eye drops wherein the active ingrédient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingrédient. The active ingrédient is preferably présent in such formulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10%, and partîcularly about 1.5% w/w.

[0131] Formulations suitable for topical administration in the mouth include lozenges comprising the active ingrédient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingrédient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingrédient in a suitable liquid carrier.

[0132] Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.

[0133] Formulations suitable for intrapulmonary or nasal administration hâve a partiele size for example in the range of 0.1 to 500 microns, such as 0.5, 1, 30, 35 etc., which is administered by rapîd inhalation through the nasal passage or by inhalation through the mouth so as to reach the alveolar sacs. Suitable formulations include aqueous or oily solutions of the active ingrédient. Formulations suitable for aérosol or dry powder administration may be prepared accordîng to conventional methods and may be delivered with other therapeutic agents such as compounds heretofore used in the treatment or prophylaxis of Filoviridae infections as described below.

[0134] Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingrédient such carriers as are known in the art to be appropriaie.

[0135] Formulations suitable for parentéral administration include aqueous and non-aqueous stérile injection solutions which may contaîn anti-oxidants, buffers, bacteriostats and solutés which render the formulation isotonie with the blood of the intended récipient; and aqueous and non-aqueous stérile suspensions which may include suspending agents and thickening agents.

[0136] The formulations are presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the stérile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from stérile powders, granules and tablets of the kînd previously described. Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the active ingrédient.

[0137] It should be understood that in addition to the ingrédients particularly mentioned above the formulations of this invention may include other agents conventîonal in the art havîng regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

[0138] The invention fùrther provides veterinary compositions comprising at least one active ingrédient as above defined together with a veterinary carrier therefor.

[0139] Veterinary carriers are materials useful for the purpose of administering the composition and may be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingrédient. These veterinary compositions may be administered orally, parenterally or by any other desired route.

[0140] Compounds of the invention are used to provide controlled release pharmaceutical formulations containing as active ingrédient one or more compounds of the invention (controlled release formulations) in which the release of the active ingrédient are controlled and regulated to allow less frequency dosing or to improve the pharmacokinetic or toxicity profile of a given active ingrédient.

IV. ROUTES OF ADMINISTRATION

[0141] One or more compounds of the invention (herein referred to as the active ingrédients) are administered by any route appropriate to the condition to be treated. Suitable routes include oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), vaginal and parentéral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and épidural), and the like. It will be apprecîated that the preferred route may vary with for example the condition of the récipient. An advantage of the compounds of this invention is that they are orally bioavailable and can be dosed orally.

[0142] In the methods of the présent invention for the treatment of Filoviridae infection, the compounds of the présent invention can be administered at any time to a human who may corne into contact with humans suffering from Filoviridae infection or is already suffering from Filoviridae infection. In some embodiments, the compounds of the présent invention can be administered prophylactically to humans coming into contact with humans suffering from Filoviridae infection. In some embodiments, administration of the compounds of the présent invention can be to humans testing positive for Filoviridae infection but not yet showing symptoms of Filoviridae infection. In some embodiments, administration of the compounds of the présent invention can be to humans upon commencement of symptoms of Filoviridae infection.

[0143] Effective dose of active ingrédient dépends at least on the nature of the condition being treated, toxicity, whether the compound is being used prophylactically (lower doses) or against an active viral infection, the method of delivery, and the pharmaceutical formulation, and will be determined by the clinician using conventional dose escalation studies. It can be expected to be from about 0.0001 to about 100 mg/kg body weight per day, typically, from about 0.01 to about 10 mg/kg body weight per day; more typically, from about .01 to about 5 mg/kg body weight per day; most typically, from about .05 to about 0.5 mg/kg body weight per day. For example, the daîly candidate dose for an adult human of approximately 70 kg body weight will range from 1 mg to 1000 mg, preferably between 5 mg and 500 mg, and may take the form of single or multiple doses.

[0144] The effective dose of a compound of the présent invention for treating the Filoviridae infection can dépend on whether the dose is to be used prophylactically or to treat a human already suffering from Filoviridae infection. Moreover, the dose can dépend on whether the human suffering from Filoviridae infection does not yet show symptoms or is already showing symptoms of Filoviridae infection. Larger doses may be necessary for treating humans testing positive for Filoviridae infection and for humans showing symptoms of Filoviridae infection as compared to humans receiving prophyîactic treatment.

[0145] Any suitable period of time for administration of the compounds of the présent invention is contemplated. For example, administration can be for from 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 for from 1 week to 15 weeks, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 weeks. Longer periods of administration are also contemplated. The time for administration can dépend on whether the compound is being administered prophylactically or to treat a human suffering from a Filoviridae infection. For example, a prophyîactic administration can be for a period of time while the human is in regular contact with other humans suffering from a Filoviridae infection, and for a suitable period of time following the last contact with a human suffering from a Filoviridae infection. For humans already suffering from a Filoviridae infection, the period of administration can be for any length of time necessary to treat the patient and a suitable period of time following a négative test for Filoviridae infection to ensure the Filoviridae infection does not retum.

V. COMBINATION THERAPY

[0146] Compositions of the invention are also used in combination with other active ingrédients. For the treatment of Filoviridae virus infections, preferably, the other active therapeutic agent is active agaînst Filoviridae virus infections, particularly Marburg virus, Ebola virus and Cueva virus infections. Non-limiting examples of these other active therapeutic agents are ribavirin, palivizumab, motavizumab, RSV-IGTV (RespiGam®), MEDI-557, A-60444, MDT-637, BMS-433771, amiodarone, dronedarone, verapamil, Ebola Convalescent Plasma (ECP), TKM-100201, BCX4430 ((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, T-705 triphosphate, FGI-106 (l-N,7-N-bîs[3(dimethylamino)propyl]-3,9-dimethylquinolino[8,7-h]quinolone-l,7-diamine), JK-05, TKMEbola, ZMapp, rNAPc2, VRC-EBOADC076-00-VP, OS-2966, MVA-BN filo, brincidofovir, Vaxart adenovirus vector 5-based ebola vaccine, Ad26-ZEBOV, FiloVax vaccine, GOVX-E301, GOVX-E302, ebola virus entry inhibitors (NPC1 înhibitors), and rVSV-EBOV, and mixtures thereof. The compounds and compositions of the présent invention may also be used in combination with phosphoramidate morpholino oligomers (PMOs), which are synthetic antisense oligonucleotide analogs designed to interfère with translational processes by forming base-pair duplexes with spécifie RNA sequences. Examples of PMOs include AVI-7287, A VI7288, AVI-7537, AVI-7539, AVI-6002, and AV1-6003. The compounds and compositions of the présent invention are also intended for use with general care provided patients with Filoviridae viral infections, including parentéral fluids (including dextrose saline and Ringer’s lactate) and nutrition, antibiotic (including metronidazole and cephalosporin antibiotics, such as ceftriaxone and cefuroxime) and/or antifungal prophylaxis, fever and pain médication, antiemetic (such as metoclopramide) and/or antidiarrheal agents, vîtamin and minerai suppléments (including Vitamin K and zinc sulfate), anti-inflammatory agents ( such as ibuprofen), pain médications, and médications for other common diseases in the patient population, such anti-malarial agents (including artemether and artesunate-lumefantrine combination therapy), typhoid (including quinolone antibiotics, such as ciprofloxacin, macrolide antibiotics, such as azithromycin, cephalosporin antibiotics, such as ceftriaxone, or aminopenicillins, such as ampicillin), or shigellosis.

[0147] It is also possible to combine any compound of the invention with one or more additional active therapeutic agents in a unitary dosage form for simuitaneous or sequential administration to a patient. The combination therapy may be administered as a simuitaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations.

[0148] Co-administration of a compound of the invention with one or more other active therapeutic agents generally refers to simuitaneous or sequential administration of a compound of the invention and one or more other active therapeutic agents, such that therapeutically effective amounts of the compound of the invention and one or more other active therapeutic agents are both présent in the body of the patient.

[0149] Co-administration includes administration of unit dosages of the compounds of the invention before or after administration of unit dosages of one or more other active therapeutic agents, for example, administration ofthe compounds ofthe invention within seconds, minutes, or hours of the administration of one or more other active therapeutic agents. For example, a unit dose of a compound of the invention can be administered first, folïowed within seconds or minutes by administration of a unit dose of one or more other active therapeutic agents. Alternatively, a unit dose of one or more other therapeutic agents can be administered first, followed by administration of a unit dose of a compound of the invention wîthin seconds or minutes. In some cases, it may be désirable to administer a unit dose of a compound of the invention first, followed, after a perîod of hours (e.g., 1-12 hours), by administration of a unit dose of one or more other active therapeutic agents. In other cases, it may be désirable to administer a unit dose of one or more other active therapeutic agents first, followed, after a period of hours (e.g., 1-12 hours), by administration of a unit dose of a compound of the invention.

[0150] The combination therapy may provide “synergy” and “synergistic”, i.e. the effect achieved when the active ingrédients used together is greater than the sum of the effects that results from using the compounds separately. A synergistic effect may be attained when the active ingrédients are: (1) co-formulated and administered or delivered simultaneously in a combined formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect may be attained when the compounds are administered or delivered sequentially, e.g. in separate tablets, pills or capsules, or by different injections in separate syrînges. In general, during alternation therapy, an effective dosage of each active ingrédient is administered sequentially, i.e. serially, whereas in combination therapy, effective dosages of two or more active ingrédients are administered together. A synergistic anti-viral effect dénotés an antiviral effect which is greater than the predicted purely additîve effects of the indivîdual compounds of the combination.

[0151] In still y et an other embodiment, the présent application pro vides for methods of inhibiting Filoviridae polymerase in a cell, comprising: contacting a cell infected with a Filovirus with an effective amount of a compound of Formula IV, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, whereby Filoviridae polymerase is inhibited.

[0152] In still yet another embodiment, the présent application provides for methods of inhibiting Filoviridae polymerase in a cell, comprising: contacting a cell infected with Filovirus with an effective amount of a compound of Formula IV, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, and at least one addîtional active therapeutic agent, whereby Filoviridae polymerase is inhibited.

[0153] In still yet another embodiment, the présent application provides for methods of inhibiting Filoviridae polymerase in a cell, comprising: contacting a cell infected with Filoviridae virus with an effective amount of a compound of Formula IV, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, and at least one additional active therapeutîc agent selected

[0154] In still yet another embodiment, the présent application provides for methods of treating Filoviridae virus infection in a human, comprising: admînistering to the patient a therapeutically effective amount of a compound of Formula IV, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof

[0155] In still yet another embodiment, the présent application provides for methods of treating Filoviridae virus infection in a human, comprising: admînistering to the patient a therapeutically effective amount of a compound of Formula IV, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, and at least one additional active therapeutîc agent, whereby Filoviridae polymerase is inhibited.

[0156] In still yet another embodiment, the présent application provides for methods of treating Filoviridae virus infection in a human, comprising: admînistering to the patient a therapeutically effective amount of a compound of Formula IV, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, and at least one additional active therapeutîc agent.

[0157] Also provided is a kit that includes a compound of Formula IV, or a pharmaceutically acceptable sait, pharmaceutically acceptable ester, stereoisomer, mixture of stereoisomers or tautomer thereof. In separate embodiments individual kits are provided includes a compound selected from Formula IV herein, as well as each subgroup and embodiment thereof, including 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 sait, pharmaceutically acceptable ester, stereoisomer, mixture of stereoisomers or tautomer thereof. In one aspect, the kit comprises a compound of Formula IV, or a pharmaceutically acceptable sait thereof Each ofthe individual kits described herein may comprise a label and/or instructions for use of the compound in the treatment of a disease or condition in a subject (e.g., human) in need thereof In some embodiments, the disease or condition is a human Filoviridae viral infection, including an Ebola viral infection or a Marburg viral infection. In other embodiments, each separate kit may also contain instructions for use of additional medical agents in combination with the compound of Formula IV in the treatment of a disease or condition in a subject (e.g., human) in need thereof. In certain of these embodiments, the disease or condition is a human Filoviridae viral infection, including an Ebola viral infection or a Marburg viral infection. In each of the kits herein there is a further embodiment in which the kit comprises individual dose units of a compound as described herein, or a pharmaceutically acceptable sait, racemate, enantiomer, diastereomer, tautomer, polymorph, pseudopolymorph, amorphous form, hydrate or solvaté thereof Examples of individual dosage units may include pills, tablets, capsules, prefilled syringes or syringe cartridges, IV bags, etc., each comprising a therapeutically effective amount of the compound in question, or a pharmaceutically acceptable sait, racemate, enantiomer, diastereomer, tautomer, polymorph, pseudopolymorph, amorphous form, hydrate or solvaté thereof. In some embodiments, the kit may contain a single dosage unit and in others multiple dosage units are présent, such as the number of dosage units required for a specified regimen or period.

[0158] Also provided are articles of manufacture that include a compound of Formula IV, or a pharmaceutically acceptable sait, pharmaceutically acceptable ester, stereoisomer, mixture of stereoisomers or tautomer thereof; and a container. In one aspect, the article of manufacture comprises a compound of Formula IV 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 sait thereof, and a container. In separate embodiments, the container of the article of manufacture may be a vial, jar, ampoule, preloaded syringe, blister package, tin, can, bottle, box, or an intravenous bag.

VI. METHODS OF INHIBITION OF A FILOVIRIDAE POLYMERASE

[0159] Another aspect of the invention relates to methods of inhibiting the activity of Filoviridae polymerase comprising the step of treating a sample suspected of containing Filoviridae with a compound or composition of the invention.

[0160] Filoviridae that can be treated using the methods of the présent invention are singlestranded négative sense RNA viruses that typically infect primates. Filoviruses are able to multiply in virtually ail cell types. The Filovirus antigens and virions are found primarily in fibroblasts and interstitium of an infected individual. There are three identified généra of Filoviruses: the Ebola virus (EBOV; five species); the Marburg virus (MARV); and the Cuevavirus, also known as the Lloviu virus (LLOV). The virions (viral partîcles) are characteristically shaped as long, cylindrical, filamentous particles which may be straight, curved, coiled, or found in a “6” or “U” shaped configuration. They are occasionally branched and the particles vary greatly in length, but the dîameter (about 80 nm) is consistent. The filovirus genome comprises seven genes that encode 4 virion structural proteins (VP30, VP35, nucleoprotein (NP), and a polymerase protein (L-pol)) and 3 membrane-as sociated proteins (VP40, glycoprotein (GP), and VP24).

[0161] The Ebola virus genus includes five known species: (1) Bumiibugyo ebolavirus, also known as Bundibugyo virus (BDBV, previously BEBOV); (2) Reston ebolavirus, also known as Reston virus or Ebola-Reston (RESTV, previously REBOV); (3) Sudan ebolavirus, also known as Sudan virus or Ebola-Sudan (SUDV, previously SEBOV); (4) Tai For est ebolavirus, also known as Tai Forest virus or Ebola-Tai (TAFV, previously CIEBOV), and (5) Taire ebolavirus, also known as Ebola virus or Ebola-Zaîre (EBOV, previously ZEBOV).

[0162] The Marburg virus genus includes the species Marburg marburgvirus, also known as Marburg virus (MARV) or Ravn virus (RAVV). The Cuevavirus genus includes the species Lloviu cuevavirus, also known as the Lloviu virus (LLOV).

[0163] Compositions of the invention may act as inhibitors of Filoviridae polymerase , as intermediates for such inhibitors or hâve other utilities as described below. The inhibitors will bind to locations on the surface or in a cavity of Filoviridae polymerase having a geometry unique to Filoviridae polymerase . Compositions binding Filoviridae polymerase may bind with varying degrees of reversibility. Those compounds binding substantially irreversibly are idéal candidates for use in this method of the invention. Once labeled, the substantially irreversibly binding compositions are useful as probes for the détection of Filoviridae polymerase. Accordingly, the invention relates to methods of detecting Filoviridae polymerase in a sample suspected of containing Filoviridae polymerase comprising the steps of treating a sample suspected of containing Filoviridae polymerase with a composition comprising a compound of the invention bound to a label; and observing the effect of the sample on the activity of the label. Suitable labels are well known in the diagnostics fîeld and include stable free radicals, fluorophores, radioisotopes, enzymes, chemilumînescent groups and chromogens. The compounds herein are labeled in conventional fashion using functional groups such as hydroxyl, carboxyl, sulfhydryl or amino.

[0164] Within the context of the invention, samples suspected of containing Filoviridae polymerase include natural or man-made materials such as living organisms; tissue or cell cultures; bîological samples such as biological material samples (blood, sérum, urine, cerebrospinal fluid, tears, sputum, saliva, tissue samples, and the like), laboratory samples; food, water, or air samples; bioproduct samples such as extracts of cells, particulariy recombinant cells synthesizing a desired glycoprotein, and the like. Typically the sample will be suspected of containing an organism which produces Filoviridae polymerase, frequently a pathogenic organism such as & Filoviridae virus. Samples can be contained in any medium including water and organic solvent\water mixtures. Samples include living organisms such as humans, and manmade materials such as cell cultures.

[0165] The treating step of the invention comprises adding the composition of the invention to the sample or it comprises adding a precursor of the composition to the sample. The addition step comprises any method of administration as described above.

[0166] If desired, the activîty of Filoviridae polymerase after application of the composition can be observed by any method including direct and indirect methods of detecting Filoviridae polymerase activîty. Quantitative, qualitative, and semiquantitative methods of determining Filoviridae polymerase activîty are ali contemplated. Typically one of the screening methods described above are applied, however, any other method such as observation of the physiological properties of a living organism are also applicable.

[0167] Organisms that contain Filoviridae polymerase include the Filoviridae virus. The compounds of this invention are useful in the treatment or prophylaxîs of Filoviridae infections in animais or in man.

[0168] However, in screening compounds capable of inhibiting human Filoviridae viruses, it should be kept in mind that the results of enzyme assays may not correlate with cell culture assays. Thus, a cell based assay should be the primary screening tool.

[0169] In another embodiment, the présent application provides for methods of treating Filoviridae virus infection in a human, comprisîng: administering to the patient a therapeutically effective amount of a compound of Formula IV, or a pharmaceutically acceptable sait, solvaté, 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 an Ebola virus. In some embodiments, the Filoviridae infection is caused by Bundibugyo ebolavinis, Reston ebolavinis, Sudan ebolavinis, Tai Forest ebolavirus, or Zaïre ebolavinis. In some embodiments, the Filoviridae infection is caused by a Marburg virus. In some embodiments, the Filoviridae infection is caused by a Lloviu virus. In some embodiments, a Filoviridae polymerase is inhibited.

