N4-hydroxycytidine and derivatives and Anti-viral uses related thereto

Abstract

To provide N4-hydroxycytidine derivatives, compositions, and methods for treatment and prophylaxis of viral infections.SOLUTION: A pharmaceutical composition is provided which comprises a pharmaceutically acceptable excipient and a compound having Formula I or a salt thereof.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to N4-hydroxycytidine nucleoside derivatives, compositions, and methods related thereto. In certain embodiments, the present disclosure relates to the treatment and prevention of viral infections.

Background Art

[0002] The causative agents of Eastern, Western, and Venezuelan equine encephalitis (EEE, WEE, and VEE, respectively) and Chikungunya fever (CHIK) are vector-borne viruses (family Togaviridae, genus Alphavirus) that can be transmitted to humans by mosquito bites. Equine encephalitis viruses are CDC category B pathogens, and CHIK virus is category C. There is considerable concern regarding the use of virulent strains of VEE virus released as an aerosol as a biological weapon against soldiers. Animal experiments have shown that aerosol exposure to VEE virus leads to rapid large-scale infection of the brain with high mortality and morbidity. See Roy et al., Pathogenesis of aerosolized Eastern equine encephalitis virus infection in guinea pigs. Virol J, 2009, 6:170.

[0003] Stuyver et al. reported that β-D-N(4)-hydroxycytidine (NHC) was found to have anti-pestivirus activity and anti-hepatitis virus activity. Antimicrob Agents Chemother, 2003, 47(1):244-54. Constantini et al. reported the evaluation of the effectiveness of 2'-C-MeC, 2'-F-2'-C-MeC, and NHC against norwalk virus. Purohit et al. J Med Chem, 2012, 55(22):9988-9997. See also Ivanov et al., Collection of Czechoslovak Chemical Communications, 2006, 71(7):1099-1106. Fox et al., JACS, 1959, 81:178-87.

[0004] The references cited herein are not an admission of prior art.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

Means for Solving the Problems

[0006] The present disclosure relates to N4-hydroxycytidine and derivatives, pharmaceutical compositions, and uses related thereto. In certain embodiments, the present disclosure relates to a compound having Formula I as defined herein,

Chemical Formula

[0007] In certain embodiments, the present disclosure contemplates derivatives of the compounds disclosed herein, such as those containing one or more identical or different substituents.

[0008] In certain embodiments, the present disclosure contemplates pharmaceutical compositions comprising a pharmaceutically acceptable excipient and a compound disclosed herein. In certain embodiments, the pharmaceutical composition is in the form of a tablet, capsule, pill, or an aqueous buffer such as saline or phosphate buffer.

[0009] In certain embodiments, the pharmaceutical composition comprises a compound disclosed herein and a propellant. In certain embodiments, the propellant is an aerosolized propellant, and the propellant is compressed air, ethanol, nitrogen, carbon dioxide, nitrous oxide, hydrofluoroalkane (HFA), 1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoropropane, or a combination thereof.

[0010] In certain embodiments, the present disclosure contemplates a pressurized or non-pressurized container comprising a compound or pharmaceutical composition described herein. In certain embodiments, the container is a manual pump spray, inhaler, metered-dose inhaler, dry powder inhaler, nebulizer, mesh nebulizer, jet nebulizer, or ultrasonic nebulizer.

[0011] In certain embodiments, the present disclosure relates to a method of treating or preventing a viral infection, the method comprising administering to a subject in need thereof an effective amount of a compound or pharmaceutical composition disclosed herein.

[0012] In certain embodiments, the viral infection is an alphavirus or a coronavirus and a flavivirus. In certain embodiments, the viral infection is an orthomyxovirus or a paramyxovirus. In certain embodiments, the viral infection is selected from Middle East respiratory syndrome coronavirus, eastern equine encephalitis virus, western equine encephalitis virus, Venezuelan equine encephalitis virus, Ross River virus, Powassan virus, Barmah Forest virus, and chikungunya virus.

[0013] In certain embodiments, the compound or pharmaceutical composition is administered orally, intravenously, or through the lungs.

[0014] In certain embodiments, the present disclosure relates to the use of a compound described herein in the manufacture of a medicament for the treatment or prevention of a viral infection.

[0015] In certain embodiments, the present disclosure relates to a method for producing a compound disclosed herein by mixing a starting material and a reagent disclosed herein under conditions such that the compound is formed. BRIEF DESCRIPTION OF THE DRAWINGS

[0016]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

[0017] Before the present disclosure is described in more detail, it is to be understood that the present disclosure is not limited to the specific embodiments described, and thus may naturally vary. Since the scope of the present disclosure is limited only by the appended claims, it is also to be understood that the terms used herein are for the purpose of describing particular embodiments only and are not limiting.

[0018] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described herein.

[0019] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated by reference to disclose and describe the relevant methods and / or materials for which the publication is cited. The citation of a publication is for its disclosure prior to the filing date and should not be construed as an admission that this disclosure has a right to antedate such a publication based on prior disclosure. Further, the date given for a particular publication may differ from the actual publication date which may need to be independently confirmed.

[0020] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features that can be readily separated from or combined with any of the features of some other embodiments without departing from the scope or spirit of the present disclosure. Any of the recited methods can be carried out in the order of recited events or in any other logically possible order.

[0021] Embodiments of the present disclosure utilize techniques in medicine, organic chemistry, biochemistry, molecular biology, pharmacology, etc., which are within the skill of the art, unless otherwise specified. Such techniques are fully described in the literature.

[0022] In certain embodiments, the pharmaceutical may be in the form of a salt or prodrug, but is administered in the methods disclosed herein as specified by weight, which refers to the weight of the recited compound. In the case of a salt or prodrug form, the weight is the molar equivalent of the corresponding salt or prodrug.

[0023] As used in this specification and the appended claims, it should be noted that the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0024] "Subject" refers to any animal, preferably a human patient, livestock, or a domesticated pet.

[0025] As used herein, the terms “prevent” and “prevention” include prevention of recurrence, spread, or onset. The present disclosure is not intended to be limited to complete prevention. In some embodiments, the onset is delayed or the severity of the disease is reduced.

[0026] As used herein, the terms “treat” and “treatment” are not limited to the case where a subject (e.g., a patient) is cured and the disease is completely eliminated. Rather, embodiments of the present disclosure also contemplate treatments that merely reduce symptoms and / or delay disease progression.

[0027] As used herein, the term “in combination with” used to describe administration with additional treatment means that the agent can be administered before, together with, or after the additional treatment, or in combinations thereof.

[0028] As used herein, “alkyl” means an acyclic straight-chain or branched-chain, unsaturated or saturated hydrocarbon, such as one containing 1 to 10 carbon atoms. “Higher alkyl” refers to an unsaturated or saturated hydrocarbon having 6 or more carbon atoms. “C6-C 16 ” refers to an alkyl containing 6 to 16 carbon atoms. Similarly, “C6-C 22" represents an alkyl group containing 6 to 22 carbon atoms. Representative saturated straight-chain alkyl groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-septyl, n-octyl, n-nonyl, and the like. On the other hand, representative saturated branched-chain alkyl groups include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Unsaturated alkyl groups contain at least one double or triple bond between adjacent carbon atoms (referred to as "alkenyl" or "alkynyl", respectively). Representative straight-chain and branched-chain alkenyl groups include ethenyl, propenyl, 1-butenyl, 2-butenyl, isobutenylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like. On the other hand, representative straight-chain and branched-chain alkynyl groups include ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.

[0029] Non-aromatic monocyclic or polycyclic alkyl groups are referred to herein as "carbocyclic" or "carbosilyl" groups. Representative saturated carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. On the other hand, representative unsaturated carbocycles include cyclopentenyl and cyclohexenyl, and the like.

[0030] The "heterocyclic carbon ring" or "heterocarbosilyl" group may be saturated or unsaturated (but not aromatic), may be monocyclic or polycyclic, the nitrogen and sulfur heteroatoms may be optionally oxidized, the nitrogen heteroatom may be optionally quaternized, and is a carbocyclic ring containing 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Heterocyclic carbon rings include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

[0031] The term "aryl" refers to an aromatic homocyclic (i.e., hydrocarbon) monocyclic, bicyclic, or tricyclic ring-containing group having preferably 6 to 12 members, such as phenyl, naphthyl, and biphenyl. Phenyl is a preferred aryl group. The term "substituted aryl" preferably refers to an aryl group substituted by one or more groups selected from alkyl, substituted alkyl, alkenyl (optionally substituted), aryl (optionally substituted), heterocyclo (optionally substituted), halo, hydroxy, alkoxy (optionally substituted), aryloxy (optionally substituted), alkanoyl (optionally substituted), aroyl, (optionally substituted), alkyl ester (optionally substituted), aryl ester (optionally substituted), cyano, nitro, amino, substituted amino, amide, lactam, urea, urethane, sulfonyl, etc., provided that optionally one or more combinations of substituents form a 3- to 7-membered ring together with the atoms to which they are attached.

[0032] As used herein, "heteroaryl" or "heteroaromatic" refers to an aromatic heterocyclic carbon ring having 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur, including both monocyclic and polycyclic ring systems and containing at least one carbon atom. The polycyclic ring system may include one or more non-aromatic rings as long as one of the rings is aromatic, but this is not necessary. Representative heteroaryls are furyl, benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl, isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl. The use of the term "heteroaryl" is intended to include N-alkylated derivatives such as 1-methylimidazol-5-yl substituents.

[0033] As used herein, "heterocyclic ring" or "heterocyclyl" refers to monocyclic and polycyclic ring systems having 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur and containing at least one carbon atom. The monocyclic and polycyclic ring systems may be aromatic, non-aromatic, or a mixture of aromatic and non-aromatic rings. Examples of heterocyclic rings include heterocyclic carbon rings and heteroaryl.

[0034] "Alkylthio" refers to an alkyl group as defined above in which the indicated number of carbon atoms are attached via a sulfur bridge. An example of alkylthio is methylthio (i.e., -S-CH3).

[0035] "Alkoxy" refers to an alkyl group as defined above in which the indicated number of carbon atoms are attached via an oxygen bridge. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n-pentoxy, and s-pentoxy. Preferred alkoxy groups are methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy.

[0036] "Alkylamino" refers to an alkyl group as defined above in which the indicated number of carbon atoms are attached via an amino bridge. An example of alkylamino is methylamino (i.e., -NH-CH3).

[0037] "Alkanoyl" refers to an alkyl (i.e., -(C=O)alkyl) as defined above in which the indicated number of carbon atoms are attached via a carbonyl bridge.

[0038] "Alkylsulfonyl" refers to an alkyl as defined above in which the indicated number of carbon atoms are attached via a sulfonyl bridge, such as mesyl (i.e., -S(=O)2alkyl), and "arylsulfonyl" refers to an aryl attached via a sulfonyl bridge (i.e., -S(=O)2aryl).

[0039] "Alkylsulfamoyl" refers to an alkyl (i.e., -NHS(=O)2alkyl) as defined above, in which the indicated number of carbon atoms are attached by a sulfamoyl bridge, and "arylsulfamoyl" refers to an alkyl (i.e., -NHS(=O)2aryl) attached by a sulfamoyl bridge.

[0040] "Alkylsulfinyl" refers to an alkyl (i.e., -S(=O)alkyl) as defined above, in which the indicated number of carbon atoms are attached by a sulfinyl bridge.

[0041] The terms "cycloalkyl" and "cycloalkenyl" refer to monocyclic, bicyclic, or tricyclic homocyclic ring groups of 3 to 15 carbon atoms that are fully saturated and partially unsaturated, respectively. The term "cycloalkenyl" includes bicyclic and tricyclic ring systems that are not aromatic as a whole but contain an aromatic moiety (e.g., fluorene, tetrahydronapthalene, dihydroindene, etc.). The rings of a polycyclic cycloalkyl group may be fused, bridged, and / or attached by one or more spiro bonds. The terms "substituted cycloalkyl" and "substituted cycloalkenyl" preferably refer to cycloalkyl and cycloalkenyl groups substituted by one or more groups selected from aryl, substituted aryl, heterocycle, substituted heterocycle, carbocycle, substituted carbocycle, halo, hydroxy, alkoxy (optionally substituted), aryloxy (optionally substituted), alkyl ester (optionally substituted), aryl ester (optionally substituted), alkanoyl (optionally substituted), aryol (optionally substituted), cyano, nitro, amino, substituted amino, amide, lactam, urea, urethane, sulfonyl, etc.

[0042] The terms "halogen" and "halo" refer to fluorine, chlorine, bromine, and iodine.

[0043] The term "substituted" refers to a molecule in which at least one hydrogen atom is replaced by a substituent. When substituted, one or more of the groups are "substituents". The molecule can be substituted in various ways. In the case of an oxo substituent ("=O"), two hydrogen atoms are substituted. Examples of substituents in this context can include halogen, hydroxy, alkyl, alkoxy, nitro, cyano, oxo, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -NRaRb, -NRaC(=O)Rb, -NRaC(=O)NRaNRb, -NRaC(=O)ORb, -NRaSO2Rb, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRb, -OC(=O)NRaRb, -ORa, -SRa, -SORa, -S(=O)2Ra, -OS(=O)2Ra, and -S(=O)2ORa. Ra and Rb in this context may be the same or different and independently are hydrogen, halogen hydroxyl, alkyl, alkoxy, alkyl, amino, alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl.

[0044] As used herein, the term "optionally substituted" means that the substitution is optional and thus the specified atom may be unsubstituted.

[0045] Compound In certain embodiments, the present disclosure relates to a compound of formula I,

Chemical formula

Chemical formula

[0046] In certain embodiments, the lipid is an aliphatic alcohol, aliphatic amine, or aliphatic thiol derived from an essential and / or non-essential fatty acid.

[0047] In certain embodiments, the lipid is an unsaturated, polyunsaturated, omega-unsaturated, or omega-polyunsaturated aliphatic alcohol, aliphatic amine, or aliphatic thiol derived from an essential and / or non-essential fatty acid.

[0048] In certain embodiments, the lipid is an aliphatic alcohol, aliphatic amine, or aliphatic thiol derived from essential and non-essential fatty acids in which one or more of its carbon units are substituted by oxygen, nitrogen, or sulfur.

[0049] In certain embodiments, the lipid is an unsaturated, polyunsaturated, omega-unsaturated, or omega-polyunsaturated aliphatic alcohol, aliphatic amine, or aliphatic thiol derived from essential and / or non-essential fatty acids in which one or more of its carbon units are substituted by oxygen, nitrogen, or sulfur.

[0050] In certain embodiments, the lipid is an aliphatic alcohol, aliphatic amine, or aliphatic thiol derived from essential and / or non-essential fatty acids that are optionally substituted.

[0051] In certain embodiments, the lipid is an unsaturated, polyunsaturated, omega-unsaturated, or omega-polyunsaturated aliphatic alcohol, aliphatic amine, or aliphatic thiol derived from essential and / or non-essential fatty acids that are optionally substituted.

[0052] In certain embodiments, the lipid is an aliphatic alcohol, aliphatic amine, or aliphatic thiol derived from essential and / or non-essential fatty acids that are optionally substituted and in which one or more of its carbon units are substituted by oxygen, nitrogen, or sulfur.

[0053] In certain embodiments, the lipid is an unsaturated, polyunsaturated, omega-unsaturated, or omega-polyunsaturated aliphatic alcohol, aliphatic amine, or aliphatic thiol derived from essential and / or non-essential fatty acids that are optionally substituted and in which one or more of its carbon units are substituted by oxygen, nitrogen, or sulfur.

[0054] In certain embodiments, the lipid is hexadecyloxypropyl.

[0055] In certain embodiments, the lipid is 2-aminohexadecyloxypropyl.

[0056] In certain embodiments, the lipid is 2-aminoarachidyl.

[0057] In certain embodiments, the lipid is 2-benzyloxyhexadecyloxypropyl.

[0058] In certain embodiments, the lipid is lauryl, myristyl, palmityl, stearyl, arachidyl, behenyl, or lignoceryl.

[0059] In certain embodiments, the lipid is of the formula:

Chemical formula

Chemical formula

Chemical formula

[0060] In certain embodiments, the R of the sphingolipid 12 is H, alkyl, methyl, ethyl, propyl, n-butyl, branched alkyl, isopropyl, 2-butyl, 1-ethylpropyl, 1-propylbutyl, cycloalkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, benzyl, phenyl, monosubstituted phenyl, disubstituted phenyl, trisubstituted phenyl, or a saturated or unsaturated C 12 ~C 19 long-chain alkyl.

[0061] In certain embodiments, the sphingolipid has the formula:

Chemical formula

[0062] In certain embodiments, the R of the sphingolipid 12 is H, alkyl, methyl, ethyl, propyl, n-butyl, branched-chain alkyl, isopropyl, 2-butyl, 1-ethylpropyl, 1-propylbutyl, cycloalkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, benzyl, phenyl, monosubstituted phenyl, disubstituted phenyl, trisubstituted phenyl, or a saturated or unsaturated C 12 ~C 19 long-chain alkyl.

[0063] Suitable sphingolipids include, but are not limited to, sphingosine, ceramide, or sphingomyelin, or 2-aminoalkyl optionally substituted with one or more substituents.

[0064] Other suitable sphingolipids include 2-aminooctadecane-3,5-diol, which may be optionally substituted by one or more substituents; (2S,3S,5S)-2-aminooctadecane-3,5-diol; (2S,3R,5S)-2-aminooctadecane-3,5-diol; 2-(methylamino)octadecane-3,5-diol; (2S,3R,5S)-2-(methylamino)octadecane-3,5-diol; 2-(dimethylamino)octadecane-3,5-diol; (2R,3S,5S)-2-(dimethylamino)octadecane-3,5-diol; 1-(pyrrolidin-2-yl)hexadecane-1,3-diol; (1S,3S)-1-((S)-pyrrolidin-2-yl)hexadecane-1,3-diol; 2-amino-11,11-difluorooctadecane-3,5-diol; (2S,3S,5S)-2-amino-11,11-difluorooctadecane-3,5-diol; 11,11-difluoro-2-(methylamino)octadecane-3,5-diol; (2S,3S,5S)-11,11-difluoro-2-(methylamino)octadecane-3,5-diol; N-((2S,3S,5S)-3,5-dihydroxyoctadecane-2-yl)acetamide; N-((2S,3S,5S)-3,5-dihydroxyoctadecane-2-yl)palmitamide; 1-(1-aminocyclopropyl)hexadecane-1,3-diol; (1S,3R)-1-(1-aminocyclopropyl)hexadecane-1,3-diol; (1S,3S)-1-(1-aminocyclopropyl)hexadecane-1,3-diol; 2-amino-2-methyloctadecane-3,5-diol; (3S,5S)-2-amino-2-methyloctadecane-3,5-diol; (3S,5R)-2-amino-2-methyloctadecane-3,5-diol; (3S,5S)-2-methyl-2-(methylamino)octadecane-3,5-diol; 2-amino-5-hydroxy-2-methyloctadecan-3-one; (Z)-2-amino-5-hydroxy-2-methyloctadecan-3-one oxime; (2S,3R,5R)-2-amino-6,6-difluorooctadecane-3,5-diol; (2S,3S,5R)-2-amino-6,6-difluorooctadecane-3,5-diol;(2S,3S,5S)-2-amino-6,6-difluorooctadecane-3,5-diol; (2S,3R,5S)-2-amino-6,6-difluorooctadecane-3,5-diol; and (2S,3S,5S)-2-amino-18,18,18-trifluorooctadecane-3,5-diol, but not limited thereto.

[0065] In certain embodiments, Q is O.

[0066] In certain embodiments, each R 7 is independently selected from hydrogen, -(C=O)O(C6-C 16 )alkyl, or -(C=O)O(C6-C 22 )alkyl.

[0067] In certain embodiments, R 1 is

Chemical formula

[0068] In certain embodiments, R 8 is hydrogen, hydroxy, or benzyloxy.

[0069] In certain embodiments, R 9 is higher alkyl, (C6-C 16 )alkyl, or (C6-C 22 )alkyl.

[0070] In certain embodiments, R 9 is tert-butyl or isobutyl.

[0071] In certain embodiments, W is O.

[0072] In certain embodiments, Z is H.

[0073] In certain embodiments, R 1is hydrogen, monophosphate, diphosphate, triphosphate,

Chem.

[0074] In certain embodiments, R 8 is hydrogen, hydroxy, or benzyloxy.

[0075] In certain embodiments, R 9 is higher alkyl, (C6 - C 16 ) alkyl, or (C6 - C 22 ) alkyl.

[0076] In certain embodiments, R 10 is isopropyl.

[0077] In certain embodiments, R 11 is methyl.

[0078] In certain embodiments, R 12 is phenyl.

[0079] In certain embodiments, R 13 is hydrogen.

[0080] In certain embodiments, R 14 is hydrogen.

[0081] In certain embodiments, R 2 is hydrogen.

[0082] In certain embodiments, R 3 is hydroxy.

[0083] In certain embodiments, R 4 is hydrogen, hydroxy, alkyl, halogen, or fluoro.

[0084] In certain embodiments, R 5is hydrogen, hydroxy, alkoxy, alkyl, methyl, ethynyl, or arylenyl.

[0085] In certain embodiments, R 6 is hydrogen.