[0170] The compounds of the présent invention can be used in the treatment of a human already suffering from a Filoviridae infection, or can be administered prophylactically to reduce or prevent the chance of a Filoviridae infection. Filoviridae infections can be characterized by hémorrhagie fever, hematemesis, dîarrhea, rétrosternal abdominal pain and prostration. The incubation period is around 21 days foilowîng contact with a human suffering from Filoviridae infection. The outcome of Filoviridae infection is typicaily death.

[0171] Also provided as separate embodiments are a compound selected from each of the Formulas herein, as well as each subgroup and embodiment thereof, including a compound selected from the group of Formula IV, or one of the spécifie compounds of the examples herein, including Compounds 1-32, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, for use in a method of treating a Filoviridae infection in a human. Also provided as separate embodiments are a compound selected from each of the Formulas herein, as well as each subgroup and embodiment thereof, including a compound selected from the group of Formula IV, or one ofthe spécifie compounds of the examples herein, including Compounds 132, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, for use in a method of treating an Ebola virus infection in a human. Also provided as separate embodiments are a compound selected from each of the Formulas herein, as well as each subgroup and embodiment thereof, including a compound selected from the group of Formula IV, or one of the spécifie compounds of the examples herein, including Compounds 1-32, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, for use in a method of treating a Marburg virus infection in a human. Within each of the embodiments herein in which the Filoviridae infection is an Ebola virus, there are further separate embodiments with them whereîn the Filoviridae infection is caused, respectively, by Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, or 7.aire ebolavirus. In some embodiments, the Filoviridae infection is caused by a Marburg virus. In some embodiments, the Filoviridae infection is caused by a Lloviu virus.

[0172] The présent invention also provides compounds of each of the Formula herein, as well as each subgroup and embodiment thereof, including a compound selected from the group of Formula (IV), or one of the spécifie compounds of the examples herein, including Compounds 1-32, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof for use in any of the methods of the invention as defined herein.

[0173] Also provided as séparaie embodiments are the uses of a compound selected from each of the Formulas herein, as well as each subgroup and embodiment thereof, including a compound selected from the group of Formula IV, or one ofthe spécifie compounds ofthe examples herein, including Compounds 1-32, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, in the préparation of a médicament for treating a Filoviridae infection in a human. Also provided as separate embodiments are the uses of a compound selected from each of the Formulas herein, as well as each subgroup and embodiment thereof, including a compound selected from the group ofFormula IV, or one ofthe spécifie compounds ofthe examples herein, including Compounds 1-32, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, in the préparation of a médicament for treating an Ebola virus infection in a human. Also provided as separate embodiments are the uses of a compound selected from each of the Formulas herein, as well as each subgroup and embodiment thereof, including a compound selected from the group of Formula IV, or one of the spécifie compounds of the exampies herein, including Compounds 1-32, or a pharmaceutically acceptable sait, solvaté, and/or ester thereof, in the préparation of a médicament for treating a Marburg virus infection in a human.

VIL SCREENS FOR FILOVIRIDAE POLYMERASE INHIBITORS.

[0174] Compositions of the invention are screened for inhibitory activity against Filoviridae polymerase by any of the conventional techniques for evaluating enzyme activity. Within the context of the invention, typically compositions are first screened for inhibition of Filoviridae polymerase in vitro and compositions showing inhibitory activity are then screened for activity in vivo. Compositions having in vitro Ki (inhibitory constants) of less than about 5 X 10'$ M and preferably less than about 1 X 10^ M are preferred for in vivo use,

[0175] Useful in vitro screens hâve been described in detail and will not be elaborated here. However, the examples describe suitable in vitro assays.

Vin. PREPARATION OF COMPOUNDS

EXAMPLES

[0176] Certain abbreviations and acronyms are used in describing the experimental details. Although most of these would be understood by one ski lied in the art, Table 1 contains a list of many of these abbreviations and acronyms.

Table 1. List of abbreviations and acronyms.

Abbreviation	Meaning
Ac2O	acetic anhydride
ABN	2,2’-azobis(2-methy Ipropionitril e)
Bn	benzyl
BnBr	benzylbromide
BSA	bis(trimethylsilyl)acetamide
BzCl	benzoyl chloride
CDI	carbonyl diimidazole
DABCO	1,4-diazabicyclo[2.2.2]octane
DBN	1,5-diazabicyclo[4.3.0]non-5-ene
DDQ	2,3-dichloro-5,6-dicyano-I,4-benzoquinone
DBU	l,5-diazabicyclo[5.4.0]undec-5-ene
DCA	dichloroacetamide
DCC	dicyclohexylcarbodiimide
DCM	dichl oromethane
DMAP	4-dimethylaminopyridine
DME	1,2-dimethoxyethane
DMTC1	dimethoxytrityl chloride
DMSO	dimethylsulfoxide
DMTr	4, 4’-dimethoxytrityl
DMF	dimethylformamîde
EtOAc	ethyl acetate
ESI	electrospray îonizatîon
EtOAc	ethyl acetate
HMDS	hexamethy Idisi 1 azane
HPLC	High pressure liquid chromatography
LDA	lithium diisopropylamide
LRMS	low resolution mass spectrum
MCPBA	meta-chloroperbenzoic acid
MeCN	acetonitrile
MeOH	methanol
MMTC	mono methoxytrityl chloride
m/z or m/e	mass to charge ratio
MH1	mass plus 1
MH'	mass minus 1
MsOH	methanesulfonic acid
MS or ms	mass spectrum
MTBE	tert-butylmethyl ether
NB S	N-bromosuccinîmide

Ph	phenyl
rt or r.t.	room température
TB AF	tetrabutyiammonium fluoride
THF	tetrahydrofuran
TMSC1	chlorotrimethylsilane
TMSBr	bromotrimethylsilane
TMSI	iodotri methy 1 si lane
TMSOTf	(trimethylsilyl)trifluorome'thylsulfonate
TEA	tri ethyl amine
TBA	tributylamine
TB AP	tributyl ammonium pyrophosphate
TB SCI	t-butyldimethylsilyl chloride
TEAB	triethylammonium bicarbonate
TFA	trifluoroacetîc acid
TLC ortie	thin layer chromatography
Tr	triphenylmethyl
Toi	4-methylbenzoyl
Turbo Grignard	1:1 mixture of isopropyl magnésium chloride and lithium chloride
δ	parts per million down field from tetramethylsilane

A. Préparation of Compounds

Example 1. (2S)-ethyl 2-(chloro(phenoxv)phosphorylamino)propanoate (Chloridate A)

TEA, DCM ------------->·

A (0177] Ethyl alanine ester hydrochloride sait (1.69 g, 11 mmol) was dissolved in anhydrous CH₂Ch (10 mL) and the mixture stirred with cooiing to 0 ^DC under N₂ (g). Phenyl dichlorophosphate (1.49 mL, 10 mmol) was added followed by dropwîse addition ofEt₃N over about 10 min. The reaction mixture was then slowly warmed to RT and stirred for about 12 h. Anhydrous Et₂O (50 mL) was added and the mixture stirred for about 30 min. The solid that formed was removed by fdtration, and the filtrate concentrated under reduced pressure. The residue was subjected to si 1 ica gel chromâtography eluting with 0-50% EtOAc in hexanes to provide intermediate A. ‘HNMR (300 MHz, CDCh) δ 7.39-7.27 (m, 5H), 4.27 (m, 3H), 1.52 (m, 3H), 1.32 (m, 3H). ³¹P NMR (121.4 MHz, CDCh) δ 8.2, 7.8.

Example 2. (2S)-2-ethylbutyl 2-(chloro(phenoxyÎphosphorvlamino)propanoate (Chloridate B)

B

[0178] The 2-ethylbutyi alanine chlorophosphoramidate ester B was prepared using the same procedure as chloridate A except substituting 2-ethylbutyl alanine ester for ethyl alanine ester. The material is used crude in the next reaction. Treatment with methanol or éthanol forms the dîsplaced product with the requîsite LCMS signal.

Example 3. (2S)-isopropyl 2-(chloro(phenoxy)phosphorviamino)propanoate (Chloridate

I

Cî

TEA, DOM

C

[0179] The isopropyï alanine chlorophosphoramidate ester C was prepared using the same procedure as chloridate A except substituting isopropyï alanine ester for the ethyl alanine ester. The material is used crude in the next reaction. Treatment with methanol or éthanol forms the dîsplaced product with the requisite LCMS signal.

Example 4. (2R, 3R, 4S, 5R)-2-(4-aminopyrrolo[l_Î2-fnL2,41triazin-7-yD-3,4-dihydroxv-5(hydroxymethyhtetrahydrofuran-2-carbonitrile (Compound 1)

[0180] The préparation of (2R, 3 R, 4S, 5R)-2-(4-aminopyrrolo[l,2-f][l,2,4]triazin-7-yl)-3,4dihydroxy-5-(hydroxymethyl)tetrahydrofiiran-2-carbonitrile is described below.

Bnô ÔBn ^AC2°

[0181] The commercial!y available lactol (10 g, 23.8 mmol) was dissolved in anhydrous DMSO (30 mL) under N₂(g). Ac₂O (20 mL) was added and the résultant reaction mixture stirred at RT for about 48 h. The reaction mixture was poured onto ice H₂O (500 mL) and the mixture stirred for 20 min. The mixture was extracted with EtOAc (3 x 200 mL) and the combîned organic extracts were then washed with H₂O (3 x 200 mL). The organic extract was dried over anhydrous MgSCL, filtered and concentrâted under reduced pressure. The residue was dissolved in CH₂C1₂ and subjected to silica gel chromatography eluting with 25% EtOAc in hexanes to provide the lactone. *H NMR (400 MHz, DMSO) Ô 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, 3.9 Ηζ,ΙΗ), 3.66 (m, 2Ή). LCMS m/z

436.1 [M+H₂0], 435.2 [M+OH]- Tr = 2.82 min. HPLC Tr = 4.59 [2-98% ACN in H2) over 5 min ai 2 mL/min flow.

[0182] The bromopyrazole (prepared according to WO2009/132135) (0.5 g, 2.4 mmol) was suspended in anhydrous THF (10 mL) under N₂ (g). The suspension was stirred and TMSC1 (0.67 mL, 5.28 mmol) was added. The mixture was stirred for 20 min. at RT and then cooled to about -78 °C after which time a solution of n-BuLi (6 mL, 1.6 N in hexanes, 9.6 mmol) was added slowly. The reaction mixture was stirred for 10 min. at about -78 °C and then the lactone (1 g, 2.4 mmol) was added via syringe. When the reaction was complété as measured by LCMS, AcOH was added to quench the reaction. The mixture was concentrated under reduced pressure and the residue dissolved in a mixture of CH₂Ch and H₂O (100 mL, Ll). The organic layer was separated and washed with H₂O (50 mL). The organic layer was then dried over anhydrous MgSCU, filtered and concentrated under reduced pressure. The residue was subjected to silica gel chromatography eluting with 0-50% EtOAc in hexanes to provide the product as a 1:1 mixture of anomers. LCMS m/z 553 [M+H],

[0183] The hydroxy nucleoside (1.1 g, 2.0 mmol) was dissolved in anhydrous CH2CI2 (40 mL) and the solution cooled with stirring to about -78 °C under N₂ (g). TMSCN (0.931 mL, 7 mmol) was added and the mixture stirred for a further 10 min. TMSOTf ( 1.63 mL, 9.0 mmol) was slowly added to the reaction and the mixture stirred for I h. The reaction mixture was then diluted with CH2CI2 (120 mL) and aqueous NaHCCh (120 mL) was added to quench the reaction. The reaction mixture was stirred for a further 10 min and the organic layer separated. The aqueous layer was extracted with CH2CI2 (150 mL) and the combined organic extracts dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The residue was dissolved in a minimal amount of CH2CI2 and subject ed to silica gel chromatography eluting with a gradient of 0-75% EtOAc and hexanes to provide the tribenzyl cyano nucleoside as a mixture of anomers. Ή 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,lH), 6.70-6.76 (m, 1H), 6.41 (bs, 2H), 5.10 (d, J = 3.9 Hz, 0.5H), 4.96 (d, J = 5.1 Hz, 0.5H), 4.31-4.85 (m, 7H), 4.09-4.18 (m, 2H), 3.61-3.90 (m, 2H). LCMS m/z 562 [M+H],

beta 1

[0184] The tribenzyl cyano nucleoside (70 mg, 0.124 mmol) was dissolved in anhydrous CH2O2 (2 mL) and cooled to about -20 °C under N₂ (g). A solution of BCh (IN in CH2CI2, 0.506 mL, 0.506 mmol) was added and the reaction mixture stirred for 1 h. at -78 ^ÜC. When the reaction was complété by LC/MS, MeOH was added to quench the reaction. The reaction mixture was allowed to warm to RT and the solvent removed under reduced pressure. The residue was subjected to C18 reverse phase HPLC, eluting for 5 min with H2O (0.1% TFA), followed by a gradient of 0-70% MeCN in H2O (0.1 % TFA) over 35 min, to elute the a-anomer, and β-anomer I. (α-anomer) ^}H NMR (300 MHz, D2O) δ 7.96 (s, 1H), 7.20 (d, J ~ 4.8 Hz, 1H), 6.91 (d, J =4.8 Hz, 1 H), 4.97 (d, J = 4.4 Hz, IH), 4.56-4.62 (m, 1 H), 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) *H NMR (400 MHz, DMSO) δ 7.91 (s, 1H), 7.80-8.00 (br s, 2H), 6.85-6.89 (m, 2H), 6.07 (d, J= 6.0 Hz, 1H), 5.17 (br s, 1H), 4.90 (br s, 1H), 4.63 (t, J= 3.9 Hz, 1H), 4.02-4.06 (m, 1H), 3.94 (br s, 1H), 3.483.64 (m,2H). LCMS m/z 292.2 [M+H], 290.0 [M-H], Tr= 0.35 min. 13C NMR (400 MHZ, DMSO), 156.0, 148.3, 124.3, 117.8, 117.0, 111.2, 101.3, 85.8, 79.0, 74.7, 70.5, 61.4. HPLC Tr = 1.32 min

Préparation of (3R,4R,5R)-2-(4-aminopyrrolo[2,l-firL2.41triazin-7-yl)-3,4-bis(benzyloxy)-5((benzyloxy)methyl)tetrahydrofuran-2-ol using LaC13-2LiCl

NH₂

THF, -20 ’C

1. TMSCI, PhMgCi, 0°C

2. /'PrMgCi, -15 °C

3. LaCI₃-2LiCI, -15 °C

[0185] A solution of 7-iodopyrrolo[2,l-f][l,2,4]triazin-4-amine (7.5 g, 28.8 mmol, 1.0 equiv) was prepared in THF (67 mL). The solution was cooled to about 0 °C, and TMSCI (3.3 mL, 30.3 mmol, 1.05 equiv) was added. The reaction mixture was stirred for about 30 min, and then PhMgCi (2 M in THF; 28 mL, 56.8 mmol, 1.97 equiv) was added while maintaining an internai température below 5 °C. The reaction mixture was agitated at about 0 °C for about 35 min, and then cooled to about -15 °C. /PrMgCl (2 M in THF, 14 mL, 30.2 mmol, 1.05 equiv) was then added while maintaining an internai température below about -10 °C. After approximatelyl5 minutes at about -15 °C, LaCh-2LiCl (0.6 M in THF, 50 mL, 14.4 mmol, 0.5 equiv) was added while maintaining an internai température below about -15 °C. The reaction mixture was agitated for about 25 min at about -20 °C.

[0186] 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 equiv) was prepared in THF (45 mL). The solution was cooled to about -20 ’C, and then transferred to the Grignard solution while maintaining an internai température below about -15 °C. The resulting reaction mixture was agitated at about -20 °C for about 30 min.

The reaction was quenched with 2 M HCl (53 mL), and the mixture warmed to about 15 °C. zPrOAc (38 mL) was added, and the organic and aqueous phases were separated. The bottom aqueous layer was discharged, and the upper organic layer was washed sequentially with 2.5 wt% NaHCCh (53 mL), 2.5 wt% NaHCO₃ (53 mL), and 10 wt%NaCÏ (53 mL).

[0187] The organic phase was concentrated to about 45 mL, and then diluted with /PrOAc (75 mL). The solution was concentrated again to about 45 mL, and then diluted with /PrOAc (23 mL). The solution was concentrated to about 45 mL, and then filtered over a pad of Celite. The filtered solution was concentrated to about 26 mL, and then diluted with MTBE (75 mL). After 2h, heptane (23 mL) was slowly added and the slurry was stirred at about 25 °C for about 2 h, and was then cooled to about -5 °C over about 8 h. The 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 no more than about 35 °C to afford (3R,4R,5R)-2-(4-aminopyrrolo[2,l-f|[l,2,4]triazin7-yï)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol.

Préparation of (3R.4R.5R)-2-(4-aminopyrrolor2.1-f1[1.2.41triazin-7-yl)-3,4-bis(benzyloxv)-5((benzyloxy)methyl)tetrahydrofuran-2-ol using CeCh

[0188] The iodopyrazole (5.02 g, 19.3 mmoi) was dissolved in THF (45 g) and the solution was cooled to about 0 °C with stirring. TMSC1 (2.04 g, 18.7 mmol) was added, and after about 1 h phenyl magnésium chloride (2.0 M in THF, 19.9 g, 38.2 mmol) was added. The reaction mixture was cooled to about -20 °C and Ao-propyl magnésium chloride (2.0 M in THF, 9.99 g, 20.5 mmol) was added slowly. After about 30 min, the reaction mixture was transferred to a mixture of anhydrous cérium 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 added slowly, and the resulting reaction mixture was stirred for about 1 h. 2 M HCl (41 g) was added, the mixture was warmed to about 15 °C, and wo-propyl acetate (35 g) was added. The layers were separated and the organic layer was washed with 2.5% NaHCOs (2 x 40 g), 10% NaCl (I x 35 g) and concentrated to about 30 mL volume. wo-Propyl acetate (44 g) was charged and the solution was concentrated to about 30 mL volume. Zw-Propyl acetate (43 g) was charged and the solution was concentrated to about 30 mL volume. The solution was filtered and the filtrate was concentrated to about 18 mL volume. /er/-Butylmethyl ether (37 g) was added followed by product seed crystals (10.7 mg). After about 14 h n-heptane (10.5 g) was added and the mixture was cooled to about -5 °C and filtered. The solids were washed with ZerAbutylmethyl ether (9 g) at about -5 °C and dried under vacuum at about 34 °C for about 15 h to provide the product.

Préparation of (3K4R.5R)-2-i4-aminopyrrolo[2J-fj[L2,41triazin-7-vl)-3,4-bis(benzyloxy)-5((benzyloxy)methyl)tetrahydrofuran-2-ol using CeCh and iPrMgCl-LÎCl

[0189] The 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). TMSC1 (2.06 g, 19.0 mmol) was added, and after about 1 h phenyl magnésium chloride (2.0 M in THF, 20.23 g, 38.8 mmol) was added. The reaction mixture was cooled to about -20 °C and zxo-propyl magnésium chloride-lithium chloride complex (2.0 M in THF, 15.37 g, 21.0 mmol) was added slowly. After about 1 h, the reaction mixture was transferred to a mixture of cérium 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 added slowly, and the resufting reaction mixture was stirred for about 1.5 h. 2 M HCl (40 g) was added, the mixture was warmed to about 15 °C and Ao-propyl acetate (35 g) was added. The layers were separated and the organic layer was washed with 2.5% NaHCCh (2 x 40 g), 10% NaCl (1 x 36 g) and concentrated to about 30 mL volume. Ao-Propyl acetate (44 g) was added and the solution was concentrated to about 30 mL volume. The solution was fdtered and the filtrate was concentrated to about 18 mL volume. fôr/-Butylmethyl ether (37 g) was added followed by product seed crystals (10.5 mg). After about 14 h π-heptane (11g) was added and the mixture was cooled to about -5 °C and filtered. The solids were washed with fër/-butylmethyl ether (9 g) at about -5 °C and dried under vacuum at about 34 °C for about 15 h to provide the product.

Préparation of (3R,4R5R)-2-(4-aminopyrrolo[2J-firL2.4]triazin-7-yl)-3,4-bis(benzyloxy)-5((benzyioxy)methyl)tetrahydrofuran-2-ol usina YCh

[0190] The iodopyrazole (4.99 g, 19.2 mmol) was dissolved in THF (44 g) and the solution was cooled to about 0 °C with stirrîng. TMSC1 (2.45 mL, 19.4 mmol) was added, and after about 30 min phenyl magnésium chloride (2.0 M in THF, 20.29 g, 39.0 mmol) was added. The réaction mixture was cooled to about -20 °C and Ao-propyl magnésium chloride (2.0 M in THF, 9.85 g, 20.1 mmol) was added slowly. After about 30 min, 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 mmi) in THF (24 g) at about -20 °C. After about 2.5 h 2 M HCl (30 g) was added, the mixture was warmed to about 15 °C, and fw-propyl acetate (22 g) was added. The layers were separated and the organic layer was washed with 2.5% NaHCOa (2 x 40 g), 10% NaCi (1 x 35 g) and concentrated to about 30 mL volume. Ao-Propyl acetate (44 g) was charged and the solution was concentrated to about 30 mL volume. Ao-Propyl acetate (45 g) was charged and the solution was concentrated to about 30 mL volume. The solution was filtered and the filtrate was concentrated to about 18 mL volume. /m-Butylmethyl ether (37 g) was added followed by product seed crystals (11.5 mg). After about 1 h 7ï-heptane (15 mL) was added and the mixture was cooled to about -5 °C and agitated for about 17 h. The slurry was filtered and the solids were washed with a ie/7-butylmethyl ether (8 g)/n-heptane (2 g) mixture precooled to about -5 °C. The resulting solids were dried under vacuum at about 34 °C for about 22 h to afford the product.

Préparation of (3R,4R,5R)-2-(4-aminopyrrolo[2.1-f][L2,41triazin-7-yl)-3,4-bis(benzvioxy)-5((benzyloxy)methyl)tetrahydrofuran-2~ol using NdCh

H₂N

BnO

Ο

BnÔ ÔBn

[0191] The 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 N₂(g). TMSC1 (2.45 mL, 19.4 mmol) was added, and after about 1 h phenyl magnésium chloride (2.0 M in THF, 19.75 g, 38.0 mmol) was added. The reaction mixture was cooled to about -20 °C and Ao-propylmagnesium chloride (2.0 M in THF, 9.40 g, 19.2 mmol) was added slowly. After about 1.5 h, the reaction mixture was transferred into a mixture of anhydrous neodymium (III) chloride (4.03 g, 16.1 mmol) and lactone (6.70 g, 16.0 mml) in THF (22 g) at about -20 °C. After about 1.5 h the reaction mixture was warmed to -10 °C and, after an additional 2 h, 2 M HCl (36 g) was added. The mixture was warmed to about 15 °C and Ao-propyl acetate (23 g) was added. The layers were separated and the organic layer was washed with 2.5% NaHCCh (2 x 44 g), 10% NaCl (1 x 41 g) and concentrated to about 30 mL volume. Ao-Propyl acetate (44 g) was charged and the solution was concentrated to about 30 mL volume. Mo-Propyl acetate (45 g) was charged and the solution was concentrated to about 30 mL volume. The solution was filtered and the filtrate was concentrated to about 18 rnL volume. fôrr-Butylmethyl ether (37 g) was added followed by product seed crystals (11.9 mg). After about 1 h «-heptane (15 mL) was added and the mixture was cooled to about -5 °C and agîtated for about 15 h. The slurry was fdtered and the solîds were washed with a tertbutylmethyl ether (8 g)Zu-heptane (11g) mixture precooled to about -5 °C. The resulting solids were dried under vacuum at about 34 °C for about 25 h to afford the product.

Préparation of (2R,3 R,4R, 5 R)-2-(4-aminopyrrolo Γ2.1 -f] [ I.2,41triazin-7-y l)-3,4-bi s(benzyloxy)5-((benzyloxy)methyl)tetrahydrofuran-2-carbonitrile

TFA TMSCN TMSOTf

ΟΗ_ξΟΙ₂

-40 °C

[0192] To a pre-cooled (-40 ^OC) solution of (3R,4R,5R)-2-(4-aminopyrrolo[2,l-f][l,2,4]triazin7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol (10.0 grains, 18.1 mmols, 1.0 equiv.) in DCM (100 mL) was charged trifluoroacetic acid (6.19 grams, 54.3 mmols, 3.0 equiv.), followed by a pre-cooled (-30 °C) solution of TMSOTf (24.1 grams, 108.6 mmols, 6.0 equiv.) and TMSCN (10.8 grams, 108.6 mmols, 6.0 equiv.) in DCM (50 mL) while maintaîning the internai température below about -25 °C. The reaction mixture was agitated at below about -30 °C for no less than 10 minutes and quenched into a pre-cooled (about -10 °C) solution of 20 wt. % KOH aq. (120 mL). The bi-phasîc mixture was warmed to ambient température. The organic layer was séparaied and washed with 10 wt. % NaCl aq. (3 X 50 mL). The organic phase was filtered, concentrated under vacuum to about 50 mL, re-diluted 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,l-f][l,2,4]triazin-7-yl)-3,4-bis(benzyloxy)-5((benzyloxy)methyl)tetrahydrofuran-2-carbonitrile at about 55 °C. Agitated at about 55 °C for about an hour and cooled to about 0 °C over about 6 hours. The solids were isolated by fdtration and the filter cake was washed with toluene (30 mL). The solids were dried under vacuum at about 50 °C.

Préparation of (2R.3R,4R.5R)-2-(4-aminopyrrolo[2,l-f|[1.2,4]triazin-7-yl)-3,4-bis(benzyloxy)5-((benzyloxy)methyÎ)tetrahydrofuran-2-carbonitrile via Flow Chemistry

[0193] Solutions of (3R,4R,5R)-2-(4-aminopyrroio[2, l-f][l,2,4]triazîn-7-yl)-3,4bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol (23.0 g in 460.07 g of DCM), TMSOTf (55.8] gin 138.07 g of DCM) and TMSCN (25.03 gin 138.10 g of DCM) were sequentiaiiy pumped, into a tube reactor at about -40 °C. The reaction mixture was collected in a fiask, kept in ice bath, containing 20% KOH aqueous solution (46.91 g KOH and 210 g of water). The layers were separated and the organic phase was sequentiaiiy washed with 10%

KOH aqueous solution (10 g KOH and 90 mL of water) and withl0% brine (2 xlOO g). The organîc phase was concentrât ed under vacuum to about 4 volumes, isopropyl alcohol was charged (162.89 g) and the mixture was concentrated under vacuum to about 10 volumes. The contents were warmed to about 60 °C, then adjusted to about 0 °C over about 6.5 h and agitated at about 0 °C for about 15.5 h. The resulting slurry was filtered, the solids were rinsed with isopropyl alcohol (61.79 g) and then dried at about 50 °C under reduced pressure ovemight to afford the product.

Préparation of (2R, 3R, 4S, 5R)-2-(4-aminopyrrolo[T,2-:firi,2.41triazin-7-yl)-3.4-dihydroxy-5(hydroxymethyl)tetrahydrofliran-2-carbonitrile

1)BCI₃, CH₂CI₂,-20 °C

2) MeOH, K₂CO₃

[0194] The tribenzyl cyano nucleoside (48.8 g, 86.9 mmol, 1.0 equiv.) was dissolved in anhydrous CH2CI2 (244 mL) and cooled to about -20 °C . A solution of BCh (IM in CH2CI2, 295 mL, 295 mmol, 3.4 equiv.) was added dropwise, maintaining the internai température below about -15 °C Following addition, the reaction mixture was stirred for 1 h at about -20 °C.

MeOH (340 ml) was added dropwise, maintaining the internai température below -15 °C. The resulting solution was distîlled to about 250 ml, then refilled with about 250 ml MeOH. The resulting solution was again distilled to about 250 ml, then refilled with about 250 ml MeOH, and finally distilled to about 125 ml. Water (125 mi) was added, followed by K2CO3 solution (20 wt% in water, 125 ml). The pH was checked, and found to be ~3. K2CO3 solution was added (20 wt% in water, 50 ml), and the pH was found to be ~8. The resulting slurry was stirred ovemight, then filtered and washed with water (50 ml) and MeOH (50 ml). The wet product cake was dried ovemight at about 40 °C ovemight. ^TH 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.3S,4R,5Rl-5-(4-aminopvrrolo|l,2-fHl,2,41triazin-7YD-5-cvano-3.4-dihvdroxYtetrahvdrofuran-2-vl)methoxy)(phenoxvïphosphorylaminojpropanoate (Compound 8)

[0195] The nucleoside I (45mg, 0.15mmol) was dissolved in anhydrous trimethyl phosphate (0.5 mL) and the solution stirred under N₂ (g) at about 0 °C. Methyl imidazole (36 pL, 0.45 mmol) was added to the solution. Chlorophosphoramidate C (69 mg, 0.225 mmol) was dissolved in anhydrous THF (0.25 mL) and added dropwise to the nucleoside mixture. When the reaction was complété by LCMS, the reaction mixture was diluted with EtOAc and washed with saturated aqueous NaHCOr solution, saturated NaCl, dried over anhydrous Na₂SO< filtered and concentrated under reduced pressure. The residue was subjected to silica gel chromatography eluting with 0-5% MeOH in CH₂C1₂ followed by préparative HPLC to give the product. ’H NMR (300 MHz, CDsOD) 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). ³¹P NMR (121.4 MHz, CD₃OD) δ 3.8. LCMS m/z 561.0 [M+H], 559.0 [M-H].

Example 12. (2S)-2-ethylbutyl 2-((«2R,3S.,4K5R)-5-(4-aminopyrrolo[L2-fHL2,4Îtriazin-7vh-5-cvano-3,4-dihvdroxvletrahvdrof»ran-2-vl)methoxv)(phenoxv)phosphorvlamino) propanoate (Compound 9)

[0196] Compound 9 can be prepared by several methods described below.

Procedure I

[0197] Prepared from Compound 1 and chloridate B according to the same method as for the préparation of compound 8. Ή NMR (300 MHz, CD₃OD) δ 7.87 (m, 1H), 7.31-7.16 (m, 5H), 6.92-6.89 (m, 2H), 4.78 (m, 1 H), 4.50-3.80 (m, 7H), 1.45-1.24 (m, 8H), 0.95-0.84 (m, 6H). ³¹P NMR (121.4 MHz, CD₃OD) δ 3.7. LCMS m/z 603.1 [M+H], 601.0 [M-H].

Procedure 2 tBuMgCI

THF, DMF

[01981 (2S)-2-ethylbutyl2-(((((2R_s3S,4R,5R)-5-(4-aminopyrrolo[2_îl-f|[l,2,4]triazin-7-yl)5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)pliosphoryI)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 a nitrogen atmosphère at RT. (2R,3R,4S,5R)-2-(4-aminopyrrolo[2,l-f][l,2,4]triazin-7-yl)-3,4-dihydroxy-5(hydroxymethyl)tetrahydrofùran-2-carbonîtrile (350 mg, 1.2 mmol) was added to the reaction mixture in one portion. A solution of z-butylmagnesium chloride in THF (IM, 1.8 mL, 1.8 mmol) was then added to the reaction dropwise over about 10 minutes. The reaction was stirred for about 2 h, at which point the reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated aqueous sodium bicarbonate solution (3x15 mL) followed by saturated aqueous sodium chloride solution (15 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting oil was purified with silica gel column chromatography (0-10% MeOH in DCM) to afford (2S)-2-ethylbutyl 2(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,l-f][l,2,4]triazin-7-yl)-5-cyano-3,4dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino) propanoate (311 mg, 43%, 1:0.4 diastereomeric mixture at phosphorus) as a whîte solid. ’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=5.4Hz, 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). ³¹P NMR (162 MHz, CD₃OD)Ô3.71, 3.65. LCMS m/z 603.1 [M+H], 600.9 [M-H], HPLC (2-98% MeCN H₂O gradient with 0.1%

TFA modifier over 8.5 min, 1.5mL/min, Column: Phenomenex Kinetex Cl8, 2.6 um 100 Â, 4.6 x 100 mm ) îr = 5.544 min, 5.601 min

Séparation of the (S) and (R) Diastereomers

[0199] (2S)-2-ethylbutyl 2-(((((2R,3 S,4R,5R)-5-(4-ammopyrrolo[2,1 -f|[l ,2,4]triazin-7-yl)-5cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino) propanoate was dissolved in acetonitrile. The resulting solution was loaded onto Lux Cellulose-2 chiral column, equilibrated in acetonitrile, and eiuted with isocratic acetonitrile/methanol (95:5 vol/vol). The first eluting diastereomer had a rétention time of 17.4 min, and the second elutîng diastereomer had a rétention time of 25.0 min.

[0200] First Eluting Diastereomer is (S)-2-ethylbutyl 2-(((R)-(((2R,3S,4R,5R)-5-(4aminopyrrolo[2,l-f][l,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydroiuran-2yl)methoxyXphenoxy)phosphoryI)amino)propanoate:

’HNMR (400 MHz, CD3OD) δ 8.05 (s, 1H), 7.36 (d, .7= 4.8 Hz, 1H), 7.29 (br t, .7 = 7.8 Hz, 2H), 7.19-7.13 (m, 3H), 7.11 (d, .7= 4.8 Hz, 1H), 4.73 (d, J = 5.2 Hz, 1H), 4.48 -4.38 (m, 2H), 4.37 - 4.28 (m, 1 H), 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 (p, J =7.3 Hz, 4H), 1.29 (d, J= 7.2 Hz, 3H), 0.87 (t, J= 7.4 Hz, 6H). ^3îPNMR (162 MHz, CD₃OD) δ 3.71 (s). HPLC (2-98% MeCN-H₂O gradient with 0.1% TFA modifier over 8.5 min, 1.5mL/min, Column: Phenomenex Kinetex C18, 2.6 um 100 Â, 4.6 x 100 mm ) /r = 5.585 min.

[0201] Second Eluting Diastereomer is (S)-2-ethylbutyl 2-(((S)-(((2R,3S,4R,5R)-5-(4aminopyrrolo[2,1 -f][l ,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2yl)methoxyXphenoxy)phosphoryl)amino)propanoate:

^]HNMR (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, ,/= 5.8 Hz, 1H), 4.08 - 3.94 (m, 2H), 3.93 -3.85 (m, 1H), 1.47 (sep, 6.2 Hz, 1H), 1.38- 1.26 (m, 7H), 0.87 (t, J= 7.5 Hz, 6H). ³¹PNMR (162 MHz, CD3OD) δ 3.73 (s). HPLC (2-98% MeCNH₂O gradient with 0.1% TFA modifier over 8.5 min, 1.5mL/min, Column: Phenomenex Kinetex C18, 2.6 um 100 Â, 4.6 x 100 mm ) 1r = 5.629 min.

Example 13. (2S)-ethyl 2-((((2R,3S.4R.5R)-5-(4-aminopvrrolo|l,2-f|| 1.2.4|triazin-7-vl)-5cyano-3,4-dihvdroxvtetrahydrofuraii-2-Yl)methoxv)(phenoxv)phosphorylamino) propanoate (Compound 10)

[0202] The préparation of (2S)-ethyi 2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,l-f][l,2,4]triazin-7-yl)5-cyano-3,4'dihydroxytetrahydrofuran-2-yl)metlioxy)(phcnoxy)phosphoryl)amino)propanoate is described below.

Procedure 1. Préparation via Chloridate A

[0203) Prepared from Compound 1 and chloridate A using same method as for the préparation of compound 8. Ή NMR (300 MHz, CD₃OD) δ 7.95 (m, 1H), 7.32-6.97 (m, 7H), 4.78 (m, 1H), 4.43-4.08 (m, 6H), 3.83 (m, 1H), 1.31-1.18 (m, 6H). ³¹P NMR (121.4 MHz, CD3OD) δ 3.7.

LCMS m/z 547.0 [M+H], 545.0 [M-H],

Procedure 2. Préparation via Nitro-Benzene Compound L

[0204] Compound 1 (50 mg, 0.17 mmol) was dissolved in NMP-THF (1:1 mL)) and cooled with ice bath. tBuMgCl (0.257 mL, 0.257 mmol) was then added over about 5 min, The resulting mixture was allowed to warm to RT and was stirred for about 30 min. Then a solution of compound L (Prepared according to US20120009147, 74.6 mg, 0.189 mmol) in THF (2 mL) was added. After about 30 min, the reaction mixture was purified by HPLC (acetonitrile 10 to 80% in water) to give compound 29 as a yellow solid. The solid was further purified with silica gel chromatography (MeOH 0 to 20% DCM) to afford compound 29. ^]H NMR (400 MHz, CD₃OD)Ô 7.76 (d, J= 6.0 Hz, 1H), 7.25-7.14 (m, 2H), 7.11 -6.99 (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, IH), 4.06 - 3.91 (m, 2H), 3.72 (ddq, 14.3, 9.3, 7.1 Hz, 1 H), 1.17 (dd, J=1A, 1.0 Hz, 1H), 1.14- 1.06 (m, 5H). ^3IP NMR (162 MHz, CD3OD) δ 3.73, 3.68. MS m/z = 547 (M+l)⁺.