[0086] In certain embodiments, each R 7 is independently selected from hydrogen, -(C=O)Oalkyl, -(C=O)alkyl, -(C=O)NHalkyl, -(C=O)Salkyl, -(C=O)O(C6-C 16 )alkyl, -(C=O)(C6-C 16 )alkyl, -(C=O)NH(C6-C 16 )alkyl, or -(C=O)S(C6-C 16 )alkyl.

[0087] In certain embodiments, the compound is 1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-(hydroxyamino)pyrimidin-2(1H)-one, 1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-((nonanoyloxy)amino)pyrimidin-2(1H)-one, and 1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-((((heptyloxy)carbonyl)oxy)amino)pyrimidin-2(1H)-one selected from.

[0088] In certain embodiments, the present disclosure relates to a compound of formula I having formula IA, [Chemical formula] or a salt thereof (X is CH2, CHMe, CMe2, CHF, CF2, or CD2; Y is H, D, F, Cl, Br, I, CH3, CD3, CF3, alkyl, acyl, alkenyl, alkynyl, hydroxyl, formyl, or SCH3; R 1 is hydrogen, monophosphate, diphosphate, triphosphate,

Chem.

[0089] In certain embodiments, the disclosure relates to a compound of Formula I having Formula IB,

Chemical formula

Chemical formula

[0090] In certain embodiments, the disclosure relates to a compound of Formula I having Formula IC,

Chemical Formula

Chemical Formula

[0091] In certain embodiments, the disclosure relates to a compound of Formula I having Formula ID,

Chemical formula

Chemical formula

[0092] In certain embodiments, the disclosure relates to a compound of formula I having formula IE, [Chemical formula] or a salt thereof (wherein Q is O, -O(C=O)-, -O(C=O)lipid, -O(C=O)V-, NH, or NR 7 ; V is O, NH, NR 7 , S, CH2, or CHR 7 ; W is CH2, NH, S, or O; X is CH2, CHMe, CMe2, CHF, CF2, or CD2; Y is N or CR’’; Z is N or CR’’; Each R’’ is independently selected from H, D, F, Cl, Br, I, CH3, CD3, CF3, alkyl, acyl, alkenyl, alkynyl, hydroxyl, formyl, or SCH3; R 2 is hydrogen, alkyl, alkenyl, alkynyl, ethynyl, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, hydroxymethyl, halogen, nitro, cyano, hydroxyl, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclic, aryl, azide, or heterocyclic, where R 2 is optionally substituted by one or more identical or different R 20 ; R 3 is hydrogen, hydroxyl, alkyl, halogen, nitro, cyano, hydroxyl, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclic, aryl, or heterocyclic, where R 3 is optionally substituted by one or more identical or different R 20 ; R 4 is hydrogen, hydroxyl, alkyl, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, halogen, nitro, cyano, hydroxyl, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclic, aryl, or heterocyclic, where R 4 is optionally substituted by one or more identical or different R 20 ; R 5is hydrogen, hydroxy, alkoxy, alkyl, alkenyl, alkynyl, ethynyl, fluoromethyl, difluoromethyl, trifluoromethyl, hydroxymethyl, arenyl, halogen, nitro, cyano, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclic, aryl, or heterocyclic, where R 5 is optionally substituted by one or more identical or different R 20 ; R 6 is hydrogen, hydroxy, alkoxy, alkyl, ethynyl, arenyl, halogen, nitro, cyano, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclic, aryl, or heterocyclic, where R 6 is optionally substituted by one or more identical or different R 20 ; Each R 7 is independently selected from none, hydrogen, -(C=O)Oalkyl, -(C=O)alkyl, -(C=O)NHalkyl, -(C=O)N-dialkyl, -(C=O)Salkyl, hydroxy, alkoxy, alkyl, higher alkyl, (C6-C 16 )alkyl, (C6-C 22 )alkyl, halogen, nitro, cyano, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclic, aryl, or heterocyclic, where each R 7 is optionally substituted by one or more identical or different R 20 ; R 15is hydrogen, -(C=O)Oalkyl, -(C=O)alkyl, -(C=O)NHalkyl, -(C=O)N-dialkyl, -(C=O)Salkyl, hydroxy, alkoxy, alkyl, higher alkyl, (C6-C 16 )alkyl, (C6-C 22 )alkyl, halogen, nitro, cyano, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclic, aryl, or heterocyclic, where R 15 is optionally substituted by one or more identical or different R 20 ; R 15’ is hydrogen, -(C=O)Oalkyl, -(C=O)alkyl, -(C=O)NHalkyl, -(C=O)N-dialkyl, -(C=O)Salkyl, hydroxy, alkoxy, alkyl, higher alkyl, (C6-C 16 )alkyl, (C6-C 22 )alkyl, halogen, nitro, cyano, amino, mercapto, formyl, carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclic, aryl, or heterocyclic, where each R 7 is optionally substituted by one or more identical or different R 20 ; R 15 and R 15’ can form a ring optionally substituted by one or more identical or different R 20 ; When Q = -O(C=O)V- and V = NR 7 , R 7 together can form a ring optionally substituted by one or more identical or different R 20 ; R 20is deuterium, alkyl, alkenyl, alkynyl, halogen, nitro, cyano, hydroxy, amino, amide, mercapto, formyl, carboxy, carbamoyl, azide, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclic, aryl, or heterocyclic, where R 13 is optionally substituted by one or more of the same or different R 21 ; and R 21 is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclic, aryl, or heterocyclic; (lipids described herein).

[0093] In certain embodiments, the disclosure relates to a compound of formula II,

Chemical formula

Chemical formula

[0094] In certain embodiments, the reference to higher alkyl, (C6-C 16 ) alkyl may be substituted by (C6-C 22 ) alkyl.

[0095] In certain embodiments, the reference to higher alkyl, (C6-C 16 ) alkyl, or (C6-C 22 ) alkyl may be substituted by polyethylene glycol or -CH2(CH2OCH2) n CH3 (wherein n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11-20, or 30-100).

[0096] Method of Use In certain embodiments, the disclosure relates to a method of treating or preventing a viral infection, the method comprising administering to a subject in need thereof an effective amount of a compound disclosed herein.

[0097] In certain embodiments, the viral infection is, or is caused by, an alphavirus, a flavivirus, or a coronavirus orthomyxovirus or paramyxovirus, or RSV, influenza, Powassan virus, or a filovirus or Ebola.

[0098] In certain embodiments, the viral infection is, or is caused by, a virus selected from Middle East respiratory syndrome coronavirus, eastern equine encephalitis virus, western equine encephalitis virus, Venezuelan equine encephalitis virus, Ross River virus, Barmah Forest virus, Powassan virus, and chikungunya virus.

[0099] In certain embodiments, the compound is administered by inhalation through the lungs.

[0100] In some embodiments, the subject is at risk of, or exhibits symptoms of, or has been diagnosed as having an influenza A virus, including subtypes H1N1, H3N2, H7N9, or H5N1, influenza B virus, influenza C virus, rotavirus group A, rotavirus group B, rotavirus group C, rotavirus group D, rotavirus group E, human coronavirus, SARS coronavirus, MERS coronavirus, human adenovirus types (HAdV-1 to 55), human papillomavirus (HPV) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59, parvovirus B19, molluscum contagiosum virus, JC virus (JCV), BK virus, Merkel cell polyomavirus, Coxsackievirus group A, norovirus, rubella virus, lymphocytic choriomeningitis virus (LCMV), dengue virus, chikungunya, eastern equine encephalitis virus (EEEV), western equine encephalitis virus (WEEV), Venezuelan equine encephalitis virus (VEEV), Ross River virus, Barmah Forest virus, yellow fever virus, measles virus, mumps virus, respiratory syncytial virus, rinderpest virus, California encephalitis virus, hantavirus, rabies virus, Ebola virus, Marburg virus, herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, roseolovirus, or Kaposi's sarcoma-associated herpesvirus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E or human immunodeficiency virus (HIV), human T-lymphotropic virus type I (HTLV-1), Friend spleen focus-forming virus (SFFV) or xenotropic murine leukemia virus-related virus (XMRV).

[0101] In certain embodiments, the subject is diagnosed with influenza A virus, including subtypes H1N1, H3N2, H7N9, H5N1 (low pathogenicity), and H5N1 (high pathogenicity), influenza B virus, influenza C virus, rotavirus group A, rotavirus group B, rotavirus group C, rotavirus group D, rotavirus group E, SARS coronavirus, MERS-CoV, human adenovirus types (HAdV-1 to 55), human papillomavirus (HPV) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59, parvovirus B19, molluscum contagiosum virus, JC virus (JCV), BK virus, Merkel cell polyomavirus, coxsackievirus group A, norovirus, rubella virus, lymphocytic choriomeningitis virus (LCMV), yellow fever virus, measles virus, mumps virus, respiratory syncytial virus, parainfluenza viruses 1 and 3, rinderpest virus, chikungunya, eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), California encephalitis virus, Japanese encephalitis virus, Rift Valley fever virus (RVFV), hantavirus, dengue virus serotypes 1, 2, 3, and 4, West Nile virus, Tacaribe virus, Funin, rabies virus, Ebola virus, Marburg virus, adenovirus, herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, roseolovirus, or Kaposi sarcoma-associated herpesvirus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, or human immunodeficiency virus (HIV).

[0102] In certain embodiments, the subject is diagnosed with gastroenteritis, acute respiratory disease, severe acute respiratory syndrome, post-viral fatigue syndrome, viral hemorrhagic fever, acquired immunodeficiency syndrome, or hepatitis.

[0103] In certain embodiments, the compounds and pharmaceutical compositions disclosed herein are contemplated to be administered in combination with other antiviral agents such as abacavir, acyclovir, acyclovir, adefovir, amantadine, amprenavir, amprigen, arbidol, atazanavir, atripla, boceprevir, cidofovir, combivir, darunavir, darunavir, dasabuvir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, famciclovir, homibursen, fosamprenavir, foscarnet, phosphonat, ganciclovir, ibacitabine, immunovir, idoxuridine, imiquimod, indinavir, inosine, type III interferon, type II interferon, type I interferon, lamivudine, ledipasvir, lopinavir, lobivud, maraviroc, moroxydine, methylthiazone, nelfinavir, nevirapine, nexavir, ombitasvir, oseltamivir, paritaprevir, peginterferon alpha-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir,simeprevir, sofosbuvir, stavudine, telaprevir, telbivudine, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, triduvir, tromantadine, truvada, valacyclovir, valganciclovir, vicriviroc, vidarabine, viramidine zalcitabine, zanamivir, or zidovudine and combinations thereof.

[0104] Formulation The pharmaceutical compositions disclosed herein may be in the form of pharmaceutically acceptable salts as generally described below. Some preferred but non-limiting examples of suitable pharmaceutically acceptable organic and / or inorganic acids are hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, acetic acid, and citric acid, and other pharmaceutically acceptable acids known per se (reference is made to the references mentioned below).

[0105] When the compounds of the present disclosure contain acidic groups as well as basic groups, the compounds of the present disclosure may also form inner salts, and such compounds are within the scope of the present disclosure. When the compound contains a hydrogen-donating heteroatom (e.g., NH), salts are contemplated to encompass isomers formed by the transfer of the hydrogen atom to a basic group or atom within the molecule.

[0106] Pharmaceutically acceptable salts of the compounds include acid addition salts and their base salts. Suitable acid addition salts are formed from acids that form non-toxic salts. Examples include acetate, adipate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, pyroglutamic acid, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate, and xinofoate. Suitable base salts are formed from bases that form non-toxic salts. Examples include aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc salts. Half salts of acids and bases, such as half sulfate and half calcium salts, may also be formed. For a general discussion of suitable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, 2002), which is incorporated herein by reference.

[0107] The compounds described herein can be administered in the form of prodrugs. A prodrug can include a covalently attached carrier that releases the active parent drug when administered to a mammalian subject. Prodrugs can be prepared by modifying the functional groups present in the compounds so that the modifying moiety is cleaved, either by conventional manipulation or in vivo, to form the parent compound. Prodrugs include, for example, compounds in which a hydroxyl group is bonded to any group that, when administered to a mammalian subject, is cleaved to form a free hydroxyl group. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate esters derivatives of alcohol functional groups in the compounds. Methods for formulating a compound as a prodrug can be found in the books by Testa and Mayer, Hydrolysis in Drug and Prodrug Metabolism, Wiley (2006). Typical prodrugs form active metabolites by conversion of the prodrug by hydrolytic enzymes, hydrolysis of amides, lactams, peptides, carboxylic acid esters, epoxides, or cleavage of esters of inorganic acids.

[0108] Pharmaceutical compositions for use in the present disclosure typically include an effective amount of a compound and a suitable pharmaceutically acceptable carrier. The formulations can be prepared by methods known per se and generally involve mixing at least one compound according to the present disclosure, optionally in sterile form, with one or more pharmaceutically acceptable carriers and, optionally, other pharmaceutically active compounds. Again, reference is made to U.S. Patent No. 6,372,778, U.S. Patent No. 6,369,086, U.S. Patent No. 6,369,087, and U.S. Patent No. 6,372,733, as well as the further references mentioned above, and standard handbooks such as the latest edition of Remington’s Pharmaceutical Sciences.

[0109] Generally, for pharmaceutical use, the compounds can be formulated as pharmaceutical preparations that include at least one compound and at least one pharmaceutically acceptable carrier, diluent, or excipient, and / or adjuvant, and optionally one or more further pharmaceutically active compounds.

[0110] The pharmaceutical preparations of the present disclosure are preferably in unit dosage forms and can be, for example, in boxes, blisters, vials, bottles, sachets, ampoules, or any other suitable single-dose or multi-dose holders or containers (which can be appropriately labeled); and can be suitably packaged together with one or more printed materials optionally including product information and / or instructions for use. Generally, such unit dosages will contain at least one compound of the present disclosure in an amount of 1 to 1000 mg, usually 5 to 500 mg, for example, about 10, 25, 50, 100, 200, 300, or 400 mg per unit dosage.

[0111] The compounds can be administered by various routes including oral, ocular, rectal, transdermal, subcutaneous, intravenous, intramuscular, or intranasal routes, depending mainly on the specific formulation to be used. The compounds are generally administered in an “effective amount,” which means an amount of the compound sufficient to obtain the desired therapeutic or prophylactic effect in the subject to which it is administered upon suitable administration. Usually, depending on the condition to be prevented or treated and the route of administration, such effective amount will generally be 0.01 to 1000 mg per kilogram of the patient's body weight per day, more frequently 0.1 to 500 mg such as 1 to 250 mg, for example, about 5, 10, 20, 50, 100, 150, 200, or 250 mg per kilogram of the patient's body weight per day, but it can be administered in divided amounts more than once a day as a single daily dose. The amount to be administered, the route of administration, and the further treatment regimen can be determined by the treating clinician depending on the age, sex, and general condition of the patient, as well as the nature and severity of the disease / symptom to be treated. Again, reference is made to U.S. Patent No. 6,372,778, U.S. Patent No. 6,369,086, U.S. Patent No. 6,369,087, and U.S. Patent No. 6,372,733, as well as the further references mentioned above, and standard handbooks such as the latest edition of Remington’s Pharmaceutical Sciences.

[0112] Depending on the method of introduction, the compounds described herein can be formulated in a variety of ways. Formulations containing one or more compounds can be prepared in a variety of pharmaceutical forms such as granules, tablets, capsules, suppositories, powders, controlled-release formulations, suspensions, emulsions, creams, gels, ointments, plasters, lotions, or aerosols. Preferably, these formulations are used in solid dosage forms that are simple for precise dosing and preferably suitable for oral administration. Solid dosage forms for oral administration include, but are not limited to, tablets, soft or hard gelatin or non-gelatin capsules, and caplets. However, liquid dosage forms such as solutions, syrups, suspensions, and shakes can also be utilized. In another embodiment, the formulation is administered topically. Suitable topical formulations include, but are not limited to, lotions, ointments, creams, and gels. In a preferred embodiment, the topical formulation is a gel. In another embodiment, the formulation is administered intranasally.

[0113] Formulations containing one or more of the compounds described herein can be prepared using pharmaceutically acceptable carriers that are composed of materials that are considered to be safe and effective and can be administered to an individual without causing undesirable biological side effects or interactions. A carrier is all the components present in a pharmaceutical formulation other than the active ingredient or ingredients. As generally used herein, "carrier" includes, but is not limited to, diluents, binders, lubricants, disintegrants, fillers, pH adjusters, preservatives, antioxidants, solubility enhancers, and coating compositions.

[0114] The carrier also includes all components of the coating composition which may contain plasticizers, pigments, colorants, stabilizers, and flow promoters. Sustained release, extended release, and / or pulsatile release dosage formulations can be prepared as described in standard references such as “Pharmaceutical dosage form tablets”, eds. Liberman et al. (New York, Marcel Dekker, Inc., 1989), “Remington - The science and practice of pharmacy”, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6th Edition, Ansel et al., (Media, PA: Williams and Wilkins, 1995). These references provide information on carriers, materials, devices, and processes for preparing sustained release dosage forms of tablets and capsules as well as tablets, capsules, and granules.

[0115] Examples of suitable coating materials include cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic polymers and copolymers, and methacrylic resins commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides, but are not limited thereto.

[0116] Furthermore, the coating material may include conventional carriers such as plasticizers, pigments, colorants, flow promoters, stabilizers, pore formers, and surfactants.

[0117] Optional pharmaceutically acceptable excipients present in the drug-containing tablets, beads, granules, or particles include, but are not limited to, diluents, binders, lubricants, disintegrants, colorants, stabilizers, and surfactants. Diluents, also referred to as "fillers," are typically necessary to increase the bulk of the solid dosage form so that it provides an actual size for tablet compression or bead and granule formation. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dried starch, hydrolyzed starch, pregelatinized starch, silicon dioxide, titanium oxide, magnesium aluminum silicate, and powdered sugar.

[0118] Binders are used to impart adhesiveness to solid dosage formulations, thereby ensuring that tablets or beads or granules remain intact after dosage form formation. Suitable binder materials include, but are not limited to, starches, pregelatinized starches, gelatin, saccharides (including sucrose, glucose, dextrose, lactose, and sorbitol), polyethylene glycol, waxes, natural and synthetic rubbers such as gum arabic, tragacanth, sodium alginate, celluloses including hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid / polymethacrylic acid, and polyvinylpyrrolidone.

[0119] Lubricants are used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glyceryl behenate, polyethylene glycol, talc, and mineral oil.

[0120] Disintegrants are used to facilitate the disintegration or "breakup" of dosage forms after administration and generally include, but are not limited to, starches, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethyl cellulose, hydroxypropyl cellulose, pregelatinized starch, clays, celluloses, arginine, gums, or cross-linked polymers such as cross-linked PVP (Polyplasdone XL from GAF Chemical Corp).

[0121] Stabilizers are used, for example, to inhibit or prevent the degradation reaction of drugs that have an oxidation reaction.

[0122] The surfactant may be anionic, cationic, amphoteric, or nonionic. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate, and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long-chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzenesulfonate; sodium dialkyl sulfosuccinates such as sodium dodecylbenzenesulfonate; sodium dialkyl sulfosuccinates such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyldimethylbenzylammonium chloride, polyoxyethylene, and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4 oleate, sorbitan acylates, sucrose acylates, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbate, polyoxyethylene octylphenyl ether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, poloxamer (registered trademark) 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfactants include sodium N-dodecyl-β-alanine, sodium N-lauryl-β-iminodipropionate, myristoamphoacetate, lauryl betaine, and lauryl sulfobetaine.

[0123] If desired, tablets, beads, granules, or particles may contain small amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, dyes, pH buffers, or preservatives.

[0124] The concentration of the compound with respect to the carrier and / or other substances can vary from about 0.5 to about 100 weight % (weight percent). For oral use, pharmaceutical formulations will generally contain from about 5 to about 100 weight % of the active material. For other uses, pharmaceutical formulations will generally contain from about 0.5 to about 50 weight % of the active material.

[0125] The compositions described herein can be formulated for modified or controlled release. Examples of controlled release dosage forms include extended release dosage forms, delayed release dosage forms, pulsatile release dosage forms, and combinations thereof.

[0126] Extended release formulations are generally prepared as diffusion or osmotic systems, for example, as described in “Remington - The science and practice of pharmacy” (20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000). Diffusion systems typically consist of two types of devices, a reservoir and a matrix, which are well known and described in the art. Matrix devices are generally prepared by compressing the drug with a polymer carrier that dissolves slowly into tablet form. The three main types of materials used in the preparation of matrix devices are insoluble plastics, hydrophilic polymers, and fatty compounds. Plastic matrices include, but are not limited to, methyl acrylate - methyl methacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymers include, but are not limited to, cellulose polymers such as methyl and ethyl cellulose, hydroxyalkyl celluloses such as hydroxypropyl - cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and Carbopol® 934, polyethylene oxide, and mixtures thereof. Fatty compounds include, but are not limited to, various waxes such as carnauba wax and glyceryl tristearate, and wax - type substances such as hydrogenated castor oil or hydrogenated vegetable oil, or mixtures thereof.

[0127] In certain preferred embodiments, the plastic material is a pharmaceutically acceptable acrylic polymer including, but not limited to, copolymers of acrylic acid and methacrylic acid, methyl methacrylate, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamine copolymer poly(methyl methacrylate), poly(methacrylic acid)(anhydride), polymethacrylate, polyacrylamide, poly(methacrylic anhydride), and glycidyl methacrylate copolymers.