7-vl)-5-cyano-3,4-dihvdroxvtetrahvdrofÎiran-2-Yl)methoxv)phosphorvI)bis(azanediyl) dipropanoate (Compound 12)

[0205] The nucleoside 1 (14.6 mg, 0.05 mmol) was dissolved in anhydrous trimethyl phosphate (0.5 mL) and stirred under N₂(g) at RT. POCh (9.2 pL, 0.1 mmol) was added and the mixture stirred for about 60 min. Alanine ethyl ester hydrochloride (61 mg, 0.4 mmol) and then EtsN (70 pL, 0.5 mmol) was added. The résultant mixture was stirred for about 15 min. and then additional Et₃N (70 pi, 0.5 mmol) was added to give a solution pH of 9-10. The mixture was stirred for about 2 h. and then diluted with EtOAc, washed with saturated aqueous NaHCO₃ solution followed by saturated aqueous NaCl solution. The organic layer was dried over anhydrous Na₂SÛ4 and concentrated under reduced pressure. The residue was subjected to préparative HPLC (Ci8 column) to yield the product 12. *H NMR (400 MHz, CD₃0D) δ 8.13 (s, 1H), 7.41 (d, .7= 4.8 Hz, IH), 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). ³¹P NMR (121.4 MHz, CD₃OD) δ 13.8. LCMS m/z 570.0 [M+H], 568.0 [M-H].

Example 18. S,S^l-2,2’~((((2R₁3S.4R,5R)-5-(4-aminopvrrolo[l,2-fl[l,2,4]triazin-7-vl)-5cyano-3.,4-dihYdroxvtetrahvdrofuran-2-vl)methoxv)phosphorvl)bis(oxv)bis(ethane-2.1diyl) bis(2,2-dimethylpropanethioate) (Compound 15)

[0206] The nucleoside 1 (0.028 g, 0.096 mmol) was dissolved in tri methylphosp hâte (1 mL).

The reaction was stirred under N₂(g) and then treated with IH-tetrazole (0.021 g, 0.29 mmol).

The réaction mixture was cooled to 0 °C and the phosphane (Nucleoside Nucléotides, Nucleic acids, 14, 3-5, 1995; 763 - 766. Lefebvre, Isabelle; Pompon, Alain; Perigaud, Christian;

Girardet, Jean-Luc, Gosselin, Gilles; et al.) (87 mg, 0.192 mmol) was added. The reaction was stirred for 2 h. and then quenched with 30% hydrogen peroxide (0.120 mL). The mixture was stirred for 30 min at RT and then treated with saturated aqueous sodium thiosulfate (1 mL). The mixture was stirred for 10 min. and then concentrated under reduced pressure, The residue was subjected to préparative HPLC to isolate the title product 15. ^lH NMR (300 MHz, CD3CN) δ 7.98 (s, 1H), 6.92 (d, 1H), 6.81 (d, 1H), 6.44 (bs, 2H), 4.82 (m, 2H), 4.47 (m, 1H), 4.24 (m, 2H), 4.00 (m, 4H), 3.80 (bs, 1 H), 3.11 (m, 4H), 1.24 (s, 9H). ³¹P NMR (121.4 MHz, CD3CN) δ-1.85 (s). LCMSm/z661 [M+H].

Example 20. f(2R, 3S, 4R, 5R)-5-(4-aminopvrrolo[l,2-flil,2,4]triazin-7-vl)-5-cvano-3,4dihydroxvtetrahvdrofuran-2-yl)methyl tetrahydrogen triphosphate (Compound 17)

[0207] Compound 17 was prepared from compound 1 using a sîmîlar procedure as previously described (WO2012012776). The product was isolated as the sodium sait. Ή 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). ³¹P NMR (121.4 MHz, D₂O) δ -5.4 (d, IP), -10.8 (d, IP), -21.1 (t, IP). LCMS m/z 530 [M-H], 531.9 [M+H] Tr= 0.22 min. HPLC ion exchange Tr=9.95 min.

Example 20-a. ((2R,3S,4R,5R)-5-(4-aminopvrrolol2J-f|[l,2,41triazin-7-vl)-5-cyano-3,4dihvdroxytetrahvdrOfuran-2-yl)inethvl phosphate (Compound 33)

[0208] A mixture of about 0.05 mmol of compound 1 and about 0.5 mL of trimethylphosphate was sealed in a container for about one to about 48 h. The mixture was cooled to about -10 to about 10 °C and about 0.075 mmol of phosphores oxychloride is added. After about one to about 24 hours, the reaction was quenched with about 0.5 mL of IM tetraethylammonium bircarbonate and the desired fractions were isolated by anion exchange chromatography to afford the tîtle compound.

[0209] Compound 33 was prepared as the bis-tri ethylammonium sait front compound 1 as previously described (WO2011150288). Ή NMR (400 MHz, D?O) δ 7.82 (s, 1H), 6.91-6.88 (m, 1H), 6.81-6.78 (m, 1H), 4.87 - 4.84 (m, 1 H), 4.40 - 4.30 (m, 2H), 3.95 - 3.77 (m, 2H), 3.10-3.00 (m, 6H), 1.20- 1.10 (m, 9H). ³ⁱP NMR (162 MHz, D₂O) δ 2.33. MS wz371.

Example20-b. ((2R,3S,4R,5R)-5-(4-aminopyr r olo [ 2,1 -f| [ 1,2,4 ]triazin-7-vl)-5-cya no-3,4dihydroxvtetrahydroftiran-2-vl)methvl trihydrogen diphosphate (Compound 34)

[0210] Compound 34 was prepared as the tri-lithium sait from compound 1 as previously described (WO2002057425). ³¹P NMR (162 MHz, D₂O) δ -5.34 (d), -9.75 (d). MS m/z 451 cvano-3,4-dihvdroxvtetrahvdrofuran-2-vI)methoxv)(phenoxv)phosphoryl)amino)-3phenylpropanoate (21)

[0211] The préparation of (2S)-ethyl 2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,lf][l,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2yl)methoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate is described below.

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

[0212] L-Phenylaianine (5 g, 30 mmol) was taken up in EtOH (30 mL). TMSC1 (6.915 mL, 54 mmol) was added to the reaction at RT. The reaction vessel was fitted with a reflux condenser and the reaction was placed in an 80 °C bath. The reaction was stirred overnight. The next day the reaction was cooled to RT, concentrated under reduced pressure and the resuiting residue was taken up in Et₂O. The resuiting slurry was filtered and the isolate solids were further washed with Et₂O. The washed solids were placed under high vacuum to yield example (S)-ethyl 2amino-3-phenylpropanoate hydrochloride. *H NMR (400 MHz, DMSO-dû) δ 8.52 (s, 3H), 7.30 (m, 5H), 4.24 (ABX 7.8 Hz, J_Bx = 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).

Préparation of (2S)-ethy1 2-(((4-nitrophenoxv)(phenoxy)phosphoryl)amino)-3-phenylpropanoate (Compound D)

[0213] (S)-ethyl 2-amîno-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)Cl₂ (0.656 mL, 4.41 mmol) was added, followed by the slow addition of EtjN (1.62 mL, 11.5 mmol) over 5 min. The cold bath was removed and the reaction was allowed to warm to RT and stir over a period of 80 min. p-NO₂PhOH (0.583 g, 4.19 mmol) was added, followed by more Et₃N (0.3 mL, 2.1 mmol). The reaction progress was monitored by LC/MS. Upon completion of the reaction, it was diluted with Et₂O, and the resulting solids were removed by filtration. The filtrate was concentrated and compound D was îsolated by si 1 ica gel column chromât ography (25 g dry load cartridge, 120 g column; eluent: 100% hexanes ramping to 55% EtOAc in hexanes). 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). ³¹P NMR (162 MHz, DMSO-d₆) δ -1.479 (s), 1.719 (s). MS m/z = 471.01 [M+1],

Préparation of (2S)-ethyl 2-(((((2R,3S.4R,5R)-5-(4-ammopyrrolo[2J-f|[L2,4]triazin-7-yl)-5cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3phenylpropanoate (Compound 21)

[0214] Compound 1 (0.030 g, 0.103 mmol) was dissolved in DMF (1 mL) and then THF (0.5 mL) was added. ABuMgCl (1M/THF, 154.5 pL, 0.154 pmol) was added to the reaction in a drop-wise manner with vigorous stîrrîng. The resulting white slurry was stirred at RT for about 30 min. A solution of compound D (0.058 g, 0.124 mmol) in THF (1 mL) was added in a dropwise manner to the reaction at RT. The reaction progress was monitored by LC/MS. When the reaction progressed to 50% conversion, the reaction was cooled in an ice bath and quenched with glacial acetic acid (70 pL). The reaction was concentrated and compound 21 was îsolated from the residue by reverse phase HPLC. ‘H NMR (400 MHz, DMSO-dô) § 7.91 (d, J= 4 Hz, 1H), 7.90 (brs, 2H), 7.09-7.30 (m, 8H), 7.01, (t, 8.2 Hz, 2H), 6.89 (d, J = 4.4 Hz, 1H), 6.82 (t, J=4.4Hz, 1H), 6.27 (m, IH),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). ³¹P NMR (162 MHz, DMSO-dr,) δ 3.69 (s), 3.34 (s). MS m/z = 623.0 [M+HJ.

Example 25. (2S)-ethvl 2-(((«2R,3S.,4R,5R)-5-(4-aminoDvrrolo[2,l-fnL2,4]triazin-7-vl)-5cyano-3,4-d ihydroxy tetra hydrofuran-2-yl) methox ylipli en oxv)Dhosphorvl)amino)-3methylbutanoate (22)

[0215] The préparation of(2S)-ethyl 2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,lf][l,2,4]triazin-7-yl)-5-cyano-3,4-dîhydroxytetrahydrofuran-2yl)methoxy)(phenoxy)phosphoryl)amîno)-3-methylbutanoate is described below.

Préparation of (2S)-ethyl 3-methyl-2-(((4-nitrophenoxv)(phenoxy)Dhosphoryl)amino) butanoate (Compound E)

PhOP(O)CI₂, Et₃N, p-NO₂PhOH

E

[0216] The (S)-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)Ch (0.287 mL, 1.932 mmol) was added, followed by the slow addition of Et₃N (1.62 mL, 11.4 mmol) over about 5 min. The coid bath was removed and the reaction was allowed to warm to RT and stir over a period of 1 h. p-NChPhOH (0.255 g, 1.836 mmol) was added, and the reaction progress was monitored by LC/MS. Upon completion of the reaction, the mixture was diluted with EtsO, and the resulting soiids were removed by filtration. The filtrate was concentrated and compound E was isolated by si 1 ica gel column chromatography (12 g dry load cartridge, 80 g column; eluent: 100% hexanes ramping to 55% EtOAc in hexanes). *H NMR (400 MHz, DMSO-dr,) δ 8.30 (d, J = 9.2 Hz, 2H), 7.48 (t, J= 9.6 Hz, 2H), 7.40 (t, J = 7.8 Hz, 2H), 7.20-7.27 (m, 3H), 6.60 (quart, J = 11.6 Hz, JH), 4.01 (m, 2H), 3.61 (m, 1H), 1.93 (m , 1H), 1.11 (m, 3H), 0.79 (m, 6H). ³¹P NMR (162 MHz, DMSO-dô) δ -0.342 (s), -0.578 (s). MS m/z = 422.9 [M+H],

Préparation of (2S)-ethyl 2-(((((2R,3S.4R,5R)-5-(4-aminopyrrolo[2, l-f][L2,4]triazin-7-yl)-5cyano-3,4-dihydroxytetrahydrofÎiran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-3methylbutanoate (Compound 22) tBuMgCI

NMP, THF

HO OH |0217] 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 /-BuMgCl (1M/THF, 425.7 pL, 0.426 pmol) was added in a drop-wise manner with vigorous stirring. The ice bath was removed and the resulting white slurry was stirred at RT for about 15 min. A solution of compound E (0.081 g, 0.192 mmol) in THF (0.5 mL) was added in a drop-wise manner to the reaction at RT. The reaction progress was monitored by LC/MS. When the reaction progressed to 50% conversion, the reaction was cooled in an ice bath and quenched with glacial acetic acid (70 pL). The réaction was concentrated and compound 22 was semi-purified from the residue by reverse phase HPLC. The semi-pure material was further purified by silica gel column chromatography (12 g dry load cartridge, 40 g column; eluent: 100% EtOAc ramping to 10% MeOH in EtOAc) to yield compound 22. ’H NMR (400 MHz, DMSO-dô) δ 7.91 (d, J ~ 1.6 Hz, 1H), 7.88 (brs, 2H), 7.32 (m, 2H), 7.15 (m, 3H), 6.90 (t, J = 4.2 Hz, 1H), 6.84 (d, J = 4.8 Hz, 1H), 6.26 (dd, J = 13.4, 6.2 Hz, 1H), 5.87 (quart. ./= 11.2 Hz, 1H), 5.35 (m, 1 H), 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). ³¹P 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-aminopvrrolo[2,l-f|[l,2,4|triazin7-yl)-5-cyano-3,4-dihytlroxvtetrahvdrofiiran-2-vl)methoxv)(phenoxv)phosDhorvl) amino)propanoate (23)

[0218] The préparation of (S)-isopropyl 2-(((R)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1 f] [ 1,2,4]tri azî n -7-yl )-5-cy ano-3,4-dihy droxytetrahy drofüran-2-yl)methoxy Xphenoxy ) phosphoryl)amino)propanoate is described below.

[0219] Compound 1 (60.0 mg, 206 pmol) was dissolved in NMP (0.28 mL). THF (0.2 mL) was added followed by tert-butyl magnésium chloride (1.0M solution in tetrahydrofuran, 0.309 mL) at RT under an argon atmosphère. 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 pmol) in THF (0.2 mL) was added, and the resulting mixture was warmed to about 50 °C. After 3 h, the reaction mixture was allowed to cool to RT and was purified direct ly by préparaiory HPLC (Phenominex Synergi 4u Hydro-RR 80Â 150 x 30 mm column, 5-100% acetonitrile/water gradient) to afford compound 23. Ή NMR (400 MHz, CD₃OD) 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 (sept, 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 (dq, .7 = 9.7, 7.1 Hz, 2H), 1.27 (dd,J= 7.1, 1.0 Hz, 3 H), 1.18 (dd, J = 6.3, 4.8 Hz, 6H). ³¹PNMR(162 MHz, CD3OD) δ 3.72 (s). LC/MS: t_R = 1.39 min, MS m/z = 561.11 [M+H], LC System: Thermo Accela 1250 UHPLC; MS System: Thermo LCQ Fleet, Column: Kinetex 2.6μ XB-C18 100A, x 4.6 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 2pl/min. HPLC: îr = 2.523 min; HPLC System: Agilent 1100 sériés., Column: Gemini 5μ Cl8 110A, 50 x 4.6 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.

Example 27. (2S)-cycIobutyl 2-(i(((2R.3S.4R,5R)-5-(4-aminopvrrolo[2,l-f|[l,2,41triazin-7vl)-5-cvano-3,4-dihvdroxvtetrahydrofuran-2-vl)methoxy)(phenoxy)phosphoryl)amino) propanoate (24)

[0220] The préparation of (2S)-cyclobutyl 2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1f] [ 1,2,4]tri azi n- 7-y l)-5-cy ano-3,4-dihydroxytetrahydrofuran-2yl)methoxy)(phenoxy)phosphoryl)amino)propanoate is described below.

Préparation of (2S)-cyclobutyl 2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (Compound G)

O

PhO-P-Cl +

I

Cl

HCl H₂N

[0221] Phenyl dichlorophosphate (1.49 mL, 10 mmol) was dissoived in 10 mL of anhydrous DCM and stirred under atmosphère nitrogen in an ice bath. L-Alanine isobutyl ester hydrochloride (0.9 g, 5 mmol) was added in one portion. Triethylamine (765 pL, 5.5 mmol) was then added dropwise. Reaction stirred for about I h. More Triethylamine (765 pL, 5.5 mmol) was added dropwise and the reaction was stirred for about 45 min. p-Nitrophenol (1.25g, 9mmol) was added in one portion and stirred for about 30 min. Triethylamine (765 pL, 5.5 mmol) was added and the reaction mixture was stirred for about 2 h. Additional p-nitrophenol (1,25g, 9 mmol) and triethylamine (765 pL, 5.5mmol) were then added, and the reaction was stirred for another about 2 h. The reaction mixture was concentrated under reduced pressure. The resulting crude was diluted with EtOAc and washed twice with 5% aqueous citric acid solution, followed with saturated aqueous sodium chloride solution. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified with silica gel column (0-20-50% EtOAc in hexanes) to give compound G. 'H NMR (400 MHz, CDîOD) 5 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). ³¹P NMR (162 MHz, CD₃OD) Ô-1.36,-1.59. MS m/z = 420.9 [M+HJ.

Préparation (2 S )-cyclobutyl 2-(((((2R,3 S.4R, 5R)-5-(4-aminopyrrolo [2.1 -fl Γ1,2,4]triazin-7-yl)-5cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxyXphenoxy)phosphoryl)amino)propanoate (Compound 24)

[0222] Compound 1 (58 mg, 0.2 mmol) was mixed with compound G (101 mg, 0.24 mmol) in 2 mL of anhydrous DMF. Magnésium chloride (42 mg, 0.44 mmol) was added in one portion. The reaction mixture was heated to about 50 °C. DIPEA (87 pL, 0.5 mmol) was added, and the reaction was stirred for about 2 h at about 50 °C. The reaction mixture was cooled to room température, was diluted with EtOAc and was washed with 5% aqueous citric acid solution followed by saturated aqueous sodium chloride solution. The organic layer was then dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified with silica gel column (0-2-5% MeOH in DCM) to afford compound 24. ^]H NMR (400 MHz, Methanol-d4) δ 7.85 (m, IH), 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, IH), 1.62 (m, 1H), 1.30- 1.16 (m, 3H). ³¹P NMR (162 MHz, cd₃od) δ 3.70, 3.65. MS m/z = 573.0 [M+H].

Example 28. (2S)-isoDroDvl 2-(((((2R,3S,4R,5R)-5-(4-amin opyrrolo [2,1-fl [1,2,4] triazin-7vl)-5-cvano-3,4-dihvdroxvtetrahvdrofuran-2-vl)methoxv)(phenoxv)phosphoryl)amino)-3phenylpropanoate (25)

[0223] The préparation of (2S)-isopropyI 2-(((((2R,3S,4R,5R)-5-(4-aminopynOlo]2,l-fi[L2,4Jtriazin7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)mcthoxy)(phenoxy)phosphoryl)amino)-3phenylpropanoate is described below.