[0128] In certain preferred embodiments, the acrylic polymer is composed of one or more ammonio methacrylate copolymers. Ammonio methacrylate copolymers are well known in the art and are described in NF XVII as completely polymerized copolymers of acrylic and methacrylic acid esters having a low content of quaternary ammonium groups.

[0129] In a preferred embodiment, the acrylic polymer is an acrylic resin lacquer such as those commercially available from Rohm Pharma under the trademark Eudragit®. In a further preferred embodiment, the acrylic polymer comprises a mixture of two acrylic resin lacquers commercially available from Rohm Pharma under the trademarks Eudragit® RL30D and Eudragit® RS30D, respectively. Eudragit® RL30D and Eudragit® RS30D are copolymers of acrylic acid esters and methacrylic acid esters having a low content of quaternary ammonium groups, and the molar ratio of ammonium groups to remaining neutral (meth)acrylic esters is 1:20 for Eudragit® RL30D and 1:40 for Eudragit® RS30D. The average molecular weight is about 150,000. Edragit® S-100 and Eudragit® L-100 are also preferred. The code names RL (high permeability) and RS (low permeability) refer to the permeability of these agents. The Eudragit® RL / RS mixture is insoluble in water and digestive fluids. However, the multiparticulate system formed to contain it is swellable and permeable in aqueous solutions and digestive fluids.

[0130] The above-mentioned polymers such as Eudragit® RL / RS can be mixed in desired ratios to ultimately obtain a sustained-release formulation having a desired dissolution profile. Desired sustained-release multiparticulate systems can be obtained, for example, from 100% Eudragit® RL, 50% Eudragit® RL and 50% Eudragit® RS, and 10% Eudragit® RL and 90% Eudragit® RS. Those skilled in the art will recognize that other acrylic polymers, such as Eudragit® L, can also be used.

[0131] Alternatively, the extended-release formulation can be prepared by utilizing an osmotic system or by applying a semipermeable coating to the dosage form. In the latter case, the desired drug release profile can be achieved by combining a low-permeability coating material and a high-permeability coating material in a suitable ratio.

[0132] The devices having the different drug release mechanisms described above can be combined into a final dosage form comprising single or multiple units. Examples of multiple units include, but are not limited to, multilayer tablets and capsules containing tablets, beads, or granules. An immediate-release layer can be added over the extended-release core using a coating or compression process, or in a multiple-unit system such as a capsule containing extended-release beads and immediate-release beads, the immediate-release portion can be added to the extended-release system.

[0133] Extended-release tablets containing hydrophilic polymers are prepared by techniques commonly known in the art, such as direct compression, wet granulation, or dry granulation. These formulations typically incorporate a polymer, a diluent, a binder, and a lubricant, as well as the pharmaceutical active ingredient. Common diluents include starches, powdered celluloses, particularly crystalline and microcrystalline celluloses, sugars such as fructose, mannitol, and sucrose, and inert powdered substances such as cereal flours and similar edible powders. Typical diluents include, for example, various types of starches, lactose, mannitol, kaolin, calcium phosphate or calcium sulfate, inorganic salts such as sodium chloride, and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders include substances such as starches, gelatin, and sugars such as lactose, fructose, and glucose. Natural and synthetic gums including gum arabic, alginates, methylcellulose, and polyvinylpyrrolidone can also be utilized. Polyethylene glycol, hydrophilic polymers, ethylcellulose, and waxes can also function as binders. Lubricants are necessary in tablet formulations to prevent the tablets and punches from sticking in the die. Lubricants are selected from slippery solids such as talc, magnesium stearate and calcium stearate, stearic acid, and hydrogenated vegetable oils.

[0134] Extended-release tablets containing a wax material are generally prepared using methods known in the art, such as direct blending, solidification, and aqueous dispersion methods. In the solidification method, the drug is mixed with the wax material and spray solidified, solidified, sieved, and processed.

[0135] The delayed-release preparation is made by coating the solid dosage form with a polymer film that is insoluble in the acidic environment of the stomach and soluble in the neutral environment of the small intestine.

[0136] The delayed release dosage unit can be prepared, for example, by coating a drug or drug-containing composition with a selected coating material. The drug-containing composition can be, for example, a tablet for incorporation into a capsule, a tablet for use as an inner core in a "coated core" dosage form, or a plurality of drug-containing beads, particles, or granules for incorporation into a tablet or capsule. Preferred coating materials include biodegradable, stepwise hydrolyzable, stepwise water-soluble, and / or enzymatically degradable polymers, and may be conventional "enteric" polymers. Enteric polymers, as recognized by those skilled in the art, become soluble in the high pH environment of the lower gastrointestinal tract or are slowly eroded as the dosage form passes through the gastrointestinal tract, while enzymatically degradable polymers are degraded by bacterial enzymes present in the lower gastrointestinal tract, particularly the colon.Suitable coating materials that provide delayed release include cellulose polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, methyl cellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, and sodium carboxymethyl cellulose; preferably acrylic acid polymers and copolymers formed from acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate, and / or ethyl methacrylate, and other methacrylic resins commercially available under the trademark Eudragit® (Rohm Pharma; Westerstadt, Germany), such as Eudragit® L30D-55 and L100-55 (soluble at pH 5.5 or higher), Eudragit® L-100 (soluble at pH 6.0 or higher), Eudragit® S (soluble at pH 7.0 or higher as a result of high esterification), and Eudragits® NE, RL, and RS (water-insoluble polymers with different degrees of permeability and swellability); vinyl polymers and copolymers such as polyvinyl pyrrolidone, vinyl acetate, vinyl acetate phthalate, vinyl acetate crotonic acid copolymer, and ethylene-vinyl acetate copolymer; enzymatically degradable polymers such as azopolymers, pectin, chitosan, amylose, and guar gum; zein and shellac, but are not limited thereto. Combinations of different coating materials can also be utilized. Multilayer coatings using different polymers are also applicable.

[0137] The preferred coating weight of a particular coating material can be readily determined by one of ordinary skill in the art by evaluating the individual release profiles of tablets, beads, and granules prepared with different amounts of various coating materials. It is the combination of the material, the method, and the form of application that produces the desired release characteristics, and this can only be determined from clinical trials.

[0138] The coating composition may contain conventional additives such as plasticizers, pigments, colorants, stabilizers, and flow promoters. Plasticizers are usually present to reduce the brittleness of the coating and are generally from about 10% to 50% by weight based on the dry weight of the polymer. Examples of typical plasticizers include polyethylene glycol, propylene glycol, triacetin, dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate, triethyl acetyl citrate, castor oil, and acetylated monoglycerides. Stabilizers are preferably used to stabilize the particles in the dispersion. Typical stabilizers are nonionic emulsifiers such as sorbitan esters, polysorbates, and polyvinylpyrrolidone. Flow promoters are recommended to reduce the adhesion effect during film formation and drying and are generally from approximately 25% to 100% by weight of the polymer weight in the coating solution. An effective flow promoter is talc. Other flow promoters such as magnesium stearate and glycerol monostearate can also be used. Pigments such as titanium dioxide can also be used. Small amounts of anti-foaming agents such as silicone (e.g., simethicone) may also be added to the coating composition.

[0139] The formulation can provide pulsed delivery of one or more compounds. "Pulsed" means that multiple drug dosages are released at time intervals. Generally, upon ingestion of the dosage form, the release of the initial dosage is substantially immediate, i.e., the first drug release "pulse" occurs within about 1 hour of ingestion. After this initial pulse, a first interval (delay time) follows, during which very little or no drug is released from the dosage form, after which a second dosage is released. Similarly, a second interval with substantially no drug release can be designed between the second and third drug release pulses. The duration of the interval with substantially no drug release can vary depending on the dosage form design, e.g., a twice-daily dosing profile, a three-times-daily dosing profile, etc. For a dosage form providing a twice-daily dosing profile, the interval with substantially no drug release has a period of approximately 3 to 14 hours between the first and second dosages. For a dosage form providing a three-times-daily profile, the interval with substantially no drug release has a period of approximately 2 to 8 hours between each of the three dosages.

[0140] In one embodiment, the pulsed release profile is achieved by a dosage form that is a closed and preferably sealed capsule containing at least two drug-containing "dosage units" in which each dosage unit within the capsule provides a different drug release profile. Control of the delayed release dosage unit is achieved by a controlled release polymer coating on the dosage unit or by incorporating the active agent into a controlled release polymer matrix. Each dosage unit can include a compressed tablet or a wet granulated tablet, but each tablet within the capsule provides different drug release. In a dosage form mimicking a twice-daily dosing profile, the first tablet releases the drug substantially immediately upon ingestion of the dosage form, while the second tablet releases the drug from approximately 3 to less than 14 hours after ingestion of the dosage form. In a dosage form mimicking a three-times-daily dosing profile, the first tablet releases the drug substantially immediately upon ingestion of the dosage form, while the second tablet releases the drug from approximately 3 to less than 10 hours after ingestion of the dosage form, and the third tablet releases the drug from at least 5 to approximately 18 hours after ingestion of the dosage form. It is possible for the dosage form to include four or more tablets. Although the dosage form generally does not include more than three tablets, dosage forms containing four or more tablets can be utilized.

[0141] Alternatively, each dosage unit in the capsule may contain a plurality of drug-containing beads, granules, or particles. As is known in the art, drug-containing "beads" refer to beads made of a drug and one or more excipients or polymers. Drug-containing beads can be produced by applying the drug to an inert carrier, for example, by creating an "inert sugar bead coated with the drug" or by creating a "core" containing both the drug and one or more excipients. Also as is known, drug-containing "granules" and "particles" contain drug particles that may or may not include one or more additional excipients or polymers. In contrast to drug-containing beads, granules and particles do not contain an inert carrier. Granules generally contain drug particles and require further processing. Generally, particles are smaller than granules and are not further processed. Beads, granules, and particles can be formulated to provide immediate release, but beads and granules are generally utilized to provide delayed release.

[0142] In one embodiment, the compound is formulated for topical administration. Suitable topical dosage forms include lotions, creams, ointments, and gels. A "gel" is a semi-solid system that contains a dispersion of an active agent, i.e., the compound, in a liquid vehicle and is made semi-solid by the action of a thickening agent or polymeric material dissolved or suspended in the liquid vehicle. The liquid can contain lipophilic components, aqueous components, or both. A portion of an emulsion can be a gel or can contain a gel component. However, some gels are not emulsions because they do not contain a homogenized blend of immiscible components. Methods for preparing lotions, creams, ointments, and gels are well known in the art.

[0143] The compounds described in this specification can be administered adjunctively together with other active compounds. These compounds include, but are not limited to, analgesics, anti-inflammatory agents, antipyretics, antidepressants, antiepileptic drugs, antihistamines, antimigraine drugs, antimuscarinic drugs, anxiolytics, analgesics, hypnotics, antipsychotics, bronchodilators, anti-asthma drugs, cardiovascular drugs, corticosteroids, dopaminergic agents, electrolytes, gastrointestinal drugs, muscle relaxants, nutrients, vitamins, parasympathomimetics, stimulants, appetite suppressants, and anti-narcolepsy drugs. "Adjunctive administration" as used herein means that the compound can be administered in the same dosage form or a different dosage form as one or more other active agents.

[0144] Specific examples of compounds that can be co-administered include aceclofenac, acetaminophen, adomexetine, almotriptan, alprazolam, amantadine, amcinonide, aminocyclopropane, amitriptyline, amolodipine, amoxapine, amphetamine, aripiprazole, aspirin, atomoxetine, azasetron, azatadine, beclomethasone, benactyzine, benoxaprofen, bellomprofen, betamethasone, bischoffazine, bromocriptine, budesonide, buprenorphine, bupropion, buspirone, butorphanol, butriptyline, caffeine, carbamazepine, carbidopa, carisoprodol, celecoxib, chlordiazepoxide, chlorpromazine, choline salicylate, citalopram, clomipramine, clonazepam, clonidine, clonitazene, clorazepate, chlortiazepam, cloxazolam, clozapine, codeine, corticosterone, cortisone, cyclobenzaprine, cyproheptadine, desipramine, desipramine, desomorphine, dexamethasone, dexanabinol, dextroamphetamine sulfate, dextromoramide, dextropropoxyphene, desocine, diazepam, dibenzepine, diclofenac sodium, diflunisal, dihydrocodeine, dihydroergotamine, dihydromorphine, dimethacrine, divalproxex, dizatriptan, dolasetron, donepezil, dotiepine, doxepin, duloxetine, ergotamine, escitalopram, estazolam, ethosuximide, etodolac, femoxetine, fenamic acid, fenoprofen, fentanyl, fludiazepam, fluoxetine, fluphenazine, flurazepam, flurbiprofen, flutazolam, fluvoxamine, frovatriptan, gabapentin, galantamine, gepirone, ginko bilboa, granisetron, haloperidol, fperidin A, hydrocodone, hydrocortisone, hydromorphine, hydroxyzine, ibuprofen, imipramine, indelpramine, indomethacin, indoprofen, iprindole, ipsapirone, ketaserin, ketoprofen,Ketorolac, Resopitron, Levodopa, Lipase, Lofepramine, Lorazepam, Loxapine, Maprotiline, Mazindol, Mefenamic Acid, Melatonin, Melitracen, Memantine, Meperidine, Meprobamate, Mesalamine, Metapramine, Metaxalone, Methadone, Methamphetamine, Methocarbamol, Methyldopa, Methylphenidate, Methyl Salicylate, Methyserpidine, Metoclopramide, Mianserin, Mifepristone, Milnacipram, Minaprine, Mirtazapine, Moclobemide, Modafinil (anti-narcolepsy drug), Molindone, Morphine, Morphine Hydrochloride, Nabumetone, Nadolol, Naproxen, Naratriptan, Nefazodone, Neurontin, Nomifensine, Nortriptyline, Olanzapine, Olsalazine, Ondansetron, Opipramol, Orphenadrine, Oxaflozan, Oxaprazin, Oxazepam, Oxitriptan, Oxycodone, Oxymorphone, Pancreatin, Parecoxib, Paroxetine, Pemoline, Pentazocine, Pepsin, Perphenazine, Phenacetin, Phentermine, Fenfluramine, Phenylbutazone, Phenytoin, Phosphatidylserine, Pimozide, Pirindole, Piroxicam, Pizotifen, Pizotyline, Pramipexole, Prednisolone, Prednisone, Pregabalin, Propranolol, Propizepine, Propoxyphene, Protriptyline, Quazepam, Quinupramine, Reboxetine, Reserpine, Risperidone, Ritanserin, Rivastigmine, Rizatriptan, Rofecoxib, Ropinirole, Rotigotine, Salsalate, Sertraline, Sibutramine, Sildenafil, Sulfasalazine, Sulindac, Sumatriptan, Tacrine, Temazepam, Tetrabenozine, Thiazide, Thioridazine, Thiothixene, Thiapride, Tiasipirone, Tizanidine, Tofenacin, Tolmetin, Troxatone, Topiramate, Tramadol, Trazodone, Triazolam, Trifluoperazine, Trimethobenzamide, Trimipramine, Tropisetron, Valdecoxib, Valproic Acid, Venlafaxine, Viloxazine, Vitamin E, Dimetindene, Diprazidone, Zolmitriptan, Zolpidem,There are zopiclone, as well as their isomers, salts, and combinations, but not limited to these.

[0145] The additional active agent can be formulated for immediate release, controlled release, or a combination thereof.

Example

[0146] Example 1. Synthesis of N4-Hydroxycytidine or 1-(3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-(hydroxyamino)pyrimidin-2-one (EIDD-01931) Following the protection of uridine by persilylation, activation of the 4-position of the nucleobase with a hindered arylsulfonyl group is carried out (see Figure 1). When this group is replaced with hydroxylamine, an N-4-hydroxy moiety is attached. Overall deprotection using any of the many fluoride sources available gives the desired product.

[0147] The compound can be produced in one step from cytidine by heating a pH-adjusted solution of hydroxylamine. Although shorter, this route has a low yield and requires purification by reverse-phase flash column chromatography, so its use is limited to small-scale production.

[0148] Example 2. General methods: All chemical reactions were carried out under a nitrogen atmosphere in glassware dried in an oven, unless otherwise specified. Chemicals and solvents were of reagent grade, purchased from commercial suppliers (typically Aldrich, Fisher, Acros, Carbosynth Limited, and Oakwood Chemical) and used as received unless otherwise specified. In particular, EIDD-1910, EIDD-1993, and EIDD-2003 were purchased from Carbosynth Limited. Solvents used in the reactions (tetrahydrofuran, methanol, acetonitrile, dichloromethane, toluene, pyridine, dimethylformamide) were anhydrous to greater than 99.9% in all cases. All reactions were followed to completion by thin layer chromatography (TLC), unless otherwise specified. TLC analysis was performed on silica gel using illumination with a UV lamp (254 nm) or staining with KMnO4 and heating. Manual flash column chromatography was performed using 40 - 60 micron (60 Å particle size) RediSep R f silica gel as the stationary phase purchased from Teledyne Isco. Automated gradient flash column chromatography was performed on a Teledyne Isco CombiFlash Companion; normal phase separations were performed using packed RediSep R f silica gel as the stationary phase, and reverse phase separations were performed using packed RediSep R f C 18 High Performance Gold stationary phase. Triphosphate purification was performed using ion exchange chromatography with DEAE (diethylaminoethyl) Sephadex A-25 as the stationary phase and an aqueous solution of TEAB (triethylammonium bicarbonate) as the mobile phase.

[0149] 11H NMR spectra were measured on a Varian 400 MHz instrument and processed using MestReNova software, version 9.0.1. Chemical shifts were measured relative to the appropriate solvent peaks: CDCl3 (δ 7.27), DMSO-d6 (δ 2.50), CD3OD (δ 3.31), D2O (δ 4.79). Couplings were described using the following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiplet, br = broad. 13 13C NMR spectra were measured on a Varian instrument at 100 MHz, and chemical shifts were measured relative to the appropriate solvent peaks: CDCl3 (δ 77.0), DMSO-d6 (δ 39.5), CD3OD (δ 49.0). 19 19F spectra were measured on a Varian instrument at 376 MHz, 31 31P spectra were measured on a Varian instrument at 162 MHz. 19 19F spectra, 31 31P spectra, and 13 13C spectra (in D2O only) chemical shifts were calibrated by MestReNova software using the absolute reference function relative to the corresponding 1 1H NMR spectra in the same solvent.

[0150] Normal-phase (low-resolution) liquid chromatography / mass spectrometry was performed using an Agilent 1200 series LC (UV absorption detector at 254 nm) and an Agilent 6120 LCMS quadrupole instrument with a Zorbax Eclipse XDB C 18 4.6 × 50 mm, 3.5 micron column eluted with a MeOH / water mixture (typically 95 / 5 no gradient). High-resolution mass spectrometry was performed by the Mass Spectrometry Center at Emory University using a Thermo LTQ-FTMS with APCI or ESI.

[0151] Example 3.

Chemical Structure

[0152] S2: 1 L round-bottom flask was charged with S1 (28 g, 47.7 mmol) and dichloromethane (700 mL). The solution was cooled to 0 °C using an ice bath; 4-DMAP (0.583 g, 4.77 mmol) and N,N-diisopropylethylamine (41.7 ml, 239 mmol) were added successively. 2,4,6-Triisopropylbenzene-1-sulfonyl chloride (28.9 g, 95 mmol) was added slowly to the flask and once the addition was complete, the flask was warmed to ambient temperature and stirred for 18 h. The dark orange solution was cooled to 0 °C using an ice bath, N,N-diisopropylethylamine (24.66 g, 191 mmol) was added via syringe followed by solid hydroxylamine hydrochloride (13.26 g, 191 mmol) added in one portion. The mixture was warmed to room temperature and stirred for 3 h. The reaction was quenched with water (200 mL) and the layers were separated. The aqueous layer was extracted with dichloromethane (200 mL), the combined organic phases were washed with brine, dried over sodium sulfate and concentrated under reduced pressure to give a dark orange oil. Purification by flash chromatography (15 - 50% gradient of EtOAc in hexanes) gave S2 (19.8 g, 69% over 2 steps) as an oil which solidified to a semi-solid upon drying in vacuo: 1 H NMR (400 MHz, CDCl3) δ 8.15 (s, 1H), 6.31 (s, 1H), 5.91 (d, J = 4.6 Hz, 1H), 5.56 (dd, J = 8.2, 2.0 Hz, 1H), 4.07 (m, 2H), 4.02 (m, 1H), 3.91 (dd, J = 11.6, 2.4 Hz, 1H), 3.73 (dd, J = 11.6, 2.4 Hz, 1H), 0.95 (s, 9H), 0.92 (s, 9H), 0.89 (s, 9H), 0.12 (s, 6H), 0.098 (s, 3H), 0.083 (s, 3H), 0.063 (s, 3H), 0.057 (s, 3H); LRMS m / z 602.3 [M+H] + .

[0153] EIDD-1931: S2 (23.3 g, 38.7 mmol) and THF (50 mL) were placed in a 50 mL round-bottom flask. Triethylamine hydrofluoride (6.30 mL, 38.7 mmol) was added all at once, and the mixture was stirred at ambient temperature for 18 h. The mixture was concentrated under reduced pressure, and the residue was dissolved in the minimal amount of MeOH, and this solution was slowly added to a Erlenmeyer flask containing rapidly stirred dichloromethane (500 mL), and the product precipitated; the mixture was stirred at room temperature for 15 min. The triturated solid was collected by vacuum filtration and washed with dichloromethane and then with ether. Drying the solid in vacuo gave the title compound (7.10 g, 71%) as a white solid: 1 H NMR (400 MHz, CD3OD) δ 7.16 (d, J = 8.2 Hz, 1H), 5.86 (d, J = 5.6 Hz, 1H), 5.59 (d, J = 8.2 Hz, 1H), 4.19 - 4.04 (m, 2H), 3.93 (q, J = 3.3 Hz, 1H), 3.77 (dd, J = 12.2, 2.9 Hz, 1H), 3.68 (dd, J = 12.1, 2.9 Hz, 1H); 1 H NMR (400 MHz, DMSO-d6) δ 9.95 (s, 1H), 9.46 (s, 1H), 7.02 (d, J = 8.2 Hz, 1H), 5.71 (d, J = 6.3 Hz, 1H), 5.54 (d, J = 7.7 Hz, 1H), 5.23 (d, J = 6.0 Hz, 1H), 5.02 (d, J = 4.6 Hz, 1H), 4.98 (t, J = 5.1 Hz, 1H), 3.95 (q, J = 5.9 Hz, 1H), 3.89 (td, J = 4.9 Hz, 3.0 Hz, 1H), 3.75 (q, J = 3.4 Hz, 1H), 3.50 (qdd, J = 11.9 Hz, 5.2 Hz, 3.5 Hz, 2H); 13 C NMR (101 MHz, DMSO-d6) δ 150.0, 143.9, 130.5, 98.89, 87.1, 85.0, 72.8, 70.8, 61.8. LRMS m / z 260.1 [M + H] + .

[0154] Example 4.

Chemical formula

[0155] Example 5.

Chemical Structure

[0156] Example 6.

Chemical Structure

[0157] EIDD-2052: To a stirred solution of S3 (0.300 g, 0.487 mmol) in MeOH (5 mL) at 0 °C under nitrogen, 1.25 M HCl in MeOH solution (2.3 mL, 2.92 mmol) was added dropwise via syringe. The mixture was stirred at room temperature for 24 h. Triethylamine (0.70 mL, 5.05 mmol) was added and the mixture was stirred for 2 h. The mixture was concentrated by rotary evaporation and flash chromatography (5 - 20% gradient of iPrOH in EtOAc) gave the title compound (85 mg, 64%) as an off-white solid: 11H NMR (400 MHz, D2O) δ 7.19 (d, J = 8.2 Hz, 1H), 5.82 (d, J = 5.4 Hz, 1H), 5.55 (d, J = 8.2 Hz, 1H), 4.15 - 4.07 (m, 2H), 3.92 (q, J = 3.5 Hz, 1H), 3.76 (dd, J = 12.2 Hz, 2.9 Hz, 1H), 3.76 (s, 3H), 3.67 (dd, J = 12.1 Hz, 3.4 Hz, 1H); 13 13C NMR (100 MHz, CD3OD) δ 151.4, 146.2, 133.0, 98.6, 89.8, 86.1, 74.7, 71.7, 62.7, 61.9, 25.2; LRMS m / z 274.1 [M + H] + .

[0158] Example 7. [Chemical formula] S4: In a round-bottom flask, 2'-methyluridine (0.850 g, 3.29 mmol), imidazole (0.896 g, 13.17 mmol), and DCM (6.5 mL) were added, and the mixture was cooled to 0 °C while stirring under nitrogen. Trimethylsilyl triflate (2.24 mL, 12.34 mmol) was added dropwise via syringe over 15 minutes. The mixture was warmed to room temperature and stirred overnight. After stirring for 16 hours, the mixture was diluted with DCM (200 mL) and poured into ice-cold water (100 mL). The organic layer was removed, and the aqueous layer was extracted with DCM (1 × 100 mL). The combined organic layers were washed with ice-cold brine (1 × 100 mL), dried over Na2SO4, filtered, and concentrated by rotary evaporation to give 1.8 g of a crude product. The substance was absorbed into hexane, and automated flash chromatography (40 g column, gradient of 5 - 20% EtOAc in hexane) gave S4 (1.50 g, 96%) as a white flaky solid: 11H NMR (400 MHz, CDCl3) δ 8.27 (d, J = 8.2 Hz, 1H), 7.92 (s, 1H), 5.92 (s, 1H), 5.64 (dd, J = 8.2 Hz, 2.3 Hz, 1H), 4.05 - 3.95 (m, 2H), 3.83 (d, J = 9.1 Hz, 1H), 3.73 (d, J = 11.2 Hz, 1H), 1.21 (s, 3H), 0.20 (s, 9H), 0.18 (s, 9H), 0.17 (s, 9H); LRMS m / z 475.2 [M+H] + .

[0159] S5: To a stirred solution of S4 (1.50 g, 3.16 mmol) and 4-DMAP (0.039 g, 0.316 mmol) in DCM (20 mL) at 0 °C under nitrogen was added N,N-diisopropylethylamine (2.75 mL, 15.80 mmol) via syringe, followed by the addition of solid 2,4,6-triisopropylbenzene-1-sulfonyl chloride (1.91 g, 6.32 mmol) all at once. The stirred mixture was allowed to warm to room temperature. After stirring at room temperature for 16 h, the mixture was cooled to 0 °C, washed with ice-cold saturated aqueous NaHCO3 (3 × 25 mL), dried over Na2SO4, filtered, and concentrated by rotary evaporation to give 4.2 g of a crude product as a brown oil. Absorbing the crude product into hexane, automated flash chromatography (80 g column, 1 - 10% gradient of EtOAc in hexane) gave the desired product of sulfonyl activation that was mostly pure (confirmed by LCMS to have the presumptive identity of 1 about 1.57 g, about 2.12 mmol) as determined by 1H NMR. The entire mixture was taken on immediately to the next step without further purification or analysis.

[0160] To a stirred solution of the freshly prepared above material (ca. 1.57 g, ca. 2.12 mmol) in MeCN (21 mL) at 0 °C under nitrogen was added triethylamine (0.89 mL, 6.35 mmol) via syringe, followed by O-methylhydroxylamine hydrochloride (0.531 g, 6.35 mmol) as a solid in one portion. The mixture was warmed to room temperature and stirred overnight. After 16 h of stirring, the mixture was poured into saturated aqueous NaHCO3 (50 mL) and extracted with DCM (3 × 50 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated by rotary evaporation. Automated flash chromatography on CombiFlash (80 g column, 5 - 15% gradient of EtOAc in hexanes) gave S5 (0.571 g, 36% over 2 steps) as a clear viscous oil that exists as a 9:1 ratio of tautomers by NMR: 1 H NMR (400 MHz, CDCl3, only major tautomer) δ 8.01 (br s, 1H), 7.59 (d, J = 8.3 Hz, 1H), 5.88 (s, 1H), 5.54 (d, J = 8.1 Hz, 1H), 4.03 - 3.93 (m, 2H), 3.84 (s, 3H), 3.82 (d, J = 9.0 Hz, 1H), 3.71 (d, J = 12.0 Hz, 1H), 1.20 (s, 3H), 0.23 - 0.15 (m, 27H); LRMS m / z 504.2 [M + H] + .

[0161] EIDD - 2054: To a round bottom flask were added S5 (0.510 g, 1.01 mmol) and a stir bar under nitrogen at room temperature. A 1% v / v solution of concentrated HCl in MeOH (10 mL, 1.20 mmol HCl) was added via syringe and the mixture was stirred at room temperature for 30 min. Solid Na2CO3 (1 g) was added in one portion and the mixture was stirred at room temperature for 30 min. Celite was added and the mixture was concentrated by rotary evaporation to give a crude product immobilized on the solid. Automated flash chromatography (12 g column, 0 - 10% gradient of MeOH in DCM) gave the title compound (0.265 g, 91%) as a white powdery solid: 11H NMR (400 MHz, CD3OD) δ 7.36 (d, J = 8.3 Hz, 1H), 5.89 (s, 1H), 5.54 (d, J = 8.2 Hz, 1H), 3.95 (dd, J = 12.5 Hz, 2.2 Hz, 1H), 3.86 (dt, J = 9.2 Hz, 2.4 Hz, 1H), 3.82 - 3.72 (m, 2H), 3.78 (s, 3H), 1.17 (s, 3H); 13 13C NMR (100 MHz, CD3OD) δ 151.3, 146.2, 132.8, 98.2, 92.6, 83.4, 79.8, 73.8, 61.9, 60.7, 20.3; LRMS m / z 288.1 [M + H] + .

[0162] Example 8.

Chemical Structure

[0163] To a stirred solution of the freshly prepared above material (ca. 1.81 g, ca. 2.44 mmol) in MeCN (25 mL) at 0 °C under nitrogen was added triethylamine (1.02 mL, 7.33 mmol) via syringe, followed by hydroxylamine hydrochloride (0.509 g, 7.33 mmol) as a solid in one portion. The mixture was warmed to room temperature and stirred for 2 h. The mixture was poured into saturated aqueous NaHCO3 (50 mL) and extracted with DCM (3 × 50 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated by rotary evaporation. Automated flash chromatography (40 g column, gradient of 5–35% EtOAc in hexanes) gave S6 (0.931 g, 54% over 2 steps) as a white flaky solid present as a 7:1 ratio of tautomers by NMR: 1 H NMR (400 MHz, DMSO-d6, major tautomer only) δ 9.99 (s, 1H), 9.57 (d, J = 2.1 Hz, 1H), 7.25 (d, J = 8.3 Hz, 1H), 5.72 (s, 1H), 5.45 (dd, J = 8.2 Hz, 2.1 Hz, 1H), 3.92 (d, J = 12.0 Hz, 1H), 3.85–3.75 (m, 2H), 3.66 (d, J = 12.0 Hz, 1H), 1.13 (s, 3H), 0.15 (s, 9H), 0.14 (s, 9H), 0.12 (s, 9H); LRMS m / z 490.0 [M + H] + .

[0164] EIDD-2053: To a round-bottom flask were added S6 (0.200 g, 0.408 mmol) and a stir bar under nitrogen at room temperature. 1% v / v MeOH solution of concentrated HCl (6 mL, 0.72 mmol HCl) was added via syringe and the mixture was stirred at room temperature for 30 min. Solid Na2CO3 (0.75 g) was added in one portion and the mixture was stirred at room temperature for 30 min. Celite was added and the mixture was concentrated by rotary evaporation to give the crude product immobilized on the solid. Automated flash chromatography (4 g column, gradient of 5–25% MeOH in DCM) gave the title compound (0.110 g, 99%) as a white powdery solid: 11H NMR (400 MHz, CD3OD) δ 7.30 (d, J = 8.3 Hz, 1H), 5.90 (s, 1H), 5.56 (d, J = 8.2 Hz, 1H), 3.95 (dd, J = 12.5 Hz, 2.1 Hz, 1H), 3.86 (dt, J = 9.2 Hz, 2.7 Hz, 1H), 3.80 (d, J = 9.2 Hz, 1H), 3.75 (dd, J = 12.5 Hz, 3.0 Hz, 1H), 1.18 (s, 3H); 13 13C NMR (100 MHz, D2O) δ 151.6, 147.3, 131.8, 98.9, 91.7, 81.9, 79.5, 73.3, 60.4, 49.5, 19.6; LRMS m / z 274.1 [M+H] + .

[0165] Example 9.

Chemical Structure

[0166] A freshly prepared Dowex (Li + form) ion-exchange column (17 mL CV) was rinsed with 5 CV of water. The prepared triethylammonium salt was absorbed into water and eluted through the ion-exchange column. The fractions containing the product were combined and lyophilized to give the title compound (0.030 g, 22%) as a fluffy, off-white solid:1 1H NMR (400 MHz, D2O) δ 7.19 (d, J = 8.3 Hz, 1H), 5.95 (d, J = 6.3 Hz, 1H), 5.82 (d, J = 8.3 Hz, 1H), 4.42 - 4.34 (m, 2H), 4.24 - 4.10 (m, 3H); 31 31P NMR (162 MHz, D2O) δ -8.5 (br s), -11.2 (d, J = 19.6 Hz), -22.0 (t, J = 19.3 Hz); LRMS m / z 498.0 [M - H] - .

[0167] Example 10.

Chem.

[0168] Example 11.

Chemical Structure

[0169] Example 12.

Chemical Structure

[0170] Example 13.

Chemical Structure

[0171] To a suspension of S9:S8 (0.566 g, 2.00 mmol) in THF (20.0 mL) was added dropwise via syringe a 1 M solution of t-butylmagnesium chloride in THF (3.00 mL, 3.00 mmol) at 0 °C under argon, and the resulting mixture was stirred at the same temperature for 1 hour. A solution of S7 (1.33 g, 3.00 mmol) in THF (20 mL) was added at 0 °C, and simultaneously the mixture was allowed to warm to room temperature and stirred for an additional 27 hours. The reaction was quenched by the addition of saturated aqueous NH4Cl at 0 °C. The resulting mixture was filtered through a Celite pad, and the pad was washed with MeOH. The filtrate was concentrated by rotary evaporation to give a brown solid, which was purified by flash chromatography (5% MeOH in DCM) to give a somewhat pure product. Further purification of the mixture by automated flash chromatography (40 g column, 0 - 25% gradient of MeOH in DCM) gave S9 (0.744 g, 67% over 2 steps) as a white solid present as a mixture of two diastereomers in a 1:2 ratio based on the integration of the P-NMR: 31 as a white solid present as a mixture of two diastereomers in a 1:2 ratio based on the integration of the P-NMR: 1 H NMR (400 MHz, CD3OD, diastereomer mixture) δ 7.61 (m, 1H), 7.34 (t, J = 7.9 Hz, 2H), 7.27 - 7.09 (m, 3H), 5.93 - 5.69 (m, 2H), 4.95 (p, J = 6.3 Hz, 1H), 4.90 (dd, J = 6.4 Hz, 2.2 Hz, 1H), 4.84 - 4.71 (m, 1H), 4.46 - 4.20 (m, 3H), 3.88 (p, J = 7.8 Hz, 1H), 2.15 (s, 1H), 1.53 (s, 3H), 1.32 (m, 6H), 1.21 (m, 6H); 1313C NMR (100 MHz, CD3OD, both diastereomers) δ 210.06, 174.62, 174.57, 174.41, 174.35, 167.89, 157.81, 152.18, 152.11, 144.64, 144.38, 130.82, 130.78, 130.77, 126.24, 126.22, 126.17, 126.16, 121.48, 121.45, 121.43, 121.40, 115.18, 115.08, 96.18, 95.96, 87.13, 87.05, 86.96, 86.88, 86.23, 82.48, 82.47, 70.14, 68.02, 51.81, 51.67, 49.64, 49.43, 49.21, 49.00, 48.79, 48.57, 48.36, 30.68, 27.46, 27.43, 25.51, 25.46, 22.00, 21.98, 21.90, 20.56, 20.49, 20.30; 31 31P NMR (162 MHz, CD3OD) δ 3.68, 3.45; C 24 H 33 O9N4NaP [M+Na] + Calculated HRMS for C20H22N4NaO9P: 575.18774, found: 575.18824.

[0172] S10: A solution of S9 (0.289 g, 0.502 mmol) in 80% aqueous HCOOH (12.40 mL) was stirred at room temperature for 3.5 h. The reaction mixture was concentrated by rotary evaporation and co-evaporated with MeOH (3 × 10 mL). The crude product S9 (0.257 g, quantitative) was obtained as a brown glassy solid and used in the next step without further purification: 1 1H NMR (400 MHz, CD3OD, diastereomer mixture) δ 8.16 (s, 1H), 7.79 (d, J = 7.5 Hz, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.50 - 7.08 (m, 5H), 6.03 - 5.68 (m, 2H), 4.96 (septet, J = 8 Hz, 1H), 4.55 - 4.24 (m, 2H), 4.23 - 4.08 (m, 2H), 4.08 - 3.99 (m, 1H), 3.97 - 3.82 (m, 1H), 1.43 - 1.26 (m, 4H), 1.26 - 1.10 (m, 6H); 1313C NMR (100 MHz, CD3OD, both diastereomers) δ 174.65, 174.61, 174.38, 174.33, 166.90, 157.46, 152.15, 152.08, 142.73, 130.89, 130.88, 130.85, 130.85, 126.28, 126.26, 121.42, 121.40, 121.37, 121.36, 96.19, 92.05, 91.97, 83.49, 83.42, 75.90, 75.84, 70.70, 70.64, 70.18, 67.14, 67.08, 51.88, 51.87, 51.71, 51.70, 49.64, 49.43, 49.21, 49.00, 48.79, 48.57, 48.36, 21.98, 21.91, 21.89, 21.80, 20.61, 20.55, 20.30; 31 31P NMR (162 MHz, CD3OD) δ 3.91, 3.76; C 21 H 30 O9N4P [M+H]+ + Calculated HRMS for C21H26N4O9P [M+H]+: 513.17449, found: 513.17413.

[0173] EIDD-2088: To a solution of S10 (0.257 g, 0.502 mmol) in THF (5 mL) was added 2N hydroxylamine at pH 6 (6.27 mL, 12.54 mmol), and the resulting mixture was stirred at 37 °C for 1.5 days. The reaction mixture was concentrated by rotary evaporation. The yellow solid obtained was redissolved in MeOH, immobilized on silica gel, and loaded onto a silica plug. Elution with 10% MeOH in CH2Cl2 through the silica plug gave a pale brown liquid after rotary evaporation of the fractions containing the product. Automated flash chromatography (12 g column, 2.5 - 15% gradient of MeOH in DCM) gave the title compound (0.155 mg, 59%) as an off-white foam: 11H NMR (400 MHz, CD3OD, diastereomer mixture) δ 7.89 (d, J = 8.0 Hz, 0.3H), 7.80 (d, J = 8.1 Hz, 0.65H), 7.48 - 7.31 (m, 2H), 7.31 - 7.13 (m, 3H), 6.02 - 5.79 (m, 2H), 4.97 (hept, J = 8 Hz, 1H), 4.55 - 4.08 (m, 6H), 3.90 (m, 1H), 1.44 - 1.26 (m, 4H), 1.22 (m, 6H); 13 13C NMR (100 MHz, CD3OD, both diastereomers) δ 174.72, 174.68, 174.36, 174.30, 155.25, 152.10, 152.03, 148.74, 148.68, 142.86, 130.92, 130.87, 126.33, 126.32, 121.43, 121.39, 91.71, 91.63, 91.58, 84.08, 84.02, 83.95, 75.48, 75.41, 70.71, 70.67, 70.20, 67.03, 51.90, 51.73, 51.71, 49.64, 49.43, 49.21, 49.00, 48.79, 48.57, 48.36, 21.98, 21.92, 21.89, 21.79, 20.59, 20.53, 20.31; 31 31P NMR (162 MHz, CD3OD) δ 3.98, 3.81; C 21 H 30 O 10 N4P [M + H] + HRMS calculated value for: 529.16941, measured value: 529.16900.

[0174] Example 14.

Chemical Structure

[0175] Example 15.

Chemical Structure

[0176] Example 16.

Chemical formula

[0177] EIDD-2107: To a stirred solution of S11 (0.910 g, 1.22 mmol) in a mixture of THF (18 mL) and DMF (6 mL) at 0 °C under nitrogen was added acetic acid (0.350 mL, 6.12 mmol), followed by the addition of solid tetraethylammonium fluoride (0.877 g, 5.88 mmol) all at once. The mixture was warmed to room temperature and stirred for 20 h. The mixture was then concentrated by rotary evaporation to give a crude product as an oil. The oil was taken up in DCM and automated flash chromatography (40 g column, 1 - 10% gradient of MeOH in DCM) gave 300 mg of a white solid as flakes consisting of the desired product and tetraethylammonium acetate. The mixture was taken up in MeOH and immobilized on celite. A second automated flash chromatography (12 g column, 1 - 10% gradient of MeOH in DCM) gave the title compound (0.228 g, 47% yield) as a white powdery solid. NMR analysis showed signals in a 5:1 ratio, presumably rotamers around one of the carbamate bonds (most of the signals related to the nucleobase are double or single but broadened): 1 H NMR (400 MHz, DMSO-d6, major rotamer only) δ 10.30 (s, 1H), 7.38 (d, J = 8.2 Hz, 1H), 6.85 (t, J = 5.8 Hz, 1H), 5.75 (d, J = 5.8 Hz, 1H), 5.69 (dd, J = 8.4 Hz, 2.2 Hz, 1H), 5.32 (d, J = 5.9 Hz, 1H), 5.10 - 5.00 (m, 2H), 3.99 (q, J = 5.6 Hz, 1H), 3.94 (q, J = 4.7 Hz, 1H), 3.83 - 3.76 (m, 1H), 3.63 - 3.46 (m, 2H), 3.04 (q, J = 6.5 Hz, 1H), 1.46 - 1.36 (m, 2H), 1.32 - 1.19 (m, 8H), 0.86 (t, J = 7.0 Hz, 3H); 13 C NMR (100 MHz, CD3OD, major rotamer peak only) δ 157.5, 150.8, 149.3, 135.3, 97.5, 89.9, 86.1, 75.0, 71.5, 64.7, 62.5, 41.9, 32.9, 30.8, 30.1, 27.7, 23.6, 14.4; C 17 H29 N4O7[M+H] + Calculated HRMS value for: 401.20308, measured value: 401.20319.