Préparation of (2S)-isopropyl 2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)-3phenylpropanoate (Compound H)

[0224] Phenyl dichlorophosphate (718 pL, 4.8 mmol) was dissolved in 10 mL of anhydrous DCM and stirred under a nitrogen atmosphère in an ice bath. L-Phenylalanine îsopropyl ester hydrochloride (1 g, 4.1 mmol) was added in one portion. Another 10 mL of anhydrous DCM was added. Triethylamine (736 pL, 5.3 mmol) was added dropwise and the reaction mixture was stirred for about 30 min. More triethylamine (736 pL, 5.3 mmol) was then added dropwise and the reaction mixture was stirred for 30 min. Additional triethylamine (736 pL, 5.3 mmol) was then added dropwise and the reaction mixture was stirred for about 15 min. p-Nitrophenol (600 mg, 4.32 mmol) was then added. The ice bath was then removed and the reaction mixture was allowed to warm to room température and stirred for about 2 h. More p-nitrophenol (50 mg) and triethylamine (736 pL, 5.3 mmol) were the added and the reaction mixture was stirred for about 1 h.

[0225] The reaction mixture was then concentrâted under reduced pressure, and was diluted with EtOAc and washed twice with 5% aqueous citric acid solution, followed with saturated aqueous sodium chloride solution. The organic layer was dried over anhydrous sodium sulfate and was concentrated under reduced pressure. The crude was purified with silica gel column (015% EtOAc in hexanes) to gîve compound H. ’H NMR (400 MHz, CDCh) 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). ³ⁱP NMR (162 MHz, cdcl3) δ-2.96,-2.98. MS m/z = 485.0 [M+H],

Préparation of (2S)-isopropyl 2-((ffi 2R,3S.4R.5R)-5-(4-aminopyrrolof2,1-flH ,2,41triazin-7-yI)5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amîno)-3phenylpropanoate (Compound 25)

[0226] 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 a nitrogen atmosphère at room température. IM tBuMgCl in THF (300 pL, 0.3 mmol) was added dropwise over 3 minutes and the reaction mixture was then stirred for about 16 h. The reaction mixture was diluted with EtOAc and washed with 5% aqueous citric acid solution, saturated aqueous sodium bicarbonate solution 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 with silica gel column (0-5% MeOH in DCM) to give compound 25. *H 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, lH),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). ³¹P NMR (162 MHz, CD₃OD)Ô3.63, 3.25. MS m/z = 637.0 [M+H],

Example 29. (S)-methyl 2-(((S)-(((2R,3S,4R,5RE5-(4-aminopvrrolol2,1 -fl[1,2.41 triazin-7vl)-5-cyano-3,4-dihvdroxytetrahvdrofuran-2-vümethoxv)(phenoxv)phosphorvl)amino) propanoate (26)

[0227] The préparation of (S)-methyl 2-(((S)-(((2R,3S,4R_s5R)-5-(4-aminopyrrolo[2,]-f][L2,4]triazin7-yI)-5-cyano~3,4-dihydroxytctrahydrofuran-2-yI)methoxy)(phenoxy)phospÎioryI)amiiLo)propanoate is described below.

[0228] Compound 1 (100 mg, 0.34 mmol) was dissolved in THF (2 mL) and cooled with an ice water bath. Then IM t-BuMgCl (0.52 mL, 0.77 mmol) was added dropwise slowly. The resulting mixture was stirred for about 30 min at room température. Then compound I (Prepared according to WO 2012142085, 219 mg, 0.52 mmol) in THF (2 mL) was added over 5 min and the resulting mixture was stirred for about 24 h at room température. The reaction mixture was then diluted with EtOAc, cooled under ice-water bath, washed with aq NaHCCE (2 mL), washed with brine, dried with sodium sulfate, and concentrated in vacuo. The resulting mixture was purified by silica gel column chromatography (MeOH 0 to 20% in DCM) and prepHPLC (acetonitrile 10 to 80% in water) to give compound 26. Ή NMR (400 MHz, CD3OD) Ô 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 (ddq, J= 10.8, 5.3, 2.7 Hz, 2H), 4.33-4.23 (m, 1 H), 4.18 (t, J = 5.5 Hz, 1H), 3.86 (dq, 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+l)\

Example 30. (S)-neopentyl 2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopvrrolo[2J-fl[L2,4]triazin7-yl)-5-cyano-3,4-dihydroxytetrahvdrofiiran-2-yl)methoxY)(phenoxv)phosphorvl)amino) propanoate (27)

[0229] The préparation of (S)-ncopcntyl 2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,l-f|[l,2,4] tnazLn-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryî)ammo) propanoate is described beîow.

[0230] Compound 1 (100 mg, 0.34 mmol) was dissoived in THF (2 mL) and cooled under ice water bath. Then IM t-BuMgCl (0.52 mL, 0.77 mmol) was added dropwise slowly. The resulting mixture was stirred for about 30 min at room température. Then compound J (Prepared according to WO2012075140, 248 mg, 0.52 mmol) was added over about 5 min and the resulting mixture was stirred for about 24 h at room température, diluted with EtOAc, cooled under ice-water bath, treated with aq NaHCOî (2 mL), washed with brine, dried with sodium sulfate, and concentrated in vacuo. The resulting mixture was purified by silica gel column chromatography (MeOH 0 to 20% in DCM) and prep-HPLC (acetonitrile 10 to 80% in water) to give Compound 27. Ή 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, 1 H), 4.38 (tdd, J= 10.0,4.9, 2.5 Hz, 2H), 4.32-4.24(m, 1H), 4.17 (t, 7= 5.6 Hz, 1H), 3.91 (dq,7=9.8, 7.1Hz, 1H),3.81 (d, J= 10.5 Hz, IH), 3.69 (d, J= 10.5 Hz, 1H), 1.31 (dd, 7 = 7.2, 1.1 Hz, 3H), 0.89 (s, 9H). MS m/z = 589 (M+l)\

100

Example 31. (2S)-cyclopentyl 2-(((((2R,3S,4R,5R)-5-(4-aininopyrrolo[2J-f|[l,2,41triazin-7vI)-5-cvano-3.4-dihvdroxvtetrahvdrofuran-2-yl)methoxv)(phenoxv)phosplioryl)amino) propanoate (28)

[0231] The préparation of (2S)-cyclopentyi 2-(((((2R,3S,4R,5R)-5-(4-atninopyrrolo[2,l-f|[l,2,4j triazin -7 - y 1) *5 -c yano - 3,4 -d ih ydr oxytetrah ydrofu ran-2 -y 1 )methoxy ) (p henoxy)p hosp ho ni ) am i no) propanoate is described below.

[0232] Compound 1 (100 mg, 0.34 mmol) was dissolved in THF (2 mL) and cooled under ice water bath. Then IM t-BuMgCl (0.52 mL, 0.77 mmol) was added dropwîse slowly. The resulting mixture was stirred for about 30 min at room température. Then compound K (Prepared according to WO2012075140, 247 mg, 0.52 mmol) in THF (2 mL) was added over about 5 min and the resulting mixture was stirred for about 24 h at room température, diluted with EtOAc, cooled under ice-water bath, treated with aq NaHCOs ( 2mL), washed with brine, dried with sodium sulfate, and concentrâted in vacuo. The resulting mixture was purified by silica gel column chromatography (MeOH 0 to 20% in DCM) and prep-HPLC (acetonitrîle 10 to 80% in water) to give example 28. *H NMR (400 MHz, CDsOD) δ 7.85 (s, 1 H), 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, 1 H), 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 (dq, 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+I)⁺.

101

EMm_JJi_Î32₁J2Shcv<didiexvLiii£<(2IL3S_J4IWRhM4_Iamù^^ vl)-5-cvano-3,4-dihvdroxvtetrahvdrofuran-2-yl)methoxv)(phenoxv)phosphorvhamino) propanoate (29)

[0233] T o a mixture of compound 1 (50 mg, 0.343 mmol), compound M (Prepared according to US20130143835, 93 mg, 0.209 mmol), and MgC12 (24.5 mg, 0.257 mmol) in DMF (1 mL) was added diisopropylethy lamine (0.075 mL, 0.43 mmol) dropwise over about 5 min at about 0 °C. The resulting mixture was stirred at about 50 °C for about 1 h. The reaction mixture was then cooled with an ice-water bath, treated with IM citric acid ( 0.5 mL), and was purified directly by prep-HPLC (ACN 0 to 70% in water) to afford compound 29. ^:H NMR (400 MHz, CD₃OD) δ 7.84 (s, IH), 7.32-7.23 (m, 2H), 7.18-7.10 (m, 3H), 6.93-6.87 (m, 2H), 4.78 (d,,7 = 5.4 Hz, IH), 4.67 (td, .7= 8.7, 4.2 Hz, IH), 4.48-4.35 (m, 2H), 4.30 (ddd, J= 10.8, 5.7, 3.7 Hz, IH), 4.20 (t, .7= 5.4 Hz, IH), 3.88-3.71 (m, IH), 1.83 -1.63 (m, 4H), 1.58-1.46 (m, IH), L46-L24(m, 5H), 1.24(s, 3H). ^3ÎP NMR (162 MHz, CD3OD) δ 3.75. MSm/z = 601 (M+l)⁺.

102

Examplc 33. Ethyl 2-(((((2R,3S,4R,5R)-5-(4-aminopvrrolo[2,Î-nil-,2,4]triazin-7-yl)-5cyano-3.4-dihvdroxytetrahvdrofuran-2-yl)methoxy)(Dhenoxy)Dhosphorvl)amino)-2methvlpropanoate (30)

[0234] The préparation of ethyl 2-(((((2R,3S,4R.5R)-5-(4-aminopyrrolo[2J-f][1,2,4]triazin-7-yl)-5cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)-2-methylpropanoate is described below.

Préparation of Ethyl 2-((tert-butoxycarbonyl)amino)-2-methylpropanoate

O

OH

PPh₃, DIAD, THF

[0235] Take up trîphenylphosphine (6.18 g, 25.00 mmol) in THF (30mL). Next charge DIAD (4.92 mL, 25.00 mmol) and stir at room température for 10 min. Dissolve 2-((tertbutoxycarbonyl)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 éthanol (2.19 mL, 37.49 mmol). Allow the reaction to stir at room température for about 1 h. The solvents were removed under reduced pressure and the crude was taken up in 1:1 Et₂O: Hexanes (120 mL). The solid trîphenylphosphine oxide was filtered off and the solvent was removed under reduced pressure. The crude was taken up in minimal CH2CI2 and purified by silica gel chromatography 0-50% EtOAc/Hex to afford ethyl 2-((tert-butoxycarbonyl)amino)-2-methylpropanoate. Ή NMR (400 MHz, Chloroform-iZ) δ 4.18 (q, J= 7.1 Hz, 2H), 1.49 (s, 6H), 1.43 (s, 9H), 1.27 (t, J= 7.1 Hz, 3H).

Préparation of Ethyl 2-amino-2-methylpropanoatehydrochloride

4N HCl in Dioxane, DCM

HCl

103

[0236] Take up ethyl 2-((tert-butoxycarbonyl)amino)-2-methyIpropanoate (2.71 g, 11.72 mmol) in CH₂C1₂ (25 mL) and slowly add 4N HCl in dioxane (25 mmol) and stir at room température. At Ih, the reaction was determined to be complété by TLC. The solvents were removed under reduced pressure and the crude was coevaporated with Et₂O two times then placed under high vacuum to afford ethyl 2-amino-2-methylpropanoate hydrochloride. ^LH NMR (400 MHz, DMSO-îZ₆) δ 8.70 (s, 3H), 4.18 (q, J= 7.1 Hz, 2H), 1.46 (s, 6H), 1.21 (t, J=1A Hz, 3H).

Préparation of Ethyl 2-methyl-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (Compound N)

no₃ o₂n n

[0237] Take up phenyl dichlorophosphate (0.97mL, 6.50mmol) and ethyl 2-amino-2methylpropanoate hydrochloride (1.09 g, 6.50 mmol) in CH₂C1₂ (50 mL). Cool the reaction mixture to about 0 °C and slowly add TEA (L75 mL, 12.45 mmol). Remove the cold baîh and allow the reaction mixture to stir at room température. After about 2 h, the addition of the amino acid was determined to be complété by ³¹P NMR. Charge p-nitrophenol (0.860 g, 6.17 mmol) followed by the addition of TEA (0.87 g, 7.69 mmol). Allow the reaction to stir at room température. After about 2 h, the reaction was determined to be complété by LCMS. The reaction was diluted with Et2O and the TEA*HC1 salis were filtered off. The crude was concentrâted and purified by silica gel chromâtography (0-50% EtOAc/Hex) to afford compound N. ‘H NMR (400 MHz, DMSO-î/6) δ 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, IH), 3.99 (q, J= 7.1 Hz, 2H), 1.39 (s, 6H), 1.08 (t,.7=7.1 Hz, 3 H). ³¹P NMR (162 MHz, DMSO-âfc) δ-2.87. LC/MS: tR = 1.65 min, MS m/z = 408.97 [M+l]., LC System: Thermo Accela 1250 UHPLC; MS System: Thermo LCQ Fleet; Column: Kinetex 2.6p XB-C18 100A, 50 x 3.00 mm; Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min-2.4 min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8mL/min.

104

Préparation of ethyl 2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,I-f|['L2,41triazin-7-vl)-5-cyano3,4-dihydroxytetrahydrofùran-2-yl)methoxy)(phenoxv)phosphoryl)amino)-2-methylpropanoate (Compound 30)

[0238] Take up compound 1 (66 mg, 0.23 mmol) in NMP (2.0 mL). Cool the mixture to about 0 °C and slowly add tBuMgCl (1.0M in THF, 0.34 mL, 0.34 mmol). Allow the reaction to stir at about 0 °C for about 30 min, then add a solution of compound N (139mg, 0.34mmol) dissolved în THF (LO mL). Remove the cold bath and place the reaction in about 50 °C preheated oil bath. After about 2 h, the reaction was cooled to room température and quenched with acetic acid and methanol. The crude was concentrated and purified by reverse phase HPLC without modifier to afford compound 30. *H NMR (400 MHz, DMSO-uk) δ 7.89 (m, 3H), 7.31 (q, 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, .7 = 11.7, 1H), 5.81 (d,,7=9.7, 1H), 5.35 (d, J = 5.6 Hz, 1H), 4.64 (dt, ,7= 9.0, 5.6 Hz, lH),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). ³¹P NMR (162 MHz, DMSO-Jî) 5 2.45, 2.41. LC/MS: t_R= 1.03 min, MS m/z = 561.03 [M+l]; LC System: Thermo Accela 1250 UHPLC; MS System: Thermo LCQ Fleet; Column: Kinetex 2.6p XB-C18 100A, 50 x 3.00 mm; Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min-2.4 min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 min2.85 min 100%-2% ACN, 2.85 mîn-3.0 min 2% ACN at 1.8mL/min.

105

Example 34, Isopropyl 2-(((((2R,3S,4K5R)-5-(4-aminopvrrolo[2,l-f|[l,2,4]triazin-7-vl)-5cyano-3,4-dihvdroxvtetrahydrofiiran-2-vl)methoxv)(phenoxv)phosphorvl)amino)-2methylpropanoate (31)

[0239] The préparation of Isopropyl 2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,I-f][l,2,4]triazin-7-yl)5-cyÎino-3,4-dihydroxytetraIiydrofuran-2-yl)meÎhoxy)(phenoxy)phosphoryl)anùno)-2-methyIpropanoate is described below.

Préparation of Isopropyl 2-((tert-butoxycarbonyl)amino)-2-methylpropanoate

O

PPh₃, DIAD, THF

[0240] Take up triphenylphosphine (6.17 g, 25.00 mmol) in THF (30 mL). Next charge DIAD (4.92 mL, 25.00 mmol) and stir at room température for about 10 min. Dissolve 2-((tertbutoxycarbonyl)amino)-2-methylpropanoic acid (5.07 g, 25.00 mmol) dissolved in THF (20mL) and add to the reaction mixture followed by the addition of isopropanol (1.91 mL, 25.00 mmol). Allow the reaction to stir at room température for about Ih. The solvents were removed under reduced pressure and the crude was taken up in 1:1 Et₂O:Hexanes (120 mL). The solid triphenylphosphine oxide was filtered off and the solvent was removed under reduced pressure. The crude was taken up in minimal CH₂C1₂ and purifîed by silica gel chromatography (0-50% EtOAc/Hex) to afford isopropyl 2-((tert-butoxycarbonyl)amino)-2-methylpropanoate. ^LH NMR (400 MHz, Chloroformé) δ 5.03 (p, J=6.2Hz, 1H), 1.48 (s, 6H), 1.40 (d, J = 6.2 Hz, 9H), 1.24 (d, .7=6.3 Hz, 6H).

Préparation of Isopropyl 2-amino-2-methylpropanoate hydrochloride

4N HCl in Dioxane, DCM

106

[0241] Take up isopropyl 2-((tert-butoxycarbonyl)amino)-2-methyïpropanoate (4.09 g, 16.67 mmol) in CH2CI2 (50 mL) and slowly add 4N HCl in dioxane (50 mmol) and stir at room température. At about 1 h, the reaction was determined to be complété by TLC. The solvents were removed under reduced pressure and the crude was coevaporated with Et₂O two limes then placed under high vacuum to afford isopropyl 2-amino-2-methylpropanoate hydrochloride. ’H NMR (400 MHz, DMSO-î4) 5 8.61 (s, 3H), 4.96 (p, .7= 6.2 Hz, IH), 1.44 (s, 6H), 1.22 (d,J = 6.2 Hz, 6H).

Préparation of Isopropvl2-methvl-2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino) propanoate (Compound O)

CH₃C1₂₁ tea, o°c

O

[0242] Take up phenyl dichlorophosphate (0.83 mL, 5.58 mmol) and isopropyl 2-amino-2methylpropanoate hydrochloride (1.01 g, 5.58 mmol) in CH2CI2 (50 mL). Cool the reaction mixture to 0 °C and slowly add TEA (1.61 mL, 11.45 mmol). Remove the cold bath and allow the reaction mixture to stir at room température. After about 2 h, the addition of the amino acid was determined to be complété by³¹P NMR. Charge p-nitrophenol (0.74 g, 5.30 mmol) foliowed by the addition of TEA (0.81, 5.84 mmol). Allow the reaction to stir at room température. After about 2 h, the reaction was determined to be complété by LCMS. The reaction was diluted with Et2O and the TEA*HC1 salts were filtered off. The crude was concentrated and purified by si 1 ica gel chromatography (0-50% EtOAc/Hex) to afford compound O. ’H NMR (400 MHz, DMSO-t&) S 8.42 - 8.19 (m, 2H), 7.55 - 7.43 (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). ³¹P NMR (162 MHz, DMSO-Æ) δ -2.84. LC/MS: tR = 1.73 min, MS m/z = 422.92 [M+l]; LC System: Thermo Accela 1250 UHPLC; MS System: Thermo LCQ Fleet, Column: Kinetex 2.6μ XB-C18 100A, 50 x 3.00 mm; Solvents: Acetonitrîle with 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min-2.4 min 2100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 min-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1.8mL/min.

107

Préparation of Isopropyl 2-(((((2R,3S,4R,5R)-5-i4-aminopyrrolor2J-f|[L2,4]triazin-7-yl)-5cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryÎ)amino)-2methylpropanoate (Compound 31)

[0243] Take up compound 1 (66 mg, 0.23 mmol) in NMP (2.0 mL). Cool the mixture to about 0 °C and slowly add tBuMgCl (1,0M in THF, 0.57mL, 0.57mmol). Allow the reaction to stir at about 0 °C for about 30 min, then add a solution of compound O (143 mg, 0.34 mmol) dissolved in THF (1.0 mL). Remove the cold bath and place the reaction in an about 50 °C preheated oil bath. After about 2 h, the reaction was cooled to room température and was quenched with acetic acid and methanol. The crude was concentrated and purifîed by reverse phase HPLC without modifier to afford compound 31. Ή NMR (400 MHz, DMSO-4) 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 (p, J=6.3 Hz, 1H), 4.71 -4.50 (m, 1H), 4.23 (m, 2H), 4.11 (m, IH), 4.03 - 3.83 (m, 1H), 1.37-1.23 (m, 6H), 1.18 - 1.04 (m, 6H). ³ⁱP NMR (162 MHz, DMSO) δ 2.47, 2.43. LC/MS: t_R = 1.08 min, MS m/z = 575.06 [M+l]; LC System: Thermo Accela 1250 UHPLC; MS System: Thermo LCQ Fleet; Column: Kinetex 2.6μ XB-C18 100A, 50 x 3.00 mm; Solvents: Acetonitrile with 0.1% formic acid, Water with 0.1% formic acid; Gradient: 0 min-2.4 min 2-100% ACN, 2.4 min-2.80 min 100% ACN, 2.8 mîn-2.85 min 100%-2% ACN, 2.85 min-3.0 min 2% ACN at 1,8mL/min.

108

Examnle 35. (S)-2-ethvlbutvl 2-a<S)-(R2R.3S.4R.5R>-544-ainmo_Pvrrolol2.1-fl 11,2,41 triazin-7-v0-5-cvano-3,4-dihvdroxvtetrahvdrofuran-2-vl)methoxy)(phenoxy)phosphorv0 amino)propanoate (32)

[0244] The préparation of (S)-2-ethylbutyl 2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,lf][l,2,4]tnazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxyXphenoxy) phosphoryl)amino)propanoate is described below.

Préparation of (3R.4R,5R)-3.4-bis(benzyloxy)-5-((benzyloxy)methyl)dihydrofuran-2(3H)-one.

TEMPO, free radical

V Y-OH Τλ°

BnO'' -- NaOCI BnO' -OBn K₂HPO₄ OBn

[0245] (3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)meÎhyl)tetrahydrofuran-2-ol (15.0 g) was combined with MTBE (60.0 mL), KBr (424.5 mg), aqueous K2HPO4 solution (2.5M, 14.3 mL), and TEMPO (56 mg). This mixture was cooled to about 1 °C. Aqueous bleach solution (7.9%wt.) was slowly charged in portions until complété consumption of starting material as indicated through a starch/iodide test. The layers were separated, and the aqueous layer was extracted with MTBE. The combined organic phase was dried over MgSÛ4 and concentrated under reduced pressure to yield the product as a solid.

Préparation (4-amino-7-iodopyrrolo[2.1-fl Γ L2,41triazine)

DMF, 0 °c

|0246] To a cold solution of 4-aminopyrrolo[2,l-f][l,2,4]-triazine (10.03 g; 74.8 mmol) in Ν,Ν-dimethyIformamide (70.27 g), N-iodosuccinimide (17.01g; 75.6 mmol) was charged in portions, while keeping the contents at about 0 °C. Upon reaction completion (about 3 h at about

109 °C), the reaction mixture was transferred into a 1 M sodium hydroxide aqueous solution (11g NaOH and 276 mL water) while keeping the contents at about 20-30 °C. The resulting slurry was agitated at about 22 °C for 1.5 h and then filtered. The solids are rinsed with water (50 mL) and drîed at about 50 °C under vacuum to yield 4-amino-7-iodopyrrolo[2,l-f[ [l,2,4]triazine as a solid. NMR (400 MHz, DMSO-d6) 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). ¹³C NMR (101 MHz, DMSO-d6) δ 155.7, 149.1, 118.8, 118.1, 104.4, 71.9. MS m/z = 260.97 [M+H],

Préparation (3R4R5R)-2-(4-aminopyrrolo[2,1 -f][ 1,2,4]triazm-7-yl)-3,4-bis(benzyloxy)-5f(benzyloxy)methyl)tetrahydrofuran-2-ol via (4-amino-7-iodopyrrolo[2,l-f] [L2,4]triazine ) nh₂

[0247] To a reactor under a nitrogen atmosphère was charged iodobase 2 (81 g) and THF (1.6 L). The resulting solution was cooled to about 5 °C, and TMSCI (68 g) was charged. PhMgCi (345mL, 1.8 M in THF) was then charged slowly while maintaining an internai température at about < 5 °C. The reaction mixture was stirred at about 0 °C for 30 min, and then cooled to about -15 °C. rPrMgCL LiCl (311 mL, 1.1 M in THF) was charged slowly while maintaining an internai température below about -12 °C. After about 10 minutes of stirring at about -15 °C, the reaction mixture was cooled to about -20 °C, and a solution of lactone 1 (130 g) in THF (400 mL) was charged. The reaction mixture was then agitated at about -20 °C for about 1 h and quenched with AcOH (57 mL). The reaction mixture was warmed to about 0 °C and adjusted to pH 7-8 with aqueous NaHCCh (5 wt%, 1300 mL). The reaction mixture was then diluted with EtOAc (1300 mL), and the organic and aqueous layers were séparaied. The organic layer was washed with IN HCl (1300 mL), aqueous NaHC0₃ (5 wt%, 1300 mL), and brine (1300 mL), and then dried over anhydrous Na2SÛ4 and concentrated to dryness. Purification by silica gel column chromatography using a gradient consisting of a mixture of MeOH and EtOAc afforded the product.

110

Préparation ((2S)-2-ethylbutyl 2~(((peifluorophenoxyXphenoxy)phosphoryi~)amino)propanoate) (mixture of Sp and RpY

1) phenyl dichlorophosphate CH₂Cl₂, -78 °C to ambient

2) pentafiuoro phénol Et₃N, 0 °C to ambient

[0248] L-Alanine 2-ethylbutyl ester hydrochloride (5.0 g, 23.84 mmol) was combined with methylene chloride (40 mL), cooied 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 min at about -78 °C and the resulting mixture was stirred at ambient température for 3h. The reaction mixture was cooied to about 0 °C and pentafluorophenol (4.4 g, 23.84 mmol) was added. Triethylamine (3.3 mL, 23.84 mmol) was added over about 60 min. The mixture was stirred for about 3h at ambient température and concentrated under reduced pressure. The residue was dissolved in EtOAc, washed with an aqueous sodium carbonate solution several times, and concentrated under reduced pressure. The residue was purified by silîca gel coiumn chromatography using a gradient of EtOAc and hexanes (0 to 30%). Product containing fractions were concentrated under reduced pressure to give (2S)-2-ethylbutyl 2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate as a solid. 'H 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, J = 7.5 Hz, 12H). ³¹P NMR (162 MHz, Chloroform-d) δ -1.52. ¹⁹F NMR (377 MHz, Chloroform-d) δ -1 53.63, -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],

Préparation ((2S)-2-ethylbutyl 2-(i(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate):

rii) Et₃N, iPAc/n-heptane

i) PhOP(O)CI₂, Et₃N, DCM ii) 4-nitrophenol, Et₃N, DCM

L-alanine-2-ethylbutylester hydrochloride (40.10 g, 0.191 mmol) was dissolved in dichloromethane (533 g) and the solution was cooied with stirring to about -15 °C under N₂(g). Phenyl dichlorophosphate (40.32 g, 0.191 mol) was added followed by slow addition of triethylamine (41.58 g, 0.411 mmol) and the reaction mixture was stirred at about -15 °C for

111 about 1.5 h. Pentafluorophenol (35.14 g, 0.191 mol) was added, followed by triethylamine (19.23 g, 0.190 mol) and the réaction mixture was stirred for about 2 h. The reaction mixture was warmed to about 0 °C and 0.5 M HCl (279.19 g) was added. The mixture was warmed to about 22 °C and the organic layer was separated and washed with 5% KHCO3 aqueous solution (281 g), then water (281 g). An aliquot ofthe organic layer (453.10 gof the 604.30 g solution) was concentrated to about 120 mL volume, isopropyl acetate (157 g) was added and the solution was concentrated to dryness. The resîdue was dîssolved in isopropyl acetate (158 g). The resulting solution was concentrated to about 120 mL volume and the température was adjusted to about 45 °C. n-Heptane (165 g) was added and the mixture was cooled to 22 °C over about 1 h. 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 mixture was stirred at 0 °C for about 17 h. The mixture was fdtered, the solids were rinsed with n-heptane (145 g) and the solids were dried under vacuum at about 40 °C for about 15 h to provide 2-ethylbutyl ((S)(penthafluorophenoxy)(phenoxy)phosphoryl)-L-alaninate.

Préparation 2-ethylbutyl ((S)-(4-nitrophenoxy)(phenoxy)phosphoryl)-L-alaninate:

i) PhOP(O)CI₂, Et₃N, iPAc \ / ü) 4-nitrophenol, Et₃N, iPAc / θ nh₂»hci _DBU, jp_Ac/_n._heptane / y 0 hn^p-oaÇ^-no₂ ÔPh

A slurry of L-alanine-2-ethylbutyIester 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 slow addition of triethyl amine (20.97 g, 207.2 mmol) and the mixture was stirred at about -20 °C for about 1 h. 4-NitrophenoI (13.23 g, 95.1 mmol) was added, followed by slow addition of triethylamine (10.01 g, 98.8 mmol) and the reaction mixture was stirred for about 1.5 h. The reaction mixture was warmed to about 0 °C and 0.5 M HCl (140 g) was added. The organic layer was separated and washed with 5% Na2CO₃ (2 x 100 g) and 10% NaCl (2 x 100 g). The organic layer was then concentrated to about 80 mL volume and isopropylacetate (4 g) was added, followed by n-heptane (110 g). Product seed crystals (0.100 g) were added followed by a second portion of n-heptane (110 g) and the mixture was cooled to about 0 °C. l,8-Diazabicycloundec-7-ene (1.49 g, 9.79 mmol) was added and the mixture was stirred at about 0 °C for about 21 h. The résultant solids were filtered and washed first with n-heptane (61 g) and then with H2O (2 x 100 g). The solids were stirred with H2O (200 g) for

112 about 1.5 h, filtered, and rinsed with H₂O (3 x 100 g), then n-heptane (61 g). The obtained solids were dried under vacuum at about 40 °C for about 19 h to provide 2-ethylbutyl ((S)-(4nitrophenoxy)(phenoxy)phosphoryl)-L-alanînate.

Préparation of Title Compound (mixture of Sp and Rp):

[0249] The nucleoside (29 mg, 0.1 mmol) and the phosphonamide (60 mg, 0.12 mmol) and N,N-dimethylformamide (2 mL) were combined at ambient température. TerAButyl magnésium chloride (IM in THF, 0.15 mL) was slowly added. After about Ih, the reaction was diluted with ethyl acetate, washed with aqueous citric acid solution (5%wt.), aqueous saturated NaHCOs solution and saturated brine solution. 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 CH₂CI₂ (0 to 5%). Product containing fractions were concentrated under reduced pressure to provide the product.

Préparation of (3aR,4R,6R.6aR)-4-(4-aminopynOÎo[2,l-f][L2,4]triazin-7-yl)-6(hydroxymethyl)-2.2-dimethvltetrahvdrofuror3,4-dirL31dioxole-4-carbonitrile:

MeO OMe

18M H₂SO₄

Acetone °C

[0250] To a mixture of (2R,3R,4S,5R)-2-(4-aminopyrrolo[2,l-f][l,2,4]triazin-7-yI)-3,4dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile (5.8g, 0.02 mol), 2,2dimethoxypropane ( 11.59 mL, 0.09 mol) and acetone (145 mL) at ambient température was added sulfuric acid (18M, 1.44 mL). The mixture was warmed to about 45 °C After about 30 min, the mixture was cooled to ambient température and sodium bicarbonate (5.8 g) and water

113

5.8 mL) were added. After 15 min, 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 extracted with ethyl acetate (2 x 50 mL). The combined organic phase was dried over sodium sulfate and concentrated under reduced pressure to give crude (2R,3R,4S,5R)-2-(4- aminopyrrolo[2,l-f][l,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2carbonitrile. ’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+l],

Préparation of (3aR,4R,6R,6aR)-4-(4-aminopyrrolo[2,1 -f|[L2,4]triazin-7-yl)-6(hydroxymethyl)-2,2-dimethyltetrahydrofÎiro[3.4-d] [ L3]dioxole-4-carbonitrile TsOH sait :

MeO OMe

p-TsOH Acetone °C

[0251] To a mixture of (2R,3R,4S,5R)-2-(4-aminopyrrolo[2,l-f][l,2,4]triazin-7-yl)-3,4dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile (5.0 g, 17.2 mmol, 1.0 equiv.), 2,2dimethoxypropane (10.5 mL, 86 mmol, 5.0 equiv.) and acetone (25 mL) at ambîent température was added p-tolylsulfonic acid (3.59 g, 1.1 equiv.). The mixture was stirred at ambient température. Aller about 30 min, isopropyl acetate (25 mL) was added over about one hour. The resulting slurry was filtered and rinsed with 2:1 heptane:isopropyl acetate (25 ml). The product was dried under vacuum at about 40 ^ÜC.

Préparation of (3aR.4R,6R,6aR)-4-(4-aminopyrroIor2, l-f|[L2,41triazin-7-yl)-6(hydroxymethyl)-2.2-dimethyitetrahydrofuro[3,4-d][L3]dioxole-4-carbonitrile:

MeO OMe

p-TsOH Acetone 23 ‘C

[0252] To a mixture of (2R,3R,4S,5R)-2-(4-aminopyrrolo[2,l-f][l,2,4]triazin-7-yl)-3,4 dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile (5 g, 17.2 mmol, 1.0 equiv.), 2,2

114 dîmethoxypropane (10,5 mL, 86 mmol, 5,0 equiv.) and acetone (25 mL) at ambient température was added/?-tolylsulfonic acide (3,59 g, L1 equiv). The mixture was stirred at ambient température. After 30 min, isopropyl acetate (25 mL) was added over one hour. The resulting slurry was filtered and rinsed with 2:1 heptane: isopropyl acetate (25 ml). The product was dried under vacuum at 40 °C. The isolated solid was added to a reactor and 5% K2CO3 solution (50 ml) and ethyl acetate (50 mL) were added. The layers were séparaied, and the aqueous layer washed with ethyl acetate (25 ml). The combined organic layers were washed with water (25 ml), then concentrated to ca.25 ml. The reactor was refilled with isopropyl acetate (25 ml) and concentrated to ca. 25 ml. The reactor was again refilled with isopropyl acetate (25 ml) and concentrated to 25 ml. The resulting solution was seeded, producing a thîck slurry. To this was added heptane (25 ml) over one hour. The resulting slurry was filtered and rinsed with 2:1 heptaneûsopropyl acetate (25 ml). The product was dried under vacuum at 40 °C. () (2R,3R,4S,5R)-2-(4-aminopyirolo[2, l-f][l,2,4]triazin-7-yl)-3,4-dihydroxy-5(hydroxymethyl)tetrahydrofuran-2-carbonitriIe. ^]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+l]_

Préparation of (2S)-2-eihylbutyl 2-(((((2R,3 S,4R,5R)-5-(4-aminopynOlo[2,l-fiïL2,4]triazin-7yl)-5-cyano-3,4-dihydroxytetrahydrofùran-2-yl)methoxy)(phenoxy)phosphoryl)amino) propanoate:

[0253] Acetonitrile (100 mL) was combined with (2S)-2-ethylbutyl 2-(((4nitrophenoxy)(phenoxy)phosphoryl)-amino)propanoate (9.6 g, 21.31 mmol), the substrate alcohol (6.6 g, 0.02 mol), ), magnésium chloride ( (1.9 g, 19.91 mmol) ai ambient température. The mixture was agitated for about 15 min and A/,V-diisopropylethylamine (8.67 mL, 49.78 mmol) was added. After about 4h, the reaction was diiuted with ethyl acetate (100 mL), cooled to about 0 °C and combined with aqueous citric acid solution (5%wl., 100 mL). The organic

115 phase was washed with aqueous cîtric acid solution (5%wt., 100 mL) and aqueous saturated ammonium chloride solution (40 mL), aqueous potassium carbonate solution (!0%wt., 2 x 100 mL), and aqueous saturated brine solution (100 mL). The organic phase was dried with sodium sulfate and concentrated under reduced pressure to provide crude product. ’H 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, IH), 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 (dq, 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+l],

Préparation of (S)-2-ethylbutyl 2-(((S)-(((2R,3S.4R,5R)-5-(4-aminopvrrolor2.1-f]rL2,41triazin7-yl)-5-cyano-3,4-dihydroxytetrahvdrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino) propanoate (Compound 32)

[0254] The crude acetonide (12.85 g) was combined with tetrahydrofuran (50 mL) and concentrated under reduced pressure. The residue was taken up in tetrahydrofuran (100 mL), cooled to about 0 °C and concentrated HCl (20 mL) was slowly added. The mixture was allowed to warm to ambient température. Aller consomption of the starting acetonide as indicated by HPLC analysis, water (100 mL) was added fbllowed by aqueous saturated sodium bicarbonate solution (200 mL). The mixture was extracted with ethyl acetate (100 mL), the organic phase washed with aqueous saturated brine solution (50 mL), dried over sodium sulfated and concentrated under reduced pressure. The residue was purifîed by silica gel column chromatography using a gradient of methanol and ethyl acetate (0 to 20%). Product containing fractions were concentrated under reduced pressure to provide the product.