[0178] Example 17

Chemical formula

[0179] S13: A suspension of 3-hexadecyloxypropan-1-ol (1.58 g, 5.26 mmol) and DIPEA (0.92 mL, 5.26 mmol) in anhydrous acetonitrile (25 mL) was added dropwise over 10 minutes to 3-((chloro(diisopropylamino)phosphino)oxy)-propanenitrile (1.2 mL, 5.26 mmol). After 18 hours at room temperature, the mixture was quenched with saturated aqueous NaHCO3 (15 mL) and extracted with ethyl acetate (2 × 100 mL). The combined organic phases were concentrated by rotary evaporation, and flash chromatography (column volume 25 mm × 140 mm, 10 - 20% gradient of EtOAc in hexane) gave S13 (1.40 g, 53%) as a white solid: 11H NMR (400 MHz, CDCl3) δ 3.89 - 3.54 (m, 6H), 3.49 (t, J = 6.3 Hz, 2H), 3.39 (t, J = 6.7 Hz, 2H), 2.64 (t, J = 6.6 Hz, 2H), 1.87 (p, J = 6.3 Hz, 2H), 1.57 (p, J = 6.3 Hz, 2H), 1.25 (s, 26H), 1.18 (dd, J = 6.8, 3.5 Hz, 12H), 0.87 (t, J = 6.6 Hz, 3H); 31 31P NMR (162 MHz, CDCl3) δ 147.40.

[0180] A solution of S14:S12 (800 mg, 2.36 mmol) and S13 (2.15 g, 4.29 mmol) in anhydrous THF (20 mL) was added dropwise to a solution of tetrazole (19 mL of a 0.45 M solution in acetonitrile, 8.59 mmol). After 19 h at room temperature, the mixture was added dropwise to a solution of tert-butyl hydroperoxide in nonane (1.9 mL of a 5.5 M solution, 10.73 mmol) and stirring was continued for an additional 1 h. The excess tert-butyl hydroperoxide was quenched with saturated sodium thiosulfate solution (50 mL), the mixture was stirred for 45 min and then extracted with ethyl acetate (2 × 100 mL). The combined organic phases were concentrated by rotary evaporation and flash chromatography (25 mm × 180 mm column volume, 0 - 5% gradient of MeOH in DCM) gave S14 (1.2 g, 80%) as a mixture of diastereomers, as a foam: 11H NMR (400 MHz, CDCl3, diastereomer mixture) δ 7.38 (d, J = 7.6 Hz, 1H, diastereomer a), 7.37 (d, J = 7.6, 1H, diastereomer b), 5.78 (d, J = 7.3 Hz, 1H), 5.54 (d, J = 5.6, 1H, diastereomer a), 5.53 (d, J = 5.6, 1H, diastereomer b), 5.14 (ddd, J = 6.5, 3.1, 1.4 Hz, 1H), 4.93 (dt, J = 7.0, 3.6 Hz, 1H), 4.34 (td, J = 7.4, 6.8, 4.8 Hz, 3H), 4.28 - 4.08 (m, 4H), 3.48 (t, J = 6.1, 2H), 3.38 (t, J = 6.8, 2H), 2.78 (t, J = 6.5 Hz, 2H, diastereomer a), 2.75 (t, J = 6.5 Hz, 2H diastereomer b), 1.93 (m, 2H), 1.55 (s, 5H), 1.34 (s, 3H), 1.25 (s, 26H), 0.87 (t, J = 6.8, 3H); 13 13C NMR (101 MHz, CDCl3, diastereomer mixture) δ 166.26, 155.40, 144.20, 144.16, 116.62, 116.59, 113.93, 97.45, 97.38, 95.74, 95.69, 86.73, 86.64, 86.54, 84.90, 84.80, 81.87, 81.66, 71.23, 67.84, 67.79, 67.69, 67.64, 66.25, 66.22, 66.03, 65.97, 62.08, 62.03, 31.90, 30.51, 30.50, 30.44, 30.43, 29.68, 29.67, 29.64, 29.61, 29.52, 29.34, 27.06, 27.04, 26.13, 25.23, 25.21, 22.67, 19.57, 19.50, 14.12; 31 31P NMR (162 MHz, CDCl3, diastereomer mixture) δ -1.75, -1.83; LRMS m / z 699.4 [M + H] + .

[0181] A solution of S15:S14 (310 mg, 0.44 mmol) in THF (4 mL) was treated at 50 °C with stirring with a 2 M aqueous solution of hydroxylamine at pH 5 (1.1 mL, 2.2 mmol). After 19 h, TLC (10% methanol in methylene chloride) showed approximately 50% conversion to the more nonpolar component. Additional hydroxylamine and extended reaction times did not increase the conversion above 50%. After cooling to room temperature, the mixture was partitioned between ethyl acetate (100 mL) and brine (10 mL). The organic phase was concentrated and flash chromatography of the crude (column volume 19 mm × 170 mm, 1 - 5% gradient of MeOH in DCM) gave S15 (70 mg, 22%) as a 1:1 mixture of diastereomers as a foam: 1 H NMR (400 MHz, CDCl3) δ 8.94 (s, 1H), 6.60 (d, J = 8.1, 1H, diastereomer a), 6.58 (d, J = 8.1, 1H, diastereomer b), 5.67 (d, J = 8.1, 1H, diastereomer a), 5.65 (d, J = 8.1, 1H, diastereomer b), 5.59 (d, J = 2.1 Hz, 1H, diastereomer a), 5.55 (d, J = 2.1 Hz, 1H, diastereomer b), 4.98 (m, 1H), 4.84 (m, 1H), 4.35 - 4.10 (m, 6H), 3.48 (t, J = 6.1 Hz, 2H), 3.38 (t, J = 6.7, 2H), 2.76 (m, 2H), 1.94 (m, 2H), 1.59 - 1.49 (m, 5H), 1.34 (s, 3H), 1.24 (s, 26H), 0.87 (t, J = 6.7 Hz, 3H); 31 P NMR (162 MHz, CDCl3, diastereomer mixture) δ -1.57, -1.64. LRMS m / z 715.3 [M + H] + .

[0182] A solution of EIDD-2108: S15 (62 mg, 0.087 mmol) in methanol (4 mL) was treated with a catalytic amount of para-toluenesulfonic acid (3.3 mg, 0.017 mmol). After stirring at room temperature for 16 h, the mixture was treated with saturated aqueous ammonium hydroxide (1.5 mL) and further stirred at room temperature for 4 h. The mixture was concentrated by rotary evaporation and the resulting residue was triturated with 5% acetonitrile in methanol (2×15 mL). Purification of the resulting white solid by flash chromatography (11 mm×45 mm column volume, 25% MeOH in DCM, 2.5% v / v saturated aqueous NH4OH) gave the title compound (25 mg, 46%) as a white solid: 1 H NMR (400 MHz, CD3OD) δ 7.21 (d, J = 8.2 Hz, 1H), 5.95 (d, J = 5.5 Hz, 1H), 5.67 (d, J = 8.2 Hz, 1H), 4.22 - 4.16 (m, 2H), 4.07 - 3.98 (m, 3H), 3.94 (q, J = 6.3 Hz, 2H), 3.52 (t, J = 6.3 Hz, 2H), 3.41 (t, J = 6.6 Hz, 2H), 1.87 (p, J = 6.3 Hz, 2H), 1.53 (q, J = 6.9 Hz, 2H), 1.28 (s, 28H), 0.92 - 0.85 (m, 3H); 13 C NMR (101 MHz, CD3OD) δ 150.45, 144.99, 130.77, 98.13, 87.51, 83.39, 83.30, 72.98, 70.72, 70.55, 66.89, 64.80, 62.51, 62.46, 31.66, 30.71, 30.63, 29.38, 29.35, 29.24, 29.07, 25.87, 22.33, 13.07; 31 P NMR (162 MHz, CD3OD) δ 0.34; C 28 H 51 N3O 10 P[M - H] - HRMS calculated for: 620.33175; found, 620.33205.

[0183] Example 18.

Chemical Structure

[0184] To a solution of S17:S16 (0.250 g, 0.508 mmol) in THF (5.1 mL) was added 2N aqueous hydroxylamine solution (6.4 mL, 12.71 mmol) at pH 6, and the resulting mixture was stirred at 55 °C for 1.5 days. After partitioning between EtOAc and H2O, the aqueous layer was separated and extracted with EtOAc (2 × 15 mL). The combined organic layers were washed with water and brine, dried over Na2SO4, filtered, and concentrated by rotary evaporation. Automated flash chromatography (24 g column, 0 - 7.5% gradient of MeOH in DCM) gave S17 (0.124 g, 48%) as a white solid: 1 H NMR (400 MHz, CDCl3) δ 8.69 (s, 1H), 8.34 (s, 1H), 6.94 (d, J = 8.2 Hz, 1H), 6.13 (dd, J = 11.0 Hz, 4.8 Hz, 1H), 5.62 (d, J = 8.3 Hz, 1H), 4.30 (dq, J = 12 Hz, 4 Hz, 1H), 3.95 (d, J = 12 Hz, 1H), 3.83 (d, J = 4 Hz, 1H), 3.77 (dd, J = 12 Hz, 4 Hz, 1H), 0.92 (s, 9H), 0.90 (s, 9H), 0.18 - 0.03 (m, 12H); 13 C NMR (100 MHz, CDCl3) δ 149.1, 144.8, 130.2, 122.1 (t, J = 259 Hz), 98.4, 83.4 (dd, J = 40 Hz, 24 Hz), 80.8 (d, J = 9 Hz), 69.8 (dd, J = 27 Hz, 18 Hz), 77.2, 60.0, 25.8, 25.5, 18.3, 18.0, 4.8, -5.3, -5.5, -5.6; 19 F NMR (376 MHz, CDCl3) δ -115.67 (dd, J = 239.5 Hz, 12.4 Hz), -117.02 (dt, J = 239.4 Hz, 10.8 Hz); C 21 H 40 O5N3F2Si2 [M + H] + HRMS calculated for: 508.24691, found: 508.24697.

[0185] A mixture of EIDD-2133: S17 (0.220 g, 0.433 mmol) and NH4F (0.128 g, 3.47 mmol) in MeOH (22 mL) was stirred under reflux overnight. The mixture was cooled to room temperature and concentrated by rotary evaporation. Flash chromatography (5 - 10% gradient of MeOH in DCM) gave a somewhat pure product. After further purification by flash chromatography twice (only the fractions where the desired product eluted with unknown impurities and could not be instantaneously stained by KMnO4 on TLC were collected), the title compound (18 mg, 15% yield) was obtained as a white solid: 1 H NMR (400 MHz, CD3OD) δ 7.05 (d, J = 8.3 Hz, 1H), 6.06 (m, 1H), 5.59 (d, J = 8.3 Hz, 1H), 4.21 (m, 1H), 3.90 (d, J = 12.6 Hz, 1H), 3.81 (td, J = 12 Hz, 4 Hz, 1H), 3.74 (dd, J = 12 Hz, 4 Hz, 1H); 13 C NMR (100 MHz, CD3OD) δ 151.1, 145.7, 131.5, 124.1 (t, J = 256 Hz), 99.3, 84.8 (dd, J = 39 Hz, 26 Hz), 82.0 (d, J = 9 Hz), 70.7 (dd, J = 26 Hz, 21 Hz), 60.6. 19 F NMR (376 MHz, CD3OD) δ 118.62 (ddd, J = 240.2 Hz, 13.4 Hz, 6.1 Hz), -119.67 (broad d, J = 240.7 Hz); C9H 12 O5N3F2 [M + H] + HRMS calculated for: 280.07395, found: 280.07347.

[0186] Example 19.

Chemical Structure

[0187] EIDD-2091: To a suspension of S18 (0.266 g, 0.500 mmol) in THF (5 mL) was added 2N aqueous hydroxylamine solution (6.3 mL, 12.49 mmol) at pH 6, and the resulting mixture was stirred at 37 °C for 1.5 days. The reaction (incomplete by TLC) was partitioned between EtOAc and H2O. The aqueous layer was extracted with EtOAc (2 × 15 mL). The combined organic layers were washed with H2O and brine, dried over Na2SO4, filtered, and concentrated by rotary evaporation. Automated flash chromatography (24 g column, 0 - 10% gradient of MeOH in DCM) gave the title compound (34 mg, 12%) as a white solid, a mixture of diastereomers. 1 1H NMR (400 MHz, CD3OD, mixture of diastereomers) δ 7.36 (t, J = 7.7 Hz, 2H), 7.28 - 7.12 (m, 3H), 6.78 (t, J = 9.0 Hz, 1H), 6.09 (q, J = 8 Hz, 1H), 5.55 (dd, J = 19.8 Hz, 8.3 Hz, 1H), 4.97 (sept, J = 6.3 Hz, 1H), 4.63 - 4.27 (m, 3H), 4.20 (m, 1H), 4.10 - 3.96 (m, 1H), 3.95 - 3.76 (m, 1H), 1.33 (t, J = 7.8 Hz, 3H), 1.22 (m, 6H); 1313C NMR (100 MHz, CD3OD, diastereomer mixture) δ 174.58, 174.54, 174.36, 174.31, 152.14, 152.07, 150.98, 145.48, 131.51, 131.34, 130.83, 126.26, 121.39, 121.37, 121.34, 121.32, 99.77, 85.24, 84.60, 80.02, 79.93, 79.88, 79.78, 71.52, 71.30, 71.05, 70.83, 70.18, 65.78, 65.72, 65.49, 65.44, 51.79, 51.66, 49.64, 49.43, 49.21, 49.00, 48.79, 48.57, 48.36, 21.97, 21.89, 20.54, 20.48, 20.39, 20.31; 19 19F NMR (376 MHz, CD3OD) δ -118.04 (dd, J = 240.8, 22.2 Hz), -119.47 (d, J = 242.6 Hz); 31 31P NMR (162 MHz, CD3OD) δ 3.76, 3.69; C 21 H 27 O8N4F2NaP [M+Na] + HRMS calculated value for: 571.13759, measured value: 571.13708.

[0188] Example 20.

Chemical Structure

[0189] S20: A solution of S19 in piperidine (51.0 mL, 25.5 mmol) was stirred at room temperature for 7 h. The reaction mixture was concentrated by rotary evaporation and then redissolved in CH2Cl2. The organic solution was washed with approximately 0.5 N ice-cold HCl (4 × 200 mL) and brine, and dried over Na2SO4. After filtration and concentration by rotary evaporation, the yellow residue was lyophilized to give S20 (8.1 g, 97%) as a pale yellow wax: 1 1H NMR (400 MHz, CDCl3) δ 12.20 (s, 1H), 5.61 (s, 2H), 5.57 (s, 2H), 1.21 (s, 18H); 1313C NMR (100 MHz, CDCl3) δ 177.2, 82.7, 38.7, 26.8; 31 31P NMR (162 MHz, CDCl3) δ -3.58; C 12 H 24 O8P[M + H] + Calculated value for positive mode HRMS for C 12 H 22 O8P[M - H] - Calculated value for negative mode HRMS: 325.10578, measured value: 325.10568.

[0190] EIDD - 2135: A solution of triethylammonium bis(POM) phosphate was prepared by adding triethylamine (0.362 mL, 2.60 mmol) to a solution of S20 (0.782 g, 2.398 mmol) in THF (8 mL). The prepared solution of triethylammonium bis(POM) phosphate was added to a solution of EIDD - 1931 (0.518 g, 1.998 mmol) in THF (32 mL) under nitrogen at room temperature and then cooled to 0 °C. DIPEA (1.392 mL, 7.99 mmol), BOP - Cl (1.017 g, 4.00 mmol) and 3 - nitro - 1H - 1,2,4 - triazole (0.456 g, 4.00 mmol) were successively added to the reaction mixture, and the resulting mixture was stirred at 0 °C for 6 hours, then warmed to room temperature and stirred overnight. The reaction mixture was partitioned between EtOAc and saturated aqueous NaHCO3. The aqueous layer was extracted with EtOAc, the combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated by rotary evaporation. Automated flash chromatography (40 g column, 0 - 10% gradient of MeOH in DCM) gave the title compound (30. mg, 2.6%) as a white foam: 1 1H NMR (400 MHz, CDCl3) δ 10.25 (s, 1H), 7.43 (d, J = 8.2 Hz, 1H), 6.83 (d, J = 8.1 Hz, 1H), 5.99 - 5.42 (m, 6H), 4.58 - 4.00 (m, 5H), 3.89 (m, 2H), 1.21 (s, 18H); 311P NMR (162 MHz, CDCl3) δ -4.77, -5.16; C 21 H 34 O 13 N3NaP [M+Na] + Calculated HRMS value for: 590.17215, found: 590.17171.

[0191] Example 21.

Chemical Structure

[0192] Example 22

Chemical Structure

[0193] S22: To a stirred solution of S21 (2.90 g, 4.94 mmol) and 4-DMAP (0.060 g, 0.49 mmol) in dichloromethane (50 mL) at 0 °C under nitrogen was added N,N-diisopropylethylamine (4.30 mL, 24.70 mmol) via syringe, followed by the addition of solid 2,4,6-triisopropylbenzene-1-sulfonyl chloride (2.99 g, 9.88 mmol) all at once. The mixture was warmed to ambient temperature, stirred for 4 h, and then recooled to 0 °C. The mixture was washed with ice-cold saturated aqueous NaHCO3 (3 × 50 mL), dried over Na2SO4, filtered, and concentrated by rotary evaporation. The crude oil was absorbed into dichloromethane, and automated flash chromatography (80 g column, 1 - 10% gradient of EtOAc in hexanes) gave S22 (3.30 g, 78%) as a clear colorless oil: 11H NMR (400 MHz, CDCl3) δ 7.92 (d, J = 7.3 Hz, 1H), 7.20 (s, 2H), 6.10 (d, J = 3.0 Hz, 1H), 6.05 (d, J = 7.3 Hz, 1H), 4.33 - 4.23 (m, 3H), 4.14 (s, 1H), 4.01 (dd, J = 8.8 Hz, 6.2 Hz, 1H), 3.80 (dd, J = 9.6 Hz, 6.2 Hz, 1H), 3.70 (t, J = 9.3 Hz, 1H), 2.90 (p, J = 7.0 Hz, 1H), 1.32 - 1.22 (m, 21H), 0.91 (s, 9H), 0.89 (s, 9H), 0.72 (s, 9H), 0.10 (s, 6H), 0.08 (s, 3H), 0.07 (s, 3H), -0.03 (s, 3H), -0.34 (s, 3H).

[0194] S23: To a stirred solution of S22 (3.30 g, 3.87 mmol) in acetonitrile (40 mL) at 0 °C under nitrogen was added triethylamine (1.08 mL, 7.73 mmol) by syringe, followed by the addition of solid hydroxylamine hydrochloride (0.537 g, 7.73 mmol) in one portion. The mixture was warmed to ambient temperature and stirred for 16 h. The mixture was recooled to 0 °C and saturated aqueous NaHCO3 (80 mL) was added. The mixture was extracted with dichloromethane (3 × 80 mL), the combined organic layers were dried over Na2SO4, filtered, and concentrated by rotary evaporation. The crude material was subjected to automated flash chromatography (80 g column, 5 - 20% gradient of EtOAc in dichloromethane) to give a somewhat pure material. A second automated flash chromatography (80 g column, 5 - 50% gradient of EtOAc in hexanes) gave S23 (1.17 g, 50%) as a white flaky solid: 11H NMR (400 MHz, CDCl3) δ 8.20 (br s, 1H), 6.90 (d, J = 8.4 Hz, 1H), 6.42 (s, 1H), 6.12 (d, J = 3.4 Hz, 1H), 5.51 (dd, J = 8.3 Hz, 1.8 Hz, 1H), 4.15 (br m, 1H), 4.07 (dd, J = 3.4 Hz, 1.4 Hz, 1H), 3.91 (dd, J = 8.2 Hz, 6.4 Hz, 1H), 3.80 (dd, J = 9.8 Hz, 5.6 Hz, 1H), 3.74 (dd, J = 9.8 Hz, 8.6 Hz, 1H), 0.91 (s, 9H), 0.90 (s, 9H), 0.86 (s, 9H), 0.12 (s, 3H), 0.11 (s, 3H), 0.08 (s, 3H), 0.07 (s, 6H), -0.02 (s, 3H); LRMS m / z 602.3 [M+H] + .