116

Préparation of (S)-2-ethylbutyl 2-((( S )-(((2R, 3 S.4R,5R)-5-(4-aminopyrrolo [2,1 -f][L2,4}triazm7-yl)-5-cyano-3,4-dihydroxvtetrahydrofliran-2-yl)methoxy)(phenoxy)phosphoryl)amino) propanoate (Compound 32)

To a vial containing (S)-2-ethylbutyl 2-(((S)-(((3aR,4R,6R,6aR)-6-(4-aminopyrrolo[2,lf][l,2,4]triazin-7-yl)-6-cyano-2,2-dimethyltetrahydrofuro[3,4-d][l,3]dîoxol-4yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (30 mg, 0.05 mmol) was added an 80% aqueous formic acid solution (1.5 mL). After 18 h at about 20 °C complété conversion was confirmed by HP LC and LC-MS. MS (m/z) = 603(M+l)”.

Préparation of (S)-2-ethylbutyl 2-(((S)-(((2R,3S.4R,5R)-5-(4-aminopyrrolo[2,l-f][L2,4]triazin7-yl)-5-cyano-3,4-dihydroxytetrahydrofùran-2-yl)methoxy)(phenoxy)phosphory])amino) propanoate (Compound 32) via Direct Coupling

MgCî₂, (/Pr)₂NEt, THF

[0255] To a mixture of (2R,3R,4S,5R)-2-(4-amînopyrrolo[2,l-f][l,2,4]triazin-7-yl)-3,4dihydroxy-5-(hydroxymethyl)tetrahydrofüran-2-carbonitrile (0.5 g, 2mmol), (S)-2-ethylbutyï 2(((S)-(4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (0.9 g, 2 mmol), and MgCl₂ (0.2 g, 2 mmol), was charged N,N-dimethylacetamide (10 mL). The resulting mixture was warmed to about 30 °C with constant stîrring. Ν,Ν-Dîisopropylethylamine (0.7 mL, 4 mmol) was then added slowly, and the reaction mixture was stirred for about 6 h. Water (10 mL) was charged HsO, followed by 2-MeTHF (10 mL), and the organic and aqueous phases were separated. The aqueous layer was then back-extracted with 2-MeTHF (10 mL). The organic layers were combined, and washed with 10 wt% citric acid solution (10 mL), followed by 10 wt% K2CO3

117 solution (10 mL), and H2O (10 mL). A small amount of brine was added to résolve émulsions in the water wash before the layers were separated. The organic layer was evaporated to dryness to afford 0.65 g of a foam. zPrOAc (2.6 mL) was added then added, and the mixture was warmed to about 40 °C to achieve dissolution. The solution was cooled to about 20 °C, and the mixture was stirred for about 3 days. The solids were isolated by filtration, and the filter cake was washed with a small amount of zPrOAc. The solids were dried to afford (S)-2-ethylbutyl 2-(((S)(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,l-f][l,2,4]triazin-7-yl)-5-cyano-3,4dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate.

[0256] To a mixture of (2R,3R,4S,5R)-2-(4-aminopyrrolo[2,l-f][l,2,4]triazin-7-yl)-3,4dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile (0.2 g, 0.7 mmol), (S)-2-ethylbutyl 2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (0.3 g, 0.7 mmol), and MgCh (0.1 g, 1 mmol), was charged N,N-dimethylacetamide (4 mL). The resulting mixture was warmed to about 30 °C with constant stirring. N,N-Diisopropylethylamine (0.3 mL, 2 mmol) was then added slowly, and the reaction mixture was stirred for 5 h. Conversion to the product was confirmed through UPLC analysis.

Préparation of (3R,4R,5R)-2-(4-aminopyrrolo[2, l-f|[L2.4]triazin-7-yl)-3,4-bis((tertbutyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-ol

TBSO

TBSÔ ÔTBS

[0257] A solution of 7-iodopyrrolo[2,l-f][l,2,4]triazin-4-amine (13.9 g, 53.5 mmol) was prepared in THF (280 mL). The solution was cooled to about 0 °C, and TMSC1 (13.6 mL, 107 mmol) was added. The reaction mixture was stirred for about 20 min, and then PhMgCl (2 M in

118

THF; 53.5 mL, 56.8 mmol) was added while maintaining an internai température below about °C. The reaction mixture was agitated at about 0 °C for about 30 min, and then cooled to about -20°C. rPrMgCl-LiCl (1.3 M in THF, 43.1 mL, 56 mmol) was then added while maintaining an internai température below about -15 °C The reaction mixture was agitated for about 30 min at about -20 °C.

[0258] In a separate flask, a solution of (3R,4R,5R)-3,4-bis((tert-butyldimethylsilyl)oxy)-5(((tert-butyidimethylsilyI)oxy)methyl)dihydrofuran-2(3H)-one (25.0 g, 50.9 mmol, 0.83 equiv) was prepared in LaCh-2LiCI (0.6 M in THF, 85 mL, 50.9 mmol). The solution was then transferred to the Grignard solution while maintaining an internai température below -20°C. The resulting réaction mixture was agitated at about -20 °C for about 4 h.

[0259] The reaction was quenched with 1 M HCl (140 mL), and the mixture warmed to ambient température. EtOAc (140 mL) was added, and the organic and aqueous phases were separated. The water layer was extracted with EtOAc (200 mL). The combined EtOAc layers were extracted sequentîalïy with saturated aqueous NaHCOa (2 x 200 mL) , water (200 mL), and brine (200 mL). The organic layer was concentrated, and then purified by silîca gel chromatography (30% EtOAc/hexane) to afford (3R,4R,5R)-2-(4-aminopyrrolo[2,lf][l,2,4]triazin-7-yl)-3,4-bis((tert-butyldimethylsiiyl)oxy)-5-(((tertbutyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-ol. ^}HNMR (300 MHz, CDCh) δ 8.15 - 7.88 (m, IH), 7.51 (d, 7= 4.8 Hz, 0.5H), 7.02 - 6.92 (m, 0.5H), 6.65- 6.57 (m, IH), 5.66-5.24 (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, IH), -0.55 (s, IH). . MS m/z = 626 (M+H).

Préparation of (2R.3R,4R,5R)-2-(4-aminopyrrolor2,l-fl[L2,41triazin-7-yl)-3,4-bis((tertbutyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofLiran-2-carbomtrile

TFA, TMSCN, TMSOTf

CH₂CI₂, -40 °C

[0260] A solution of(3R,4R,5R)-2-(4-aminopyrrolo[2,l-f][l,2,4]triazm-7-yl)-3,4-bis((tertbutyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyI)ietrahydrofuran-2-ol (1.50 g, 2.40 mmol) in CH2CI2 (15 mL) was cooled to about -40 °C. Trifluoroacetic acid (0.555 mL, 7.20

119 mmol) was added keeping the température below -20°C. In a separate flask, trimethylsilyl trifluoromethanesulfonate (2.60 mL, 14.4 mmol) was added to 5 ml of CH2CI2 (5 mL) at about 15 °C, followed by trimethylsilyl cyanide (1.92 mL, 14.4 mmol), and the solution was cooied to about -30 °C. The cooied solution was added to the solution of (3R,4R,5R)-2-(4aminopyrrolo[2,l-f][l,2,4]triazin-7-yl)-3,4-bis((tert-butyldimethylsilyl)oxy)-5-(((tertbutyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-oi while keeping the température below -25 °C. The réaction 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 warmed to about 0 °C. Water (50 mL) was added while keeping the température below about 20 °C. When the addition was complété the mixture was stirred for 15 min at room température. The layers were separated and the organic layer was washed sequentially with KOH (20 mL), water (20 mL), and brine (20 mL). The organic layer was dried over NaiSCL, concentrated, and then purified by silica gel chromatography (30% EtOAc / hexane) to afford the product as a 3.SJ mixture of diastereomers). The mixture was purified further by prep-HPLC (ACN 0 to 95% in water) to afford the product as a single diastereomer. Ή NMR (400 MHz, DMSO-d6) δ 8.14-7.92 (m, 2H), 7.89 (s, 1H), 6.95 (d, J=4.8Hz, 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, 1.4 Hz, 1H), 4.12 (ddd, J = 5.9, 4.1, 1.4 Hz, 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).

Préparation of (S)-2-ethylbutyl 2-(((SL(((2R.3R,4R,5R)-5-(4-aminopyrrolo[2J-firL2,4]triazin7-yl)-3,4-bis((tert-butvldimethyÎsilyl)oxy)-5-cyanotetrahydroiuran-2-yl)methoxy)(phenoxy) phosphoryllaminolpropanoate

To a mixture of (2R,3R,4R,5R)-2-(4-aminopyrrolo[2,l-f|[l,2,4]triazin-7-yl)-3,4-bis((tertbutyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile (16 mg, 0.03 mmol), (S)-2-ethylbutyl 2-((( S)-(4-nitrophenoxy)(phenoxy)phosphoryI)amino)propanoate (17 mg, 0.04 mmol), and MgCh (4 mg, 0.05 mmol), was charged THF (0.3 mL). The resulting mixture was warmed to about 50 °C with constant stirring. N,N-Diisopropylethy lamine (0.013 mL, 0.08

120 mmol) was then added, and the reaction mixture was stirred for 21 h. Conversion to the product was confirmed through UPLC and LC-MS analysis. MS m/z — 831 (M+H).

A solution of (2R, 3 R,4R, 5 R)-2-(4-ami nopyrrolo[2,1 -f] [ 1, 2,4]triazin-7-y l)-3,4-bi s((tertbutyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofùran-2-carbonitrile (16 mg, 0.03 mmol) in THF (0.3 mL) was cooled to -10 °C. /BuMgCl was added dropwise (0.07 mL, 0.07 mmol), followed by a solution of (S)-2-ethy1butyl 2-(((S)(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (22 mg, 0.04 mmol) in THF (0.15 mL). The reaction mixture was warmed to 5 °C, and stirred for 16 h. The reaction was quenched with MeOH, concentrated, and then purified by silica gel chromatography (EtOAc /hexanes) to afford the product . 1H NMR (400 MHz, CDCb) 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, 1 H), 4.41-4.35 (m, 1H), 4.32 - 4.26 (m, 1 H), 4.24 (dd, 1 = 4.5, 1.7 Hz, JH), 4.10-3.99 (m, 2H), 3.96 (dd, J = 10.9, 5.7 Hz, 1H), 3.80 - 3.72 (m, 1H), 1.48 (h, J = 6.2 Hz, IH), 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). 3 IP NMR (162 MHz, CDC13) δ 2.74 (s). MS m/z = 831 (M+H).

Préparation of (S)-2-ethylbutyl 2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolor2,1 -f|[~l,2.41triazin7-yl)-5-cyano-3,4-dihydroxvtetrahydrofuran-2-vl)methoxyXphenoxy)phosphoryl)amino) propanoate

HCl ’C

A crude solution of (S)-2-ethylbutyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-aminopyrrolo[2,lfj [ 1,2,4]triazin-7-yl )-3,4-bi s((tert-butyl dimethy isi 1 y l)oxy)-5 -cyanotetrahy drofuran-2121 yl)methoxy)(phenoxy)phosphoryl)amino)propanoate was cooled to about 0 °C and conc HCl (0.05 mL, 0.62 mmol) was slowly added. The reaction mixture was stirred for about 72 hours at about 20 *’C. Conversion to the product was confirmed through UPLC and LC-MS analysis. MS m/z = 603 (M+H).

deprotection

A solution of (S)-2-ethylbutyl 2-(((S)-(((2R,3R,4R,5R)-5-(4-aminopyrrolo[2,l-f][l,2,4]triazin-7yl)-3,4-bis((tert4?utyldimethylsilyl)oxy)-5-cyanotetrahydrofuran-2yl)methoxy)(phenoxy)phosphoryl)amino)propanoate in a fluoride or acid can deprotect to a solution of (S)-2-ethylbutyl 2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,l-f][l,2,4]triazin-7yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2yl)methoxy)(phenoxy)phosphoryl)amino)propanoate. Représentative fluorides include, but are not limited to TBAF, KF, pyridinium hydrofluorîde, triethylammonium hydrofluoride, hydrogen fluoride, hydrochloric acid, toluenesulfonic acid, or any other suitable fluoride source.

Représentative acids include, but are not limited to those found in Greene, T. W.; Wuts, P. G. M. Protective Groups In Organic Synthesis, 4th Ed, John Wiley & Sons: New York, 2006.

Example 35-a. ((((2R,3S,4R,5R)-5-(4-aminoDvrrolo[2J-f!H.,2,41 triazin-7-yl)-5-cvano-3.4dihvdroxytetrahvdrofuran-2-yl)methoxY)oxidoDhosDhoryl)alaninate (Compound 35)

]0261] 2-ethylbutyl ((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,l-f][l,2,4]triazin-7-yl)-5cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate (130 mg, 0.216 mmol) was dissolved in mixture of acetonitrile (6 mL) and water (2 mL). Aqueous sodium hydroxide solution (2N, 0.5 mL) was added dropwise over 5 min at rt and the reaction

122 mixture was stirred. After 2 h, the resuiting mixture was concentrated under reduced pressure and the residue was purified by HPLC on a C18 column eluting with water to afford the desired product as the bis-sodium sait. ^]H NMR (400 MHz, D2O) δ 7.79 (s, 1H), 6.86 (d, J= 4.7 Hz, 1H), 6.80 (d, ,7= 4.7 Hz, 1H), 4.86 (d, .7= 5.4 Hz, IH), 4.40 - 4.34 (m, 1H), 4.30 (dd, J= 5.3, 3.0 Hz, 1H), 3.75 (qdd, .7= 11.6,4.5, 3.1 Hz, 2H), 3.20 (dq, <7= 8.6, 7.1 Hz, 1H), 0.86 (d, ,7= 7.0 Hz, 3H). ³¹P NMR (162 MHz, D2O) δ 7.30. LCMS m/z 442 95 [M+H], HPLC (2-98% MeCN-H₂0 gradient with 0.1% TFA modifier over 8.5 min, 1.5mL/min, Column: Phenomenex Kinetex C18, 2.6 um 100 Â, 4.6 x 100 mm )tR = 2.694 min.

B, Antiviral Activity

[0262] Another aspect of the invention relates to methods of inhibiting viral infections, comprising the step of treating a sampie or subject suspected of needing such inhibition with a composition ofthe invention.

[0263] Within the context of the invention samples suspected of containing a virus include natural or man-made materials such as living organisms; tissue or cell cultures, biological samples such as biological materîal samples (blood, sérum, urine, cerebrospinal fluîd, tears, sputum, saliva, tissue samples, and the like); laboratory samples; food, water, or air samples; bioproduct samples such as extracts of cells, particularly recombinant cells synthesizîng a desired glycoprotein; and the like. Typically the sample will be suspected of containing an organism which induces a viral infection, frequentiy a pathogenîc organism such as a tumor virus. Samples can be contained in any medium including water and organic solvent\water mixtures. Samples include living organisms such as humans, and manmade materials such as cell cultures.

]0264] If desired, the anti-virus activity of a compound of the invention after application of the composition can be observed by any method including direct and indirect methods of detecting such activity. Quantitative, qualitative, and semiquantitative methods of determining such activity are ali contemplated. Typically one ofthe screening methods described above are applied, however, any other method such as observation of the physiological properties of a living organism are also applicable.

[0265] The antiviral activity of a compound of the invention can be measured using standard screening protocole that are known. For example, the antiviral activity of a compound can be measured using the following general protocols.

123

Attorney Docket No. 1106.1P2F

or a pharmaceutically acceptable sait or hydrate thereof.

17. The compound of daim 16, having the structure:

or a pharmaceutically acceptable sait or hydrate thereof.

18. A pharmaceutical composition comprising a therapeutically effective amount of a compound of the formula:

144

or a pharmaceutically acceptable sait or hydrate thereof.

19. A compound as described in any one of claims 1-7 and 16-17, or a pharmaceutically acceptable sait thereof, for use in treating a Filoviridae virus infection in a human.

20. A compound as described in any one of claims 1 -7 and 16-17, or a pharmaceutically acceptable sait thereof, for use in treating an ebolavirus infection in a human.

21. A compound as described in any of claims 1-7 and 16-17, or a pharmaceutically acceptable sait or ester thereof, for use in treating a Marburg virus infection in a human.

22. Use of a compound as described in any of claims 1-7 and 16-17, or a pharmaceutically acceptable sait or ester thereof, for the préparation of a médicament for treating a Filoviridae virus infection in a human.

23. Use of a compound as described in any of claims 1-7 and 16-17, or a pharmaceutically acceptable sait, hydrate, or ester thereof, for the préparation of a médicament for treating an ebolavirus infection in a human.

24. Use of a compound as described in any of claims 1-7 and 16-17, or a pharmaceutically acceptable sait, hydrate, or ester thereof, for the préparation of a médicament for treating a Marburg virus infection in a human.

145

Virus	Cell line	Plate format	Cell number	MOI (pfu/cell)	Incubation (Days)	Rcad oui	Values
EBOV (Zaïre)	Hela	384	4000	0.5	2	HCS	EC50
EBOV (Zaïre)	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: EBOV-GFP: EBOV-LUC: MARV-GFP: NiV: NiV-GFP: NiV-LUC:

Ebola virus strain Zaïre

Ebola reporter virus expressing green fluorescent protein

Ebola reporter virus expressing luciferase

Marburg virus expressing green fluorescent protein

Nipah virus

Nipah reporter virus expressing green fluorescent protein

Nipah reporter virus expressing luciferase

HCS: GFP: LUC:	High content imaging (immuno-staining of ebolavinis GP-protein) Green fluorescent protein Luciferase
CPE	Cytopathic effects measured by cell titer glo (CTG) reagent
Hela:	Hela épithélial cell (cervical carcinoma)
HFF-1:	Human foreskin fibroblast
Huh-7: HVMEC-TERT:	Hépatocyte Human microvascular endothélial cells i mmortalized with the telomerase catalytîc protein

Example 36. Ebola virus antiviral activîty and cytotoxicitv assavs

[0266] Antiviral activîty of Compound 1 and Compound 9 was measured against Ebola virus (EBOV), Marburg virus (MARV) (Table 2), and Nipah virus (NiV) (Table 3) using fully replicating reporter viruses expressing luciferase or green fluorescent protein (GFP) (Uebelhoer, L.S., 2014. AVR; Hoenen, T., 2013. AVR). Further antiviral activîty of Compound 1 and Compound 9 was measured against Ebola virus (EBOV), Marburg virus (MARV) (Table 2-a), using fully replicating reporter viruses expressing luciferase or green fluorescent protein (GFP) (Uebelhoer, L.S., 2014. AVR; Hoenen, T , 2013. AVR).Ail studies were conducted in biosafety level-4 contain ment (BSL-4) at the Cent ers for Disease Control and Prévention (CDC). Ebola virus antiviral assays were conducted in primary human microvascular endothélial cells

124 immortalized with the telomerase catalytic proteîn (HMVEC-TERT) and in Huh-7 cells (Shao, R., 2004, BBRC). Nipah virus antiviraï activity was measured in HMVEC-TERT and Hela cells.