[0195] EIDD-02200: To a stirred solution of S23 (0.602 g, 1.00 mmol) in THF (8 mL) at room temperature under nitrogen was added dropwise triethylamine trihydrofluoride (0.163 mL, 1.00 mmol) via syringe. The mixture was stirred at ambient temperature for 4 days. Celite was added to the reaction mixture, and the crude was immobilized on the Celite by rotary evaporation. Automated flash chromatography (24 g column, 5 - 25% gradient of MeOH in dichloromethane) gave 600 mg of a somewhat pure product. The mixture was absorbed into water, and automated reverse-phase flash chromatography (43 g column, 0 - 15% gradient of acetonitrile in water) gave the desired product free of impurities. Dissolving the solid in water, freezing in a dry ice / acetone bath, and lyophilizing gave the title compound (0.164 g, 63% yield) as a white fluffy solid: 1 1H NMR (400 MHz, CD3OD) δ 7.13 (d, J = 8.3 Hz, 1H), 6.07 (d, J = 4.4 Hz, 1H), 5.51 (d, J = 8.3 Hz, 1H), 4.10 (dd, J = 4.5 Hz, 1.3 Hz, 1H), 4.03 (t, J = 3.4 Hz, 1H), 3.87 - 3.72 (m, 3H); 11H NMR (400 MHz, D2O) δ 7.08 (d, J = 8.3 Hz, 1H), 6.09 (d, J = 5.6 Hz, 1H), 5.67 (d, J = 8.3 Hz, 1H), 4.33 (t, J = 5.4 Hz, 1H), 4.06 (t, J = 5.6 Hz, 1H), 3.89 - 3.86 (m, 2H), 3.76 (dd, J = 13.1 Hz, 6.1 Hz, 1H); 13 13C NMR (100 MHz, D2O) δ 150.9, 146.8, 132.8, 97.0, 84.1, 82.1, 75.8, 74.8, 60.4; LRMS m / z 260.1 [M+H] + .

[0196] Example 23.

Chem.

[0197] S25: To a stirred suspension of S24 (0.831 g, 2.78 mmol) in dichloromethane (14 mL) at room temperature under nitrogen was added triethylamine (0.58 mL, 4.16 mmol) and 4-DMAP (3.4 mg, 0.028 mmol), and the mixture was stirred at room temperature for 15 minutes. A solution of 4,4'-dimethoxytrityl chloride (0.988 g, 2.92 mmol) in dichloromethane (14 mL) was added dropwise, and the mixture was stirred overnight at room temperature. The reaction mixture was washed with brine (1×30 mL), dried over Na2SO4, filtered, and concentrated by rotary evaporation. Flash chromatography (9:1 hexane:EtOAc, 2.5% v / v Et3N) gave S25 (1.39 g, 83%) as a yellow foam: 1 H NMR (400 MHz, CD3OD) δ 7.35 - 7.20 (m, 10H), 7.01 (d, J = 8.3 Hz, 1H), 6.85 - 6.80 (m, 4H), 5.80 (d, J = 3.0 Hz, 1H), 5.52 (d, J = 8.2 Hz, 1H), 4.84 (dd, J = 6.4 Hz, 3.0 Hz, 1H), 4.77 (dd, J = 6.4 Hz, 3.6 Hz, 1H), 4.10 (q, J = 4.0 Hz, 1H), 3.73 (dd, J = 11.9 Hz, 3.6 Hz, 1H), 3.68 (dd, J = 12.0 Hz, 4.6 Hz, 1H), 1.53 (s, 3H), 1.34 (s, 3H).

[0198] S27: To a stirred solution of S26 (0.523 g, 2.56 mmol) and N,N-diisopropylethylamine (0.46 mL, 2.64 mmol) in acetonitrile (5 mL) at 0 °C under nitrogen was added S25 (0.300 g, 0.499 mmol). The resulting mixture was warmed to room temperature, stirred for 22 hours, then diluted with EtOAc (50 mL), washed with brine (2×50 mL), dried over Na2SO4, and concentrated by rotary evaporation. The crude residue was carried over directly to the next step without further purification.

[0199] EIDD-2207: The whole of the crude S27 prepared in the previous step was mixed with 80% w / w aqueous formic acid solution (10 mL), and the mixture was stirred at room temperature for 20 hours. The mixture was concentrated by rotary evaporation, and automated flash chromatography (40 g column, 0 - 15% gradient of methanol in dichloromethane) gave the title compound (0.104 g, 48% in two steps) as a yellow foam and as an approximately 1:1 diastereomer mixture with phosphorus: 1 H NMR (400 MHz, CD3OD, diastereomer mixture) δ 7.41 - 7.35 (m, 1H), 7.26 - 7.18 (m, 2H), 7.12 (d, J = 8.3 Hz, 1H), 6.75 (d, J = 8.3 Hz, 0.5x1H), 6.69 (d, J = 8.3 Hz, 0.5x1H), 5.79 (d, J = 4.8 Hz, 0.5x1H), 5.75 (d, J = 4.8 Hz, 0.5x1H), 5.54 - 5.42 (m, 2H), 5.46 (d, J = 8.2 Hz, 0.5x1H), 5.32 (d, J = 8.2 Hz, 0.5x1H), 4.56 - 4.25 (m, 2H), 4.13 - 4.02 (m, 3H); 31 P NMR (162 MHz, CD3OD, diastereomer mixture) δ -9.13, -9.33; C 16 H 18 N3O9PNa [M+Na] + Calculated HRMS value for: 450.06729; Measured value: 450.06777.

[0200] Example 24.

Chemical Structure

[0201] Example 25. [Chem.] To a sealable pressure tube were added uridine (1.00 g, 4.09 mmol), K2CO3 (0.679 g, 4.91 mmol), and deuterium oxide (8.2 mL). The mixture was purged with nitrogen for 15 minutes, the tube was sealed, and the contents were heated at 95 °C for 16 hours with stirring. The mixture was cooled to room temperature, the tube was opened, and the mixture was transferred to a round-bottom flask and concentrated by rotary evaporation. The resulting crude product was co-evaporated with MeOH (×3) to remove water. NMR analysis showed greater than 95% deuterium incorporation at the 5-position of the nucleobase. The pale brown solid S28 (1.00 g, 100%) was used in the next step without further purification: 1 1H NMR (400 MHz, CD3OD) δ 7.76 (s, 1H), 5.88 (d, J = 4.2 Hz, 1H), 4.17 - 4.12 (m, 2H), 4.00 - 3.96 (m, 1H), 3.84 (dd, J = 12.3 Hz, 2.8 Hz, 1H), 3.72 (dd, J = 12.3 Hz, 3.5 Hz, 1H); 13 13C NMR (100 MHz, CD3OD) δ 185.6, 177.4, 160.4, 141.1, 91.8, 85.8, 75.9, 71.2, 62.4.

[0202] S29: To a round-bottom flask were added S28 (1.00 g, 4.09 mmol) and dichloromethane (8 mL) under nitrogen. The resulting mixture was cooled to 0 °C, and 4-DMAP (0.050 g, 0.408 mmol) and imidazole (1.11 g, 16.3 mmol) were added all at once. TBSCl (2.15 g, 14.3 mmol) was added as a solid all at once, the mixture was warmed to ambient temperature, and stirred for 16 hours. Water (25 mL) was added to the reaction mixture, the layers were separated, and the aqueous layer was extracted with dichloromethane (2 × 25 mL). The combined organic layers were washed with brine (1 × 25 mL), dried over Na2SO4, filtered, and concentrated by rotary evaporation. Automated flash chromatography (40 g column, 0 - 35% gradient of EtOAc in hexane) gave S29 (2.52 g, 84%) as an off-white foam: 11H NMR (400 MHz, CDCl3) δ 8.08 (br s, 1H), 8.03 (s, 1H), 5.89 (d, J = 3.6 Hz, 1H), 4.12 - 4.06 (m, 3H), 3.99 (dd, J = 11.5 Hz, 1.8 Hz, 1H), 3.76 (d, J = 12.0 Hz, 1H), 0.96 (s, 9H), 0.92 (s, 9H), 0.90 (s, 9H), 0.14 (s, 3H), 0.13 (s, 3H), 0.10 (s, 3H), 0.09 (s, 3H), 0.08 (s, 3H), 0.07 (s, 3H); 13 13C NMR (100 MHz, CDCl3) δ 163.7, 150.3, 140.3, 89.0, 84.3, 76.1, 70.5, 61.6, 26.0 (3C), 25.8 (3C), 25.7 (3C), 18.4, 18.3, 17.9, -4.2, -4.6, -4.8, -4.9, -5.4, -5.6; C 27 H 54 DN2NaO6Si [M + Na] + Calculated HRMS for: 610.32446, Found: 610.32482.

[0203] S30: To a stirred solution of S29 (0.840 g, 1.43 mmol) in acetonitrile (14.3 mL) at 0 °C under nitrogen was added sequentially p-toluenesulfonyl chloride (0.545 g, 2.86 mmol), 4-DMAP (0.175 g, 1.43 mmol), and triethylamine (0.80 mL, 5.71 mmol). The mixture was stirred at 0 °C for 2.5 h, at which point hydroxylamine hydrochloride (0.993 g, 14.3 mmol) was added as a solid in one portion. The mixture was heated at 50 °C for 3 days and then cooled to room temperature. The reaction mixture was diluted with EtOAc (100 mL) and then washed with water (2 × 100 mL) and brine (1 × 100 mL), dried over Na2SO4, filtered, and concentrated by rotary evaporation. Automated flash chromatography (40 g column, 5 - 35% gradient of EtOAc in hexanes) gave a mixture of starting material and the desired product. A second automated flash chromatography (24 g column, 10 - 40% gradient of EtOAc in hexanes) gave S30 (0.332 g, 39%) as an off-white foam: 11H NMR (400 MHz, CDCl3) δ 8.37 (br s, 1H), 5.92 (d, J = 4.6 Hz, 1H), 4.10 - 4.05 (m, 2H), 4.04 - 4.00 (m, 1H), 3.91 (dd, J = 11.6 Hz, 2.4 Hz, 1H), 3.73 (dd, J = 11.6 Hz, 1.8 Hz, 1H), 0.95 (s, 9H), 0.92 (s, 9H), 0.89 (s, 9H), 0.12 (s, 6H), 0.10 (s, 3H), 0.08 (s, 3H), 0.06 (s, 3H), 0.05 (s, 3H).

[0204] EIDD - 2261: In a round - bottom flask, S30 (0.332 g, 0.551 mmol), tetramethylammonium fluoride (0.196 g, 2.64 mmol), THF (8.25 mL), and DMF (2.75 mL) were placed at 0 °C under nitrogen. Acetic acid (0.157 mL, 2.75 mmol) was added all at once by syringe. The mixture was warmed to 45 °C and heated with stirring for 4 days, then concentrated by rotary evaporation. Automated flash chromatography (40 g column, 0 - 20% gradient of MeOH in DCM) gave the title compound (0.106 g, 74%) as a white solid. Final NMR analysis showed greater than 95% deuterium incorporation at the 5 - position of the nucleobase: 1 1H NMR (400 MHz, D2O) δ 7.16 (s, 1H), 5.85 (d, J = 5.6 Hz, 1H), 4.14 (t, J = 5.5 Hz, 1H), 4.10 (dd, J = 5.6 Hz, 3.8 Hz, 1H), 3.93 (q, J = 3.4 Hz, 1H), 3.77 (dd, J = 12.2 Hz, 2.9 Hz, 1H), 3.68 (dd, J = 12.2 Hz, 3.4 Hz, 1H); 13 13C NMR (100 MHz, CD3OD) δ 151.8, 146.3, 132.1, 89.7, 86.1, 74.6, 71.8, 62.8; C9H 13 DN3O 6[ M + H] + HRMS calculated for [M + H]: 261.09399, found: 261.09371.

[0205] Example 26.

Chemical Structure

[0206] S32: To a stirred solution of S31 (2.61 g, 7.37 mmol) in EtOD (75 mL) at room temperature under nitrogen, NaBD4 (1.234 g, 29.5 mmol) was added in one portion. The mixture was stirred at room temperature for 1 hour, heated at 55 °C for 6 hours, and then left at room temperature overnight. The mixture was cooled to 0 °C and the excess reagent was quenched with AcOD. Concentration of the mixture by rotary evaporation gave crude S32 (2.57 g), which was carried directly on to the next step without further purification.

[0207] S33: To a stirred suspension of crude S32 (2.00 g of impure material, ca. 5.74 mmol) in dichloromethane (70 mL) at 0 °C was added solid imidazole (1.90 g, 27.9 mmol) and 4-DMAP (0.171 g, 1.40 mmol). Solid t-butyldimethylsilyl chloride (2.11 g, 14.0 mmol) was added and the mixture was warmed to room temperature and stirred for 4 days. The mixture was washed successively with water and brine (1×70 mL each), dried over Na2SO4, filtered and concentrated by rotary evaporation. Automated flash chromatography (120 g column, 0 - 35% gradient of EtOAc in hexane) gave S33 (1.42 g, 66% over 2 steps) as a white solid: 1 H NMR (400 MHz, CDCl3) δ 8.30 (br s, 1H), 7.72 (m, 1H), 5.99 (d, J = 2.8 Hz, 1H), 5.69 (dd, J = 8.2 Hz, 2.3 Hz, 1H), 4.77 (dd, J = 6.1 Hz, 2.9 Hz, 1H), 4.69 (dd, J = 6.2 Hz, 2.8 Hz, 1H), 4.33 (d, J = 3.0 Hz, 1H), 1.60 (s, 3H), 1.37 (s, 3H), 0.91 (s, 9H), 0.11 (s, 3), 0.10 (s, 3H); 13 C NMR (100 MHz, CDCl3) δ 162.7, 149.9, 140.5, 114.1, 102.1, 91.9, 86.5, 85.4, 80.3, 27.4, 25.9 (3C), 25.4, 18.4, -5.4, -5.5; C 18 H 29 D2N2O6Si [M + H] + HRMS calcd for: 401.20714, found: 401.20663.

[0208] S34: To a stirred solution of S33 (1.42 g, 3.55 mmol) in acetonitrile (35 mL) at 0 °C under nitrogen was added p-toluenesulfonyl chloride (1.35 g, 7.09 mmol), 4-DMAP (0.433 g, 3.55 mmol), and triethylamine (9.88 mL, 70.9 mmol) successively. The resulting mixture was stirred at 0 °C for 2.5 h. Hydroxylamine hydrochloride (2.46 g, 35.5 mmol) was added and the mixture was stirred at 50 °C for 2 days with stirring. The mixture was re-cooled to room temperature, diluted with EtOAc (100 mL), then washed with water (2 × 50 mL) and brine (1 × 50 mL), dried over Na2SO4, filtered, and concentrated by rotary evaporation. Automated flash chromatography (120 g column, 1 - 3.5% gradient of methanol in dichloromethane) gave S34 (0.416 g, 28%) as an off-white solid: 1 H NMR (400 MHz, CDCl3) δ 8.36 (br s, 1H), 7.00 (m, 1H), 5.97 (d, J = 3.1 Hz, 1H), 5.58 (d, J = 8.2 Hz, 1H), 4.77 (dd, J = 6.2 Hz, 3.2 Hz, 1H), 4.68 (dd, J = 6.3 Hz, 3.2 Hz, 1H), 4.22 (d, J = 3.2 Hz, 1H), 1.59 (s, 3H), 1.36 (s, 3H), 0.92 (s, 9H), 0.11 (s, 3H), 0.10 (s, 3H); 13 C NMR (100 MHz, CDCl3) δ 149.0, 145.4, 131.4, 114.1, 98.3, 90.8, 85.5, 84.5, 80.2, 27.4, 25.9(3C), 25.5, 18.4, -5.4, -5.5; C 18 H 29 D2N3O6Si [M + H] + HRMS calculated for: 416.21804, found: 416.21827.

[0209] S35: To a stirred solution of S34 (0.416 g, 1.00 mmol) in THF (5 mL) at 0 °C under nitrogen was added a 1.0 M THF solution of TBAF (1.50 mL, 1.5 mmol), and the resulting mixture was kept at 0 °C for 24 h. The reaction mixture was concentrated by rotary evaporation, and automated flash chromatography (40 g column, 0–8% gradient of methanol in dichloromethane) gave S35 (0.257 g, 85%) as a white solid: 1 H NMR (400 MHz, CD3OD) δ 7.02 (m, 1H), 5.81 (d, J = 3.2 Hz, 1H), 5.58 (d, J = 8.2 Hz, 1H), 4.86 (dd, J = 6.4 Hz, 3.2 Hz, 1H), 4.79 (dd, J = 6.5 Hz, 3.6 Hz, 1H), 4.09 (d, J = 3.7 Hz, 1H), 1.54 (s, 3H), 1.34 (s, 3H); 13 C NMR (100 MHz, CD3OD) δ 151.3, 146.2, 133.4, 115.2, 99.4, 92.9, 87.2, 84.9, 82.1, 27.6, 25.6; C 12 H 16 D2N3O6 [M + H] + HRMS calculated for: 302.13157, found: 302.13130.

[0210] EIDD-2345: To a stirred solution of S35 (0.140 g, 0.465 mmol) in methanol (8.4 mL) and water (0.93 mL) at room temperature was added Dowex 50WX8 in the hydrogen form (0.30 g), and the mixture was stirred at room temperature for 24 h. The reaction mixture was filtered, and the filtrate was concentrated by rotary evaporation. Automated flash chromatography (40 g column, 5–20% gradient of methanol in dichloromethane) gave the title compound (0.050 g, 41%) as an off-white solid: 1 H NMR (400 MHz, CD3OD) δ 7.17 (m, 1H), 5.86 (d, J = 5.6 Hz, 1H), 5.60 (d, J = 8.2 Hz, 1H), 4.15 (t, J = 5.5 Hz, 1H), 4.11 (dd, J = 5.6 Hz, 3.5 Hz, 1H), 3.94 (d, J = 3.8 Hz, 1H); 1313C NMR (100 MHz, CD3OD) δ 151.8, 146.3, 132.2, 99.3, 89.7, 86.0, 74.6, 71.7, C9H 10 D2N3O6 [M+H] + HRMS calculated value for: 260.08571, measured value: 260.08578.

[0211] Example 27.

Chemical Structure

[0212] EIDD - 2356: To a stirred solution of S36 (0.120 g, 0.138 mmol) in THF (2.75 mL) at 0 °C under nitrogen was added a 1 M THF solution of TBAF (0.483 mL, 0.483 mmol). The solution was stirred at 0 °C for 5 h and then concentrated by rotary evaporation. Automated flash chromatography (12 g column, 0 - 10% gradient of MeOH in dichloromethane) gave the title compound (0.055 g, 76%) as an off - white solid: 1 1H NMR (400 MHz, CDCl3 containing 1 drop of CD3OD) δ 7.26 (d, J = 8.2 Hz, 1H), 5.62 (d, J = 4.4 Hz, 1H), 5.55 (d, J = 8.2 Hz, 1H), 4.14 - 4.06 (m, 2H), 3.96 - 3.92 (m, 1H), 3.82 - 3.76 (m, 1H), 3.65 (m, 1H, covered by MeOH - d4), 3.15 (t, 7.0 Hz, 2H), 1.56 (m, 2H), 1.30 - 1.11 (br s, 28H), 0.79 (t, J = 6.9 Hz, 3H); C 26 H 47 N4O7[M + H] + HRMS calculated value for: 527.34393, measured value: 527.34396.

[0213] Example 28.

Chemical Structure

[0214] EIDD-2357: To a stirred solution of S37 (0.128 g, 0.143 mmol) in THF (2.85 mL) at 0 °C under nitrogen was added a 1 M solution of TBAF in THF (0.499 mL, 0.499 mmol). The solution was stirred at 0 °C for 5 h and then concentrated by rotary evaporation. Automated flash chromatography (12 g column, 0 - 10% gradient of MeOH in dichloromethane) gave the title compound (0.059 g, 74%) as an off-white solid: 1 H NMR (400 MHz, CDCl3) δ 10.70 (br s, 1H), 7.47 (d, J = 8.2 Hz, 1H), 6.56 (t, J = 6.2 Hz, 1H), 5.76 (s, 1H), 5.60 (d, J = 8.2 Hz, 1H), 4.32 - 4.20 (br m, 2H), 4.12 - 4.02 (br m, 2H), 3.90 (d, J = 11.7 Hz, 1H), 1.56 (m, 2H), 1.26 (br s, 30H), 0.89 (t, J = 7.0 Hz, 3H); C 28 H 51 N4O7 [M + H] + HRMS calculated for: 555.37523, found: 555.37531.

[0215] Example 29.

Chemical formula

[0216] EIDD-2422: To a stirred solution of S38 (0.330 g, 0.500 mmol) in THF (3.75 mL) and DMF (1.25 mL) at 0 °C was added acetic acid (0.143 mL, 2.50 mmol), followed by tetraethylammonium fluoride (0.359 g, 2.40 mmol) added in one portion. The mixture was warmed to ambient temperature and stirred for 24 h. The mixture was concentrated by rotary evaporation and the crude material was taken up in dichloromethane. Automated flash chromatography (12 g column, 1 - 25% gradient of MeOH in dichloromethane) gave 80 mg of a somewhat pure material. This material was taken up in water and automated reverse-phase flash chromatography (30 g column, 0 - 100% gradient of acetonitrile in water) gave the desired product free of impurities. The solid was dissolved in water, frozen in a dry ice / acetone bath and lyophilized to give the title compound (0.057 g, 36% yield) as a white fluffy solid. NMR analysis showed signals in a ratio of 13:1 in D2O and 8:1 in MeOH-d4, indicating the ratio of solvent-dependent rotamers of a single pure compound: 1 H NMR (400 MHz, CD3OD, major rotamer) δ 7.45 (d, J = 8.2 Hz, 1H), 5.86 (d, J = 5.1 Hz, 1H), 5.69 (d, J = 8.2 Hz, 1H), 4.16 - 4.08 (m, 2H), 3.96 (q, J = 3.2 Hz, 1H), 3.79 (dd, J = 12.2 Hz, 2.8 Hz, 1H), 3.69 (dd, J = 12.2 Hz, 3.3 Hz, 1H), 2.79 (s, 3H); 1 H NMR (400 MHz, D2O, major rotamer) δ 7.27 (d, J = 8.2 Hz, 1H), 5.84 (d, J = 5.4 Hz, 1H), 5.80 (d, J = 8.2 Hz, 1H), 4.28 (t, J = 5.2 Hz, 1H), 4.17 (t, J = 5.2 Hz, 1H), 4.05 (q, J = 4.2 Hz, 1H), 3.82 (dd, J = 12.8 Hz, 3.1 Hz, 1H), 3.73 (dd, J = 12.8 Hz, 4.6 Hz, 1H), 2.76 (s, 3H); 1313C NMR (100 MHz, D2O) δ 157.6, 150.2, 148.8, 134.0, 97.1, 88.4, 84.1, 73.1, 69.7, 61.0, 26.9; LRMS m / z 315.1 [M-H] - .