[0267] Antiviral assays were conducted in 96well plates. Eight to ten concentrations of compound were diluted in 3-fold serial dilution incréments in media and 100 uL/well of each dilution was transferred in triplicate onto plates containing preseeded cell monolayers. The plates were transferred to BSL-4 containment and the appropriate dilution of virus stock, previously determined by titration and prepared in cell culture media, was added to test plates containing cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as 0% and 100% virus inhibition control, respectively. Following the infection, test plates were încubated for 3 to 4 days in a tissue culture incubât or. After the incubation, virus réplication was measured in an Envîsion plate reader by direct fluorescence for GFP reporter viruses or after subséquent addition of luciferase substrate for lucîferase reporter viruses. For virus yield assays, media from infected cells was removed and a portion was used to quantify viral RNA by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The remaining media was serially diluted and the amount of infectious virus was measured by using the diluted media to infect fresh cell monolayers to détermine the tissue culture infectious dose that caused 50% cytopathic effects (TCID50) using Cell TiterGlo reagent (Promega, Madison, WI). For virus cytopathic effect (CPE) assays, viability of infected cells was measure using Cell TiterGlo reagent.

[0268] The percentage inhibition was calculated for each tested concentration relative to the 0% and 100% inhibition Controls and the ECsû value for each compound was determined by non-linear régression as the effective concentration of compound that inhibited virus réplication by 50%.

Example 37. EBOV-GFP HMVEC-TERT cells

[0269] HMVEC-TERT cells were seeded in 96 well plates. Eight to ten concentrations of compound were diluted in 3-fold serial dilution incréments in media and 100 uL/well of each dilution was transferred in triplicate onto 96 well plates containing preseeded HMVEC-TERT monolayers. The plates were transferred to BSL-4 containment and the appropriate dilution of EBOV-GFP virus stock, previously determined by titration and prepared in cell culture media, was added to test plates containing cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as 0% and

125

100% virus inhibition control, respectively. Following the infection, test plates were incubated for 3 to 4 days in a tissue culture incubator. After the incubation, virus réplication was measured in an Envision plate reader by direct fluorescence to measure GFP expression from the reporter virus. The percentage inhibition was calculated for each tested concentration relative to the 0% and 100% inhibition Controls and the ECso value for each compound was determined by nonlînear régression as the effective concentration of compound that inhibited virus réplication by 50%.

Example 38. EBOV-GFP Huh-7 cells

[0270] Huh-7 cells were seeded in 96 well plates. Eight to ten concentrations of compound were diluted in 3-fold serial dilution incréments in media and 100 uL/well of each dilution was transferred in triplicate onto 96 well plates containing preseeded Huh-7 monolayers. The plates were transferred to BSL-4 containment and the appropriate dilution of EBOV-GFP virus stock, previously determined by titration and prepared in cell culture media, was added to test plates containing cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as 0% and 100% virus inhibition control, respectively. Following the infection, test plates were incubated for 3 to 4 days in a tissue culture incubator. After the incubation, virus réplication was measured in an Envision plate reader by direct fluorescence to measure GFP expression from the reporter virus. The percentage inhibition was calculated for each tested concentration relative to the 0% and 100% inhibition Controls and the ECso value for each compound was determined by non-linear régression as the effective concentration of compound that inhibited virus réplication by 50%.

Example 39. EBOV-Luc Huh-7 cells

[0271] Huh-7 cells were seeded in 96well plates. Eight to ten concentrations of compound were diluted in 3-fold serial dilution incréments in media and 100 uL/well of each dilution was transferred in triplicate onto plates containing preseeded cell monolayers. The plates were transferred to BSL-4 containment and the appropriate dilution of the EBOV-Luc virus stock, previously determined by titration and prepared in cell culture media, was added to test plates containing cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as 0% and 100% virus inhibition control, respectively. Following the infection, test plates were incubated for 3 to 4 days in a tissue culture incubator. After the incubation, virus réplication was measured in an Envision

126 plate reader after subséquent addition of luciferase substrate. The percentage inhibition was calculated for each tested concentration relative to the 0% and 100% inhibition Controls and the EC;o value for each compound was determined by non-linear régression as the effective concentration of compound that inhibited virus réplication by 50%.

Examnle 40. MARV-GFP Huh-7 cells

102721 Huh-7 cells were seeded in 96 well plates. Eight to ten concentrations of compound were diluted in 3-fold serial dilution incréments in media and 100 uL/well of each dilution was transferred in triplicate onto 96 well plates containing preseeded Huh-7 monolayers. The plates were transferred to BSL-4 contaminent and the appropriate dilution of MARV-GFP virus stock, previously determined by titration and prepared in cell culture media, was added to test plates containing cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as 0% and 100% virus inhibition control, respectively. Following the infection, test plates were incubated for 3 to 4 days in a tissue culture incubât or. After the incubation, virus réplication was measured in an Envision plate reader by direct fluorescence to measure GFP expression from the reporter virus. The percentage inhibition was calculated for each tested concentration relative to the 0% and 100% inhibition Controls and the EC50 value for each compound was determined by non-linear régression as the effective concentration of compound that inhibited virus réplication by 50%.

Example 41. Ebola Huh-7 (RNA)

[0273] Huh-7 cells were seeded in 96 well plates. Eight to ten concentrations of compound were diluted in 3-fold serial dilution incréments in media and 100 uL/well of each dilution was transferred in triplicate onto plates containing preseeded Huh-7 cell monolayers. The plates were transferred to BSL-4 containment and the appropriate dilution of EBOV virus stock, previously determined by titration and prepared in cell culture media, was added to test plates containing cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as 0% and 100% virus inhibition control, respectively. Following the infection, test plates were incubated for 3 to 4 days in a tissue culture incubator. After the incubation, media from infected cells was removed and a portion was used to quantify viral RNA by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The percentage inhibition was calculated for each tested concentration relative to the 0% and 100% inhibition Controls and the EC50 value for each compound was determined by

127 non-linear régression as the effective concentration of compound that inhibited virus réplication by 50%.

Example 42. Ebola Huh-7 (Yield)

[0274] Huh-7 cells were seeded in 96 well plates. Eight to ten concentrations of compound were diluted in 3-fold serial dilution incréments in media and 100 uL/well of each dilution was transferred in triplicate onto plates containîng preseeded Huh-7 cell monolayers. The plates were transferred to BSL-4 containment and the appropriate dilution of EBOV virus stock, previously determined by titration and prepared in cell culture media, was added to test plates containîng cells and serially diluted compounds. Each plate inciuded three weils of infected untreated cells and three weils of uninfected cells that served as 0% and 100% virus inhibition control, respectively. Following the infection, test plates were incubated for 3 to 4 days in a tissue culture incubator. After the incubation, media ftom infected cells was removed and diluted in 10-fold serial dilutions. The amount of infectious virus was measured by using the diluted media to infect fresh cell monolayers to détermine the tissue culture infectious dose that caused 50% cytopathic effects (TCID50) using Cell TiterGlo reagent (Promega, Madison, WI). The percentage inhibition was calculated for each tested concentration relative to the 0% and 100% inhibition Controls and the ECso value for each compound was determined by non-linear régression as the effective concentration of compound that inhibited virus réplication by 50%.

Example 43. Ebola HeLa cells

[0275] The antiviral activity of selected compounds was measured against ebolavirus (EBOV) strain Zaïre conducted in bîosafety level-4 containment (BSL-4) at the US Army Medical Research Institute for Infections Disease (USAMRIED) Hela cells were seeded in 384 well plates at 5000 cells / well. The antiviral activity of each compound was measured in quadruplicate. Eight to ten concentrations of compound were added directly to the cell cultures using the HP300 digital dispenser in 3-fold serial dilution incréments 2h prior to infection. The plates were transferred to BSL-4 containment and the appropriate dilution of virus stock, previously determined by titration and prepared in cell culture media, was added to test plates containîng cells and serially diluted compounds. Each plate inciuded three weils of infected untreated cells and three weils of uninfected cells that served as 0% and 100% virus inhibition control, respectively. Following the infection, test plates were incubated for 2 days in a tissue culture incubator. After the incubation, the cells were fixed in formalin solution and virus

128 réplication was measured by quantifying Ebola glycoprotein ievels after immunostaining and high content imaging using the Perkîn Elmer Opéra confocal microscopy instrument. The percentage inhibition was calculated for each tested concentration relative to the 0% and 100% inhibition Controls and the ECso value for each compound was determined by non-linear régression as the effective concentration of compound that inhibited virus réplication by 50%.

Example 44. Ebola Macrophage cultures

[0276] The antiviral activity of selected compounds was measured against ebolavirus (EBOV) strain Zaire conducted in biosafety level-4 containment (BSL-4) at the US Army Medical Research Institute for Infections Disease (USAMRIID). Macrophage cultures were isolated from fresh human PBMCs and differentiated in the presence of 5ng/ml GM-CSF and 50uM Bmercaptoethanol. The media was changed every 2 days and cells that adhered to the tissue culture plate after 7 days were removed with 0.5M EDTA in Ix PBS, concentrated by centrifugation at 200 x g for 10 minutes and plated in 384 well assay plates at 40,000 cells / well. The antiviral activity of each compound was measured in quadruplicate. Eight to ten concentrations of compound were added directly to the cell cultures using the HP300 digital dispenser in 3-fold serial dilution incréments 2h prior to infection. The plates were transferred to BSL-4 containment and the appropriate dilution of virus stock, previously determined by titration and prepared in cell culture media, was added to test plates containing cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as 0% and 100% virus inhibition control, respectively. Following the infection, test plates were incubâted for 2 days in a tissue culture incubator. After the incubation, the cells were fixed in formalin solution and virus réplication was measured by quantifying Ebola glycoprotein Ievels after immunostaining and high content imaging using the Perkin Elmer Opéra confocal microscopy instrument. The percentage inhibition was calculated for each tested concentration relative to the 0% and 100% inhibition Controls and the ECso value for each compound was determined by non-linear régression as the effective concentration of compound that inhibited virus réplication by 50%.

Example 45. Nipah-GFP HMVEC-TERT cells

[0277] HMVEC-TERT cells were seeded in 96 well plates. Eight to ten concentrations of compound were diluted in 3-fold serial dilution incréments in media and 100 uL/well of each dilution was transferred in triplicate onto 96 well plates containing preseeded HMVEC-TERT

129 monolayers. The plates were transferred to BSL-4 contaminent and the appropriate dilution of NiV-GFP virus stock, previously determîned by titration and prepared in cell culture media, was added to test plates containing cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as 0% and 100% virus inhibition control, respectively. Following the infection, test plates were incubated for 3 to 4 days in a tissue culture incubator. After the incubation, virus réplication was measured in an Envision plate reader by direct fluorescence to measure GFP expression from the reporter virus. The percentage inhibition was calculated for each tested concentration relative to the 0% and 100% inhibition Controls and the EC50 value for each compound was determîned by non-linear régression as the effective concentration of compound that inhîbited virus réplication by 50%.

Example 46. NiV-Luc HMVEC-TERT

[0278] HMVEC-TERT cells were seeded in 96well plates. Eight to ten concentrations of compound were diluted in 3-fold serial dilution incréments in media and 100 uL/welI of each dilution was transferred in triplicate onto plates containing preseeded cell monolayers. The plates were transferred to BSL-4 containment and the appropriate dilution of the Niv-Luc virus stock, previously determîned by titration and prepared in cell culture media, was added to test plates containing cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as 0% and 100% virus inhibition control, respectively. Following the infection, test plates were incubated for 3 to 4 days in a tissue culture incubator. After the incubation, virus réplication was measured in an Envision plate reader after subséquent addition of luciferase substrate. The percentage inhibition was calculated for each tested concentration relative to the 0% and 100% inhibition contrais and the EC50 value for each compound was determîned by non-linear régression as the effective concentration of compound that inhîbited virus réplication by 50%.

Example 47. NiV Hela (YieltD

[0279] Hela cells were seeded in 96 well plates. Eight to ten concentrations of compound were diluted in 3-fold serial dilution incréments in media and 100 uL/well of each dilution was transferred in triplicate onto plates containing preseeded Hela cell monolayers. The plates were transferred to BSL-4 containment and the appropriate dilution of Niv virus stock, previously determîned by titration and prepared in cell culture media, was added to test plates containing cells and serially diluted compounds. Each plate included three wells of infected untreated cells

130 and three wells of uninfected cells that served as 0% and 100% virus inhibition control, respectively. Following the infection, test plates were incubated for 4 days in a tissue culture incubator. After the incubation, media from infected cells was removed and diluted in 10-fold sériai dilutions. The amount of infectious virus was measured by using the diluted media to infect fresh cell monolayers to détermine the tissue culture infectious dose that caused 50% cytopathic effects (TCID50) using Cell TiterGlo reagent (Promega, Madison, WI). The percentage inhibition was calculated for each tested concentration relative to the 0% and 100% inhibition Controls and the EC50 value for each compound was determined by non-linear régression as the effective concentration of compound that inhibited virus réplication by 50%.

Example48. NivHela(RNA)

[0280] Huh-7 cells were seeded in 96 well plates. Eight to ten concentrations of compound were diluted in 3-fold serial dilution incréments in media and 100 uL/well of each dilution was transferred in triplicate onto plates containing preseeded Hela cell monolayers. The plates were transferred to BSL-4 containment and the appropriate dilution of Niv virus stock, previously determined by titration and prepared in cell culture media, was added to test plates containing cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as 0% and 100% virus inhibition control, respectively. Following the infection, test plates were incubated for 3 to 4 days in a tissue culture incubator. After the incubation, media from infected cells was removed and a portion was used to quantify viral RNA by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The percentage inhibition was calculated for each tested concentration relative to the 0% and 100% inhibition Controls and the EC50 value for each compound was determined by non-linear régression as the effective concentration of compound that inhibited virus réplication by 50%.

131

Table 2: Ebola and Marburg virus antiviral assays

	ECso (nM)
Assay	Reporter Virus	RNA	Yield	Antigen expression (high content imaging)
Virus	EBOV-GFP	EVOVLuc	MARVGFP	Ebola
Cell line	HMVECTERT	Huh-7	Huh-7	Huh-7	Huh-7	Hela	Macrophage
Compound 1	771	1492	3126	1726	ND	ND	>20,000	>20,000
Compound 9	121	90	ND	ND	1	1029	290	501
(R)-Diastereomer of Compound 9	62	70	ND	ND	ND	ND
(S)-Diastereomer of Compound 9 (Compound 32)	40	81	ND	ND	ND	ND
Compound 10
Compound 15	630	271	ND	ND	ND	ND
Compound 21	905							270
Compound 22	ND	ND	ND	ND	ND	ND
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						>20,000	1230

EBOV-GFP: Ebola virus expressing the GFP reporter gene EBOV-Luc: Ebola virus expressing he luciferase reporter gene MARV-GFP: Marburg virus expressing the GFP reporter gene Ebola: Ebolavims strain 2014

132

Table 2-a: Ebola and Marburg virus antiviral assays

	ECso (nM)
Assay	Reporter Virus	RNA	Yield	Autigen expression (high content imaging)
Virus	EBOV-GFP	EVOVLuc	MARVGFP	Ebola
Cell line	HMVECTERT	Huh-7	Huh-7	Huh-7	Huh-7	Hela	Macrophage
Compound 1	771	1492	3126	1726	ND	ND	>20,000	>20,000
Compound 9	121	90	ND	ND	1	1029	290, 270	501,70
(R)-Diastereomer of Compound 9	62	70	ND	ND	ND	ND	210	112
(S)-Diastcreomer of Compound 9 (Compound 32)	40	81	ND	ND	ND	ND	100	87
Compound 10							3200
Compound 15	630	271	ND	ND	ND	ND	520	501
Compound 21	905,473							270
Compound 22	ND	ND	ND	ND	ND	ND	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							>20,000, >10000

EBOV-GFP: Ebola virus expressing the GFP reporter gene EBO V-Luc: Ebola virus expressing he luciferase reporter gene MARV-GFP: Marburg virus expressing the GFP reporter gene Ebola: Ebolavirus strain 2014

133

Table 3: Nipah and Hendra virus antiviral assays

	ECso (nM)
Assay	Reporter Virus	CPE	Yield
Virus	NiV GFP	NiV Luc	NiV
Cell line	HMVEC-TERT	Hela
Compound 1	13420	3500	1484	1000
Compound 9	60	30	ND	ND

NiV GFP: Nipah virus expressing the GFP reporter gene

NiV-Luc: Nipah virus expressing the luciferase reporter gene

NiV: Nipah virus

[0281] Ail publications, patents, and patent documents cited herein above are incorporated by reference herein, as though individually incorporated by reference.

[0282] The invention has been described with reference to varions spécifie and preferred embodiments and techniques. However, one skilled in the art will understand that many variations and modifications may be made whîle remaining within the spirit and scope of the invention.

Claims (5)

1. A compound having the structure:

   or a pharmaceutically acceptable sait thereof
2. 2. A pharmaceutical composition comprising a therapeutically effective amount of a compound ofthe formula:

   or a pharmaceutically acceptable sait thereof
3. 3. A pharmaceutical composition comprising the compound of claim 1 or a pharmaceutically acceptable sait thereof. and eue or more pharmaceutically acceptable caméra and optionally other therapeutic agents.
4. 4. The pharmaceutical composition ofclaim 3, wherein the other therapeutic agent is nbavmn, palivizumab, motavizumab, RSV-IGIV (ResprGam*). MEDI-557, A-60444. MDT-637. BMS-433771, amiodarone, dronedarone, verapamil, Ebola Convalescent Plasma (ECP) TKM-100201, BCX4430 ((2_S,3_S,4R,_5R).2.(4-amino.₅H^^ (h_ydroxymelh_yl)p_yrrolidine-3,4-diol), favrpnavir (also known as T-705 or Avigan),T-705 monophosphate. T-705 diphosphate, T-705 triphosphate, FGI-106 (l-N,7-N-bis[3- ’ (dimeth_ylamino)prop_yl]-3,9-dimeth_ylquinolmo[8,7-h]quinolone-l ,7-diamine). JK-05 TKM-Ebola, ZMapp. rNAPc2, VRC-EBOADC076-00-VP, OS-2966. MVA-BN fi_lo> bnnctdofovm Vaxart

   135 adenovirus vector 5-based ebola vaccine, Ad26-ZEBOV, FiloVax vaccine, GOVX-E301, GOVXE302, ebola virus entry inhibitors (NPC1 inhibitors), or rVSV-EBOV or mixtures thereof.
5. 5. The pharmaceutical composition of ctaim 4, wherein the other therapeutic agent is ZMapp.