[0217] Example 30.

Chemical Structure

[0218] EIDD-2423: To a stirred solution of S39 (1.16 g, 1.72 mmol) in THF (12.9 mL) and DMF (4.3 mL) at 0 °C was added acetic acid (0.493 mL, 8.62 mmol), followed by the addition of tetraethylammonium fluoride (1.24 g, 8.27 mmol) all at once. The mixture was warmed to ambient temperature and stirred for 16 h. The mixture was concentrated by rotary evaporation and the crude was taken up in dichloromethane. Automated flash chromatography (80 g column, 1 - 15% gradient of MeOH in dichloromethane) gave 400 mg of a somewhat pure material. This material was taken up in water and automated reverse-phase flash chromatography (100 g column, 0 - 100% gradient of acetonitrile in water) gave the desired product free of impurities. The solid was dissolved in water, frozen in a dry ice / acetone bath, and lyophilized to give the title compound (0.200 g, 35% yield) as a white fluffy solid. NMR analysis showed a 9:1 ratio of signals in D2O and a 5:1 ratio in MeOH-d4, indicating the ratio of solvent-dependent rotational isomers of a single pure compound: 1 H NMR (400 MHz, CD3OD, major rotational isomer) δ 7.46 (d, J = 8.3 Hz, 1H), 5.85 (d, J = 4.8 Hz, 1H), 5.72 (d, J = 8.2 Hz, 1H), 4.18 - 4.11 (m, 2H), 3.97 (q, J = 3.5 Hz, 1H), 3.80 (dd, J = 12.1 Hz, 2.8 Hz, 1H), 3.70 (dd, J = 12.2 Hz, 3.2 Hz, 1H), 3.05 (br s, 3H), 2.98 (br s, 3H); 1 H NMR (400 MHz, D2O, major rotational isomer) δ 7.27 (d, J = 8.3 Hz, 1H), 5.84 (d, J = 5.4 Hz, 1H), 5.80 (d, J = 8.3 Hz, 1H), 4.28 (t, J = 5.4 Hz, 1H), 4.17 (d, J = 5.2 Hz, 1H), 4.05 (q, J = 4.3 Hz, 1H), 3.82 (dd, J = 12.7 Hz, 3.2 Hz, 1H), 3.73 (dd, J = 12.7 Hz, 4.5 Hz, 1H), 2.99 (br s, 3H), 2.91 (br s, 3H); 1313C NMR (100 MHz, D2O) δ 156.2, 150.1, 149.4, 133.9, 97.2, 88.3, 84.1, 73.0, 69.7, 61.0, 36.5, 35.7; LRMS m / z 329.0 [M-H] - .

[0219] Example 31.

Chemical Structure

[0220] EIDD-2474: The whole of the crude product S40 prepared as described above was stirred with formic acid (10 mL) at room temperature for 12 hours. The solvent was removed by rotary evaporation and the crude product was purified by flash column chromatography using methanol and dichloromethane to give the title compound (0.140 g, 42% in two steps) as a colorless solid: 11H NMR (400 MHz, DMSO-d6) δ 10.05 (s, 1H), 9.61 (s, 1H), 6.85 (d, J = 8.1 Hz, 1H), 5.75 (d, J = 5.8 Hz, 1H), 5.57 (d, J = 8.1 Hz, 1H), 5.42 (d, J = 5.8 Hz, 1H), 5.30 (d, J = 5.0 Hz, 1H), 4.31 (dd, J = 11.7 Hz, 3.2 Hz, 1H), 4.20 (dd, J = 11.8 Hz, 5.4 Hz, 1H), 4.14 - 4.08 (m, 1H), 4.02 (q, J = 5.7 Hz, 1H), 3.97 - 3.90 (m, 2H), 3.10 (m, 1H), 1.61 - 1.18 (m, 10H), 0.90 - 0.86 (m, 3H); 13 13C NMR (100 MHz, DMSO-d6) δ 154.9, 149.9, 143.6, 130.3, 99.2, 87.9, 81.0, 72.1, 70.4, 68.2, 67.8, 45.9, 31.6, 28.5, 25.6, 22.5, 14.4; LRMS m / z 402.1 [M + H] + .

[0221] Example 32. [Chemical formula] S41: A solution of S25 (0.40 g, 0.66 mmol) in anhydrous dichloromethane (5 mL) in a 50 mL round-bottom flask was cooled to 0 °C under nitrogen by an ice bath, treated with pyridine (0.10 mL, 1.33 mmol) and DMAP (0.080 g, 0.66 mmol), followed by the addition of heptyl isocyanate (0.16 mL, 0.99 mmol), and stirred at 40 °C for 12 hours. After completion of the reaction, the reaction mixture was diluted with dichloromethane (25 mL) and washed with 5% hydrochloric acid (25 mL) and aqueous sodium hydrogen carbonate solution (25 mL). The organic layer was dried over Na2SO4 and concentrated by rotary evaporation to give crude S41. The crude product was carried over directly to the next step without further purification.

[0222] EIDD-2475: The whole of the crude S41 prepared as described above was stirred with formic acid (10 mL) at room temperature for 12 hours. The solvent was removed by rotary evaporation, and the crude product was purified by flash column chromatography using methanol and dichloromethane, giving the title compound (0.150 g, 56% in two steps) as a colorless solid: 1 H NMR (400 MHz, DMSO-d6) δ 9.98 (s, 1H), 9.53 (s, 1H), 7.26 (t, J = 5.5 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 5.71 (d, J = 6.3 Hz, 1H), 5.52 (d, J = 8.2 Hz, 1H), 4.19 - 3.77 (m, 5H), 2.94 (q, J = 6.2 Hz, 2H), 1.48 - 1.10 (m, 10H), 0.83 (t, J = 6.6 Hz, 3H); 13 C NMR (101 MHz, DMSO-d6) δ 156.3, 150.0, 143.7, 130.4, 99.1, 87.4, 81.9, 72.1, 70.6, 64.2, 31.7, 29.9, 28.9, 26.6, 22.5, 14.4; LRMS m / z 401.1 [M + H] + .

[0223] Example 33.

Chemical Structure

[0224] EIDD-2476: The whole of the crude S42 prepared above was stirred with formic acid (5 mL) at room temperature for 12 h. The solvent was removed by rotary evaporation and the crude product was purified by flash column chromatography using methanol and dichloromethane to give the title compound (0.080 g, 54% over 2 steps) as a colorless solid: 1 H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 9.54 (s, 1H), 6.81 (d, J = 8.3 Hz, 1H), 5.69 (d, J = 5.6 Hz, 1H) (dd, J = 8.2 Hz, 1.8 Hz, 1H), 5.35 (d, J = 5.8 Hz, 1H), 5.22 (d, J = 5.1 Hz, 1H), 4.25 - 4.02 (m, 2H), 4.03 - 3.78 (m, 3H), 2.35 - 2.20 (m, 2H), 1.58 - 1.42 (m, 2H), 1.22 (m, 10H), 0.83 (t, J = 3.3 Hz, 3H); 13 C NMR (100 MHz, DMSO-d6) δ 173.2, 149.9, 143.7, 130.3, 99.2, 88.0, 81.1, 72.3, 70.4, 64.3, 33.8, 31.7, 29.1, 29.0, 28.9, 24.9, 22.5, 14.4; LRMS m / z 400.2 [M + H] + .

[0225] Example 34.

Chemical Structure

[0226] S44: An aqueous solution (solution volume of 36.4 mL) of hydroxylamine hydrochloride (12.7 g, 182 mmol) at about 5 N was prepared and adjusted to pH = 6 with a small amount of aqueous NaOH solution (10% w / w). This solution, S43 (3.82 g, 9.11 mmol), and THF (18 mL) were placed in a sealable pressure tube, the flask was sealed, and the mixture was heated at 37 °C for 5 days while stirring. The mixture was cooled to room temperature, transferred to a round-bottom flask, and concentrated by rotary evaporation. The crude material was absorbed into methanol and immobilized on celite. Automated flash chromatography (80 g column, 0 - 10% gradient of MeOH in dichloromethane) gave S44 (2.28 g, 58%) as a white solid in flakes: 1 1H NMR (400 MHz, CDCl3) δ 8.58 (br s, 1H), 7.72 (br s, 1H), 6.68 (d, J = 8.2 Hz, 1H), 5.69 (d, J = 2.5 Hz, 1H), 5.63 (dd, J = 7.8 Hz, 1.1 Hz, 1H), 4.93 (dd, J = 6.4 Hz, 2.4 Hz, 1H), 4.85 (dd, J = 6.5 Hz, 3.6 Hz, 1H), 4.30 - 4.20 (m, 3H), 4.20 - 4.10 (m, 5H), 1.57 (s, 3H), 1.35 (s, 3H), 1.35 (tdd, J = 7.0 Hz, 4.1 Hz, 1.0 Hz, 6H); 31P NMR (162 MHz, CDCl3) δ -1.09; LRMS m / z 436.1 [M+H] + .

[0227] EIDD-2503: A solution of S44 (0.25 g, 0.57 mmol) was stirred with formic acid (5 mL) at room temperature for 12 hours under nitrogen. After completion of the reaction, the solvent was removed by rotary evaporation, and the crude product was purified by flash column chromatography using methanol and dichloromethane, giving the title compound (0.180 g, 79%) as a colorless solid: 1 H NMR (400 MHz, DMSO-d6) δ 10.00 (s, 1H), 9.57 (s, 1H), 6.83 (d, J = 8.2 Hz, 1H), 5.71 (d, J = 5.9 Hz, 1H), 5.54 (dd, J = 8.2 Hz, 2.0 Hz, 1H), 5.38 (d, J = 5.8 Hz, 1H), 5.24 (d, J = 4.7 Hz, 1H), 4.16 - 3.86 (m, 8H), 1.30 - 1.15 (m, 5H). 13 C NMR (100 MHz, DMSO-d6) δ 149.9, 143.7, 130.3, 110.0, 99.1, 87.8, 82.0, 72.1, 70.2, 67.2, 63.9, 16.4; 31 P NMR (162 MHz, DMSO-d6) δ -1.12; LRMS m / z 396.1 [M+H] + .

[0228] Example 35.

Chemical Structure

[0229] S46: Lithium bromide (1.44 g, 16.56 mmol) was added to a solution of S45 (3.00 g, 8.28 mmol) in anhydrous tetrahydrofuran (60 mL) at room temperature under nitrogen, and the reaction mixture was refluxed for 6 hours. After completion of the reaction, it was concentrated by rotary evaporation, and the crude product was partitioned between dichloromethane (60 mL) and water (60 mL). The aqueous layer was removed, the organic layer was washed with brine (60 mL), dried over Na2SO4, and concentrated by rotary evaporation. Purification of the crude product by flash column chromatography using ethyl acetate and hexane gave S45 (2.30 g, 80%) as a colorless solid: 11H NMR (400 MHz, CDCl3) δ 9.24 (s, 1H), 7.34 (d, J = 8.2 Hz, 1H), 5.76 (d, J = 8.2 Hz, 1H), 5.66 (d, J = 2.2 Hz, 1H), 5.01 (dd, J = 6.5 Hz, 2.3 Hz, 1H), 4.88 (dd, J = 6.5 Hz, 3.7 Hz, 1H), 4.38 (td, J = 5.7 Hz, 3.8 Hz, 1H), 3.68 (dd, J = 10.6 Hz, 6.2 Hz, 1H), 3.56 (dd, J = 10.6 Hz, 5.2 Hz, 1H), 1.57 (s, 3H), 1.36 (s, 3H); LRMS m / z 348.9 [M+H] + .

[0230] S47: To a suspension of S46 (2.0 g, 5.76 mmol) in anhydrous toluene (40 mL) at room temperature under nitrogen was added ethanol (5 mL), followed by tributyltin hydride (3.11 mL, 11.52 mmol) and AIBN (0.94 g, 5.76 mmol). The reaction mixture was refluxed for 6 h. After completion of the reaction, the solvent was removed under reduced pressure, and the crude product was dissolved in dichloromethane (50 mL) and vacuum filtered through a glass frit. The filtrate was concentrated by rotary evaporation and the crude product was purified by flash column chromatography using ethyl acetate and hexane to give S47 (1.10 g, 71%) as a colorless foam: 1 1H NMR (400 MHz, CDCl3) δ 9.81 (s, 1H), 7.26 (d, J = 8.0 Hz, 1H), 5.73 (d, J = 8.0 Hz, 1H), 5.62 (d, J = 2.2 Hz, 1H), 4.94 (dd, J = 6.5 Hz, 2.2 Hz, 1H), 4.54 (dd, J = 6.5 Hz, 4.6 Hz, 1H), 4.19 (qd, J = 6.4 Hz, 4.7 Hz, 1H), 1.54 (s, 3H), 1.37 (d, J = 6.5 Hz, 3H), 1.32 (s, 3H). LRMS m / z 269.1 [M+H] + .

[0231] A solution of S48:S47 (1.00 g, 3.73 mmol) in anhydrous dichloromethane (30 mL) was cooled to 0 °C with stirring under nitrogen. To this solution were added N,N-diisopropylethylamine (3.25 mL, 18.64 mmol) and 4-DMAP (46 mg, 0.37 mmol), followed by 2,4,6-triisopropylbenzenesulfonyl chloride (1.69 g, 5.59 mmol). After disappearance of the starting material, hydroxylamine hydrochloride (0.648 g, 9.32 mmol) was added and the mixture was stirred at room temperature for an additional 12 h. After completion of the reaction, the reaction mixture was diluted with dichloromethane (70 mL), washed with 5% hydrochloric acid (100 mL), followed by aqueous sodium bicarbonate (100 mL) and brine (100 mL). The organic layer was dried over Na2SO4, filtered, and concentrated by rotary evaporation. Purification of the crude product by flash column chromatography using ethyl acetate and hexane gave S48 (0.59 g, 55.9%) as a colorless solid: 1 H NMR (400 MHz, DMSO-d6) δ 10.03 (s, 1H), 9.62 (d, J = 1.8 Hz, 1H), 6.85 (d, J = 8.2 Hz, 1H), 5.66 (d, J = 2.8 Hz, 1H), 5.55 (dd, J = 8.1 Hz, 2.1 Hz, 1H), 4.86 (dd, J = 6.6 Hz, 2.8 Hz, 1H), 4.47 (dd, J = 6.5 Hz, 4.9 Hz, 1H), 3.97 - 3.84 (m, 1H), 1.44 (s, 3H), 1.30 - 1.15 (m, 5H); LRMS m / z 284.1 [M+H] + .

[0232] A solution of EIDD-2524:S48 (0.250 g, 0.88 mmol) was stirred in formic acid (5 mL) at room temperature for 12 h. After completion of the reaction, the mixture was concentrated by rotary evaporation and the crude product was purified by flash column chromatography using methanol and dichloromethane to give the title compound (0.150 g, 70%) as a colorless solid: 11H NMR (400 MHz, DMSO-d6) δ 9.94 (s, 1H), 9.46 (s, 1H), 6.75 (d, J = 8.2 Hz, 1H), 5.59 (d, J = 5.1 Hz, 1H), 5.51 (d, J = 8.2 Hz, 1H), 5.20 (s, 1H), 4.98 (s, 1H), 3.94 (s, 1H), 3.78 - 3.65 (m, 1H), 3.59 (dd, J = 5.5 Hz, 3.9 Hz, 1H), 1.17 (d, J = 6.4 Hz, 3H). 13 13C NMR (100 MHz, DMSO-d6) δ 149.9, 143.8, 130.8, 99.1, 88.5, 79.0, 74.8, 72.5, 19.3; LRMS m / z 244.1 [M+H] + .

[0233] Example 36. Assay Protocol (1) Screening assay for DENV, JEV, POWV, WNV, YFV, PTV, RVFV, CHIKV, EEEV, VEEV, WEEV, TCRV, PCV, JUNV, MPRLV Primary cytopathic effect (CPE) reduction assay. Conduct CPE inhibition assays at four concentrations. Prepare a confluent or nearly confluent cell culture monolayer in a 96-well disposable microplate. Maintain the cells in MEM or DMEM supplemented with FBS as required for each cell line. Use the same medium for the antiviral assay, but reduce the FBS to 2% or less and supplement with 50 μg / ml gentamicin. Prepare the test compound at four log 10 final concentrations, usually 0.1, 1.0, 10, and 100 μg / ml or μM. Virus control and cell control wells are present in all microplates. In parallel, test a known active agent as a positive control using the same method as applied to the test compound. Test the positive control with each run of the assay. Prepare the assay by first removing the growth medium from the cells in the 96-well plate. Then apply the test compound to the wells at 2× concentration in a volume of 0.1 ml. The virus is usually at a volume of 0.1 ml and a 50% cell culture infectious dose (CCID 50) However, it is placed in the wells designated for virus infection. A medium without virus is placed in the toxicity control wells and the cell control wells. The virus control wells are similarly treated with the virus. The plate is incubated at 37 °C and 5% CO2 until maximum CPE is observed in the virus control wells. The plate is then stained with 0.011% neutral red for approximately 2 hours at 37 °C in a 5% CO2 incubator. The neutral red medium is removed by complete aspiration, and the cells may be rinsed once with phosphate-buffered saline (PBS) to remove residual dye. After completely removing the PBS, the incorporated neutral red is eluted with 50% Zensen's citrate buffer / 50% ethanol (pH 4.2) for at least 30 minutes. The neutral red dye penetrates living cells, so the stronger the red color, the greater the number of living cells present in the well. The dye content in each well is quantified using a 96-well spectrophotometer at a wavelength of 540 nm. Using a Microsoft Excel computer-based spreadsheet, the dye content in each set of wells is converted to a percentage relative to the dye present in the untreated control wells. Then, the 50% effective (EC 50 , virus inhibitory) concentration and the 50% cytotoxic (CC 50 , cell inhibitory) concentration are calculated by linear regression analysis. The quotient of CC 50 divided by EC 50 gives the selectivity index (SI) value.

[0234] Secondary CPE / virus yield reduction (VYR) assay. This assay involves a method similar to that described in the previous paragraph using a 96-well microplate of cells. Differences are described in this section. Eight semi-log 10The concentration inhibitor is tested for antiviral activity and cytotoxicity. After sufficient viral replication has occurred, supernatant samples are taken from each infected well (storing three replicate wells), and if necessary, saved for the VYR portion of this test. Alternatively, separate plates may be prepared and frozen for VYR assay. After maximum CPE is observed, viable plates are stained with neutral red dye. Quantify the incorporated dye content as described above. The data resulting from this part of the test are the neutral red EC 50 , CC 50 , and SI values. Compounds observed to be active above are further evaluated by VYR assay. The VYR test directly determines how much the test compound inhibits viral replication. Titrate the virus replicated in the presence of the test compound and compare it to the virus from an untreated infected control. Titrate the saved virus sample (collected as described above) by the end-point dilution method. This is achieved by titrating the log 10 dilutions of the virus by end-point dilution using 3 or 4 microwells per dilution on a fresh monolayer of cells. After individual CPE (measured by neutral red uptake) is measured, the wells are scored for the presence or absence of virus. Plotting the log 10 of the inhibitor concentration against the log 10 of the virus generated at each concentration allows calculation of the 90% (1 log 10 ) effective concentration by linear regression. Dividing the EC 90 obtained in part 1 of the assay by the CC 50 gives the SI value for this test.

[0235] Example 37. (2) Screening Assay for Lassa Fever Virus (LASV) Primary Lassa fever virus assay. In a 12-well disposable cell culture plate, prepare a confluent or nearly confluent cell culture monolayer. Maintain the cells in DMEM supplemented with 10% FBS. For the antiviral assay, use the same medium but reduce the FBS to 2% or less and supplement with 1% penicillin / streptomycin. Test the test compound at four log 10 final concentrations, usually 0.1, 1.0, 10, and 100 μg / ml or μM. Perform virus and cell controls in parallel with each test compound. Additionally, test a known active agent as a positive control using the same experimental protocol described for the virus and cell controls. Test the positive control with each run of the assay. Prepare the assay by removing the growth medium from the cells in the 12-well plate and infecting the cells with 0.01 MOI of the LASV strain Josiah. Incubate the cells at 90 min: 500 μl inoculum / M12 well, 37 °C, 5% CO2, with constant gentle shaking. Remove the inoculum and wash the cells twice with medium. Then apply the test compound to the total volume of 1 ml of medium. Collect the tissue culture supernatant (TCS) at the appropriate time point. Then use the TCS to determine the compound inhibitory effect on virus replication. Titrate the virus replicated in the presence of the test compound and compare it to the virus from untreated, infected controls. For titration of the TCS, prepare 10-fold serial dilutions and use them to infect fresh monolayers of cells. Cover the cells with a 1:1 mixture of 1% agarose and 2×MEM supplemented with 10% FBS and 1% penicillin and determine the number of plaques. Plot the log 10 of the inhibitor concentration against the log 10 of the virus produced at each concentration. A 90% (1 log 10 ) effective concentration can be calculated by linear regression.

[0236] Secondary Lassa fever virus assay. The secondary assay involves a method similar to that described in the previous paragraph using cells in a 12-well plate. The differences are explained in this section. The cells are infected as described above, but this time are diluted 1:1 with 2×MEM, supplemented with 2% FBS and 1% penicillin / streptomycin, and overlaid with 1% agarose supplemented with the corresponding drug concentration. The cells are incubated at 37 °C in 5% CO2 for 6 days. Then, after removing the overlay, the plates are stained with 0.05% crystal violet in 10% buffered formalin for approximately 20 minutes at room temperature. The plates are then washed, dried, and the number of plaques is counted. The number of plaques in each set of compound dilutions is converted to a percentage relative to the untreated virus control. Then, the 50% effective (EC 50 , virus-inhibiting) concentration is calculated by linear regression analysis.

[0237] Example 38. (3) Screening assay for Ebola virus (EBOV) and Nipah virus (NIV) Primary Ebola / Nipah virus assay. A 4-concentration plaque reduction assay is performed. A confluent or nearly confluent cell culture monolayer is prepared in a 12-well disposable cell culture plate. The cells are maintained in DMEM supplemented with 10% FBS. The same medium is used for the antiviral assay, but the FBS is reduced to 2% or less and supplemented with 1% penicillin / streptomycin. The test compound is at 4 logs 10Prepare at final concentrations, usually 0.1, 1.0, 10, and 100 μg / ml or μM. Virus controls and cell controls are performed in parallel with each test compound. Further, a known active agent is tested as a positive control using the same experimental protocol as described for the virus and cell controls. The positive control is tested with each run of the assay. The assay is prepared by removing the growth medium from the cells in a 12-well plate. The test compound is then applied to the wells at twice the concentration in a 0.1 ml volume. The virus is usually approximately 200 plaque-forming units in a 0.1 ml volume and is placed in the wells designated for virus infection. Medium without virus is placed in the toxicity control wells and cell control wells. The virus control wells are treated similarly with virus. The plate is incubated at 37 °C in 5% CO2 for 1 hour. Except for the virus compound inoculum, the cells are washed, diluted 1:1 with 2X MEM, supplemented with 2% FBS and 1% penicillin / streptomycin, and covered with 1.6% tragacanth supplemented with the corresponding drug concentration. The cells are incubated at 37 °C in 5% CO2 for 10 days. Then, except for the cover, the plate is stained with 0.05% crystal violet in 10% buffered formalin for approximately 20 minutes at room temperature. The plate is then washed, dried, and the number of plaques is counted. The number of plaques in each set of compound dilutions is converted to a percentage relative to the untreated virus control. Then, the 50% effective (EC 50 , virus-inhibitory) concentration is calculated by linear regression analysis.

[0238] Secondary Ebola / Nipah virus assay with VYR component. The secondary assay includes methods similar to those described in the previous paragraph using cells in a 12-well plate. The differences are described in this section. Eight semi-log 10An inhibitor of concentration is tested for antiviral assays. One positive control drug per batch of compound to be evaluated is tested. In this assay, cells are infected with a virus. The cells are infected as described above, but this time are incubated with DMEM supplemented with 2% FBS and 1% penicillin / streptomycin and the corresponding drug concentration. The cells are incubated at 37 °C with 5% CO2 for 10 days, and the number of green fluorescent cells is observed daily under a microscope. An aliquot of the supernatant from the infected cells is taken daily, and three replicate assay wells are set aside. The supernatant set aside is then used to determine the inhibitory effect of the compound on virus replication. The virus replicated in the presence of the test compound is titrated and compared with the virus from untreated infected controls. For titration of the virus samples set aside, 10-fold serial dilutions are prepared and used to infect fresh monolayers of cells. The cells are overlaid with tragacanth, and the number of plaques is determined. The log of the inhibitor concentration 10 and the log of the virus generated at each concentration 10 are plotted, and the 90% (1 log 10 ) effective concentration can be calculated by linear regression.

[0239] Example 39. Antidengue virus cell protection assay: Cell preparation - BHK21 cells (Syrian golden hamster kidney cells, ATCC catalog number CCL-I 0), Vero cells (African green monkey kidney cells, ATCC catalog number CCL-81), or Huh-7 cells (human hepatocellular carcinoma) are passaged in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin in T-75 flasks before use in antiviral assays. The day before the assay, the cells are split 1:2 to ensure they are in the exponential growth phase at the time of infection. Total cell and viability quantification was performed using a hemocytometer and trypan blue dye exclusion. The cell viability was greater than 95% for the cells intended for use in the assay. The cells are seeded at 3×10 3 (5×10 for Vero cells and Huh-7 cells 5) The cells were resuspended in tissue culture medium and added to a flat-bottom microtiter plate in a volume of 100 μL. The plate was incubated overnight at 37 °C / 5% CO2 to allow cell adhesion. The monolayer was observed to be approximately 70% confluent.

[0240] Virus preparation - Dengue virus type 2 New Guinea C strain was obtained from ATCC (catalog number VR-1584) and propagated in LLC-MK2 (rhesus monkey kidney cells; catalog number CCL-7.1) cells for the production of a stock virus pool. An aliquot of the virus that had been pre-titered in BHK21 cells was removed from the freezer (-80 °C) and thawed slowly to room temperature in a biosafety cabinet. The virus was resuspended and diluted in assay medium (DMEM supplemented with 2% heat-inactivated FBS, 2 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin) such that the amount of virus added to each well in a volume of 100 μL was determined to result in 85 - 95% cell killing 6 days post-infection.

[0241] Plate format - Each plate included cell control wells (cells only), virus control wells (cells and virus), triple drug toxicity wells per compound (cells and drug only), and triple experimental wells (drug, cells, and virus).

[0242] Efficacy and Toxicity After incubation in a 5% CO2 incubator at 37°C, the test plates were stained with the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide). XTT-tetrazolium is metabolized by the mitochondrial enzymes of metabolically active cells into a soluble formazan product, enabling rapid quantitative analysis of the inhibition of virus-induced cell killing by antiviral test substances. The XTT solution was prepared daily as a 1 mg / mL stock in RPMI 1640. The phenazine methosulfate (PMS) solution was prepared at 0.15 mg / ml in PBS and stored at -20°C in the dark. The XTT / PMS stock was prepared immediately before use by adding 40 μL of PMS per ml of the XTT solution. 50 microliters of XTT / PMS was added to each well of the plate, and the plate was reincubated at 37°C for 4 hours. The plate was sealed with an adhesive plate sealer and gently shaken or inverted several times to mix the soluble formazan product, and the plate was read spectrophotometrically at 450 / 650 nm using a Molecular Devices Vmax plate reader.

[0243] Data Analysis Raw data were collected from Softmax Pro 4.6 software and imported into a Microsoft Excel spreadsheet for analysis. The percent reduction in the cytopathic effect of the virus relative to the untreated virus control was calculated for each compound. The percent cell control values were calculated for each compound by comparing drug-treated uninfected cells to uninfected cells in medium only.

[0244] Example 40. Anti-RSV Cell Protection Assay: Cell Preparation - HEp2 cells (human epithelial cells, ATCC catalog number CCL-23) were passaged in T-75 flasks in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 1 mM sodium pyruvate, and 0.1 mM NEAA before use in the antiviral assay. The day before the assay, the cells were split 1:2 to ensure that they were in the exponential growth phase at the time of infection. Total cell and viability quantification was performed using a hemocytometer and trypan blue dye exclusion. The cell viability was over 95% for the cells to be used in the assay. The cells were resuspended in tissue culture medium at 1×10 4 cells per well and added to flat-bottom microtiter plates in a volume of 100 μL. The plates were incubated overnight at 37°C / 5% CO2 to allow cell attachment. Virus Preparation - RSV strains Long and 9320 were obtained from ATCC (catalog numbers VR-26 and VR-955, respectively) and grown in HEp2 cells for the production of stock virus pools. An aliquot of the virus, whose titer had been previously measured, was removed from the freezer (-80°C) and thawed slowly to room temperature in a safety cabinet. The virus was resuspended and diluted in assay medium (DMEM supplemented with 2% heat-inactivated FBS, 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 1 mM sodium pyruvate, and 0.1 mM NEAA) such that the amount of virus added to each well in a volume of 100 μL was the amount determined to result in 85 - 95% cell killing 6 days post-infection. Efficacy and Toxicity The XTT-plates were stained and analyzed as described previously for the dengue cytoprotection assay.

[0245] Example 41. Anti-influenza virus cytoprotection assay: Cell Preparation - Mock cells (Canine kidney cells, ATCC catalog number CCL - 34) were passaged in T - 75 flasks in DMEM supplemented with 10% FBS, 2 mM L - glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 1 mM sodium pyruvate, and 0.1 mM NEAA before being used in the antiviral assay. One day before the assay, the cells were split 1:2 to ensure that they were in the exponential growth phase at the time of infection. Total cell and viability quantification was performed using a hemocytometer and trypan blue dye exclusion. The cell viability was over 95% for the cells intended to be used in the assay. The cells were resuspended in tissue culture medium at 1×10 4 cells per well and added to flat - bottom microtiter plates in a volume of 100 μL. The plates were incubated overnight at 37°C / 5% CO2 to allow cell attachment.

[0246] Virus Preparation - Influenza A / PR / 8 / 34 (ATCC #VR - 95), A / CA / 05 / 09 (CDC), A / NY / 18 / 09 (CDC), and A / NWS / 33 (ATCC #VR - 219) strains were obtained from ATCC or the Centers for Disease Control and propagated in MDCK cells for the preparation of stock virus pools. An aliquot of the virus, whose titer had been previously measured, was removed from the freezer (-80°C) and allowed to thaw slowly to room temperature in a safety cabinet. The virus was resuspended and diluted in assay medium (DMEM supplemented with 0.5% BSA, 2 mM L - glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 1 mM sodium pyruvate, 0.1 mM NEAA, and 1 μg / ml TPCK - treated trypsin) such that the amount of virus added to each well in a volume of 100 μL was the amount determined to result in 85 - 95% cell killing 4 days post - infection. The XTT - plate was stained for efficacy and toxicity and analyzed as described previously for the dengue cytoprotection assay.

[0247] Example 42. Hepatitis C Virus Antiviral Assay: The cell line Huh-luc / neo-ET was obtained by ImQuest BioSciences from Dr. Ralf Bartenschlager (Department of Molecular Virology, Hygiene Institute, University of Heidelberg, Germany) under a special license agreement. This cell line contains a firefly luciferase gene-ubiquitin-neomycin phosphotransferase fusion protein and an EMCV IRES-driven NS3-5B HCV coding sequence containing ET tissue culture-adaptive mutations (E1202G, T1202I, and K1846T), and is a persistently replicating 389 luc-ubi-neo / NS3-3’ / ET replicon. The stock culture of Huh-luc / neo-ET was propagated by culturing in DMEM supplemented with 10% FCS, 2 mM glutamine, penicillin (100 U / mL) / streptomycin (100 μg / mL), and 1× non-essential amino acids plus 1 mg / mL G418. The cells were split 1:4 and subcultured twice in the same medium with 250 μg / mL G418 added. The cells were treated with trypsin, counted by staining with trypan blue, and seeded at a cell culture density of 7.5×10 3 cells per well in 96-well tissue culture plates and incubated at 37°C in 5% CO2 for 24 hours. After 24 hours of incubation, the medium was removed and replaced in triplicate with the same medium without G418 but with the test compound added. Six wells in each plate were filled with medium only as untreated controls. The cells were incubated for a further 72 hours at 37°C in 5% CO2, and then the anti-HCV activity was measured by luciferase endpoint. For the assessment of cytotoxicity by XTT staining, duplicate plates were treated and incubated in parallel.

[0248] Cell viability - The cell culture monolayer from the treated cells was stained with the tetrazolium dye XTT to evaluate the cell viability of the Huh-luc / neo-ET reporter cell line in the presence of the compound.

[0249] Measurement of viral replication - HCV replication from the replicon assay system was measured by luciferase activity using the britelite plus luminescence reporter gene kit according to the manufacturer's instructions (Perkin Elmer, Shelton, CT). Briefly, one vial of britelite plus lyophilized substance was solubilized in 10 mL of britelite reconstitution buffer, inverted, and gently mixed. After incubating at room temperature for 5 minutes, britelite plus reagent was added to a 96-well plate at 100 μL per well. The plate was sealed with an adhesive film and incubated at room temperature for approximately 10 minutes to lyse the cells. The contents of the wells were transferred to a white 96-well plate, and luminescence was measured within 15 minutes using a Wallac 1450 Micorbeta Trilux liquid scintillation counter. The data was imported into a customized Microsoft Excel 2007 spreadsheet for determination of the 50% viral inhibitory concentration (EC 50 ).

[0250] Example 43. Anti-parainfluenza-3 cell protection assay: Cell preparation - HEp2 cells (human epithelial cells, ATCC catalog number CCL-23) were passaged in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 1 mM sodium pyruvate, and 0.1 mM NEAA in a T-75 flask before use in the antiviral assay. One day before the assay, the cells were split 1:2 to ensure that they were in the exponential growth phase at the time of infection. Total cell and viability quantification was performed using a hemocytometer and trypan blue dye exclusion. The cell viability was greater than 95% for the cells to be used in the assay. The cells were resuspended in tissue culture medium at 1×10 4 cells per well and added to a flat-bottom microtiter plate in a volume of 100 μL. The plate was incubated overnight at 37°C / 5% CO2 to allow cell attachment.

[0251] Virus preparation - Parainfluenza virus type 3 strain SF4 was obtained from ATCC (Catalog No. VR-281) and propagated in HEp2 cells for the production of a stock virus pool. An aliquot of the virus, whose titer had been previously measured, was removed from the freezer (-80 °C) and slowly thawed to room temperature in a biosafety cabinet. The virus was resuspended and diluted in assay medium (DMEM supplemented with 2% heat-inactivated FBS, 2 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin) such that the amount of virus added to each well in a volume of 100 μL was the amount determined to result in 85 - 95% cell killing 6 days post-infection.

[0252] Plate Format - Each plate contains cell control wells (cells only), virus control wells (cells plus virus), triple drug toxicity wells per compound (cells plus drug only), and triple experimental wells (drug plus cells plus virus). Efficacy and Toxicity After incubation at 37 °C in a 5% CO2 incubator, the test plates were stained with the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide). XTT - tetrazolium is metabolized by the mitochondrial enzymes of metabolically active cells to soluble formazan products, enabling rapid quantitative analysis of the inhibition of virus-induced cell killing by antiviral test substances. The XTT solution was prepared daily as a 1 mg / mL stock in RPMI 1640. The phenazine methosulfate (PMS) solution was prepared at 0.15 mg / mL in PBS and stored at -20 °C in the dark. The XTT / PMS stock was prepared immediately before use by adding 40 μL of PMS per mL of the XTT solution. 50 microliters of XTT / PMS was added to each well of the plate, and the plate was reincubated at 37 °C for 4 hours. The plate was sealed with an adhesive plate sealer and gently shaken or inverted several times to mix the soluble formazan products, and the plate was read spectrophotometrically at 450 / 650 nm using a Molecular Devices Vmax plate reader.

[0253] Data Analysis - Raw data were collected from Softmax Pro 4.6 software and imported into a Microsoft Excel spreadsheet for analysis. The percent reduction in the viral cytopathic effect relative to the untreated virus control was calculated for each compound. The percent cell control values were calculated for each compound by comparing drug-treated uninfected cells to uninfected cells in medium only.

[0254] Example 44. Influenza Polymerase Inhibition Assay: Virus preparation - Purified influenza virus A / PR / 8 / 34 (1 ml) was obtained from Advanced Biotechnologies, Inc. (Columbia, MD), thawed, and divided into five aliquots for storage at -80°C until use. On the day of assay preparation, 20 μL of 2.5% Triton N-101 was added to 180 μL of purified virus. The disrupted virus was diluted 1:2 in a solution containing 0.25% Triton and PBS. Disruption provided a source of influenza ribonucleoprotein (RNP) containing influenza RNA-dependent RNA polymerase and template RNA. Samples were stored on ice until used in the assay.

[0255] Polymerase reaction - Each 50 μL polymerase reaction contained the following: 5 μL of disrupted RNP, 100 mM Tris-HCl (pH 8.0), 100 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 0.25% Triton N-101, 5 μCi of [α- 32 P] GTP, 100 μM ATP, 50 μM each of (CTP, UTP), 1 μM GTP, and 200 μM adenylyl (3'-5') guanosine. To test inhibitors, reactions contained the inhibitor and were done the same with a reaction containing a positive control (2'-deoxy-2'-fluoroguanosine-5'-triphosphate). Other controls included a reaction mixture of RNP+ and RNP+ 1% DMSO. The reaction mixture without ApG primer and NTP was incubated at 30°C for 20 minutes. When ApG and NTP were added to the reaction mixture, the samples were incubated at 30°C for 1 hour and then immediately followed by transferring the reaction to a glass fiber filter plate and then precipitating with 10% trichloroacetic acid (TCA). The plate was then washed 5 times with 5% TCA followed by 1 wash with 95% ethanol. When the filter was dry, incorporation of [α- 32 P] GTP was measured using a liquid scintillation counter (Micro beta).

[0256] Plate format - Each test plate contained triplicate samples of three compounds (at six concentrations), in addition to triplicate samples of RNP+ reaction mixture (RNP only), RNP+ 1% DMSO, and reaction mixture only (without RNP).

[0257] Data analysis - Raw data was collected from a Micro Beta scintillation counter. Incorporation of radioactive GTP is directly correlated with the level of polymerase activity. The "percent inhibition value" was obtained by dividing the mean value of each test compound by the RNP+ 1% DMSO control. The mean obtained at each concentration of 2DFGTP was compared to the RNP+ reaction control. The data was then imported into a Microsoft Excel spreadsheet and IC 50 values were calculated by linear regression analysis.

[0258] Example 45. HCV polymerase inhibition assay: The activity of the compounds that inhibit HCV polymerase was evaluated using the previously reported method (Lam et al. 2010. Antimicrobial Agents and Chemotherapy 54(8):3187-3196). The HCV NS5B polymerase assay was performed in a 96-well reaction plate with a volume of 20 μL. Each reaction contained 40 ng / μL of purified recombinant NS5BΔ22 genotype-1b polymerase, 20 ng / μL of the HCV genotype-1b complimentary IRES template, 1 μM of each of the four natural ribonucleotides, 1 U / mL Optizyme RNase inhibitor (Promega, Madison, WI), 1 mM MgCl2, 0.75 mM MnCl2, and 2 mM dithiothreitol (DTT) in 50 mM HEPES buffer (pH 7.5). The reaction mixture was assembled in two steps on ice. Step 1 consisted of combining all reaction components except the natural nucleotides and labeled UTP in the polymerase reaction mixture. 10 microliters (10 μL) of the polymerase mixture was dispensed into individual wells of a 96-well reaction plate on ice. A polymerase reaction mixture without NS5B polymerase was also included as an enzyme-free control. Serial half-log dilutions of the test compounds and control compounds, 2'-O-methyl-CTP and 2'-O-methyl-GTP (Trilink, San Diego, CA), were prepared in water, and 5 μL of the serial diluted compounds or water only (compound-free control) was added to the wells containing the polymerase mixture. Then, 5 microliters of the nucleotide mix (natural nucleotides and labeled UTP) was added to the reaction plate wells, and the plate was incubated at 27 °C for 30 minutes. The reaction was quenched by the addition of 80 μL of stop solution (12.5 mM EDTA, 2.25 M NaCl, and 225 mM sodium citrate), and the RNA product was applied to a Hybond-N+ membrane (GE Healthcare, Piscataway, N.J) using a dot blot apparatus under vacuum. The membrane was removed from the dot blot apparatus, washed four times with 4XSSC (0.6 M NaCl and 60 mM sodium citrate), then once with water and once with 100% ethanol.The membrane was air-dried, exposed to a phosphorimaging screen, and the image was captured using a Typhoon 8600 Phospho imager. After image capture, the membrane was placed in a Micro beta cassette with scintillation fluid, and the CPM in each reaction was counted with a Micro beta 1450. The CPM data was imported into a custom Excel spreadsheet for the determination of compound IC. 50

[0259] Example 46. NS5B RNA-dependent RNA polymerase reaction conditions Compounds were assayed for inhibition of NS5B-δ21 from HCV GT-1b Con-1. The reaction contained purified recombinant enzyme, 1 u / μL minus-strand HCV IRES RNA template, and 32 P]-CTP or 32 P]-UTP in a 1 μM NTP mixture. The assay plates were incubated at 27 °C for 1 h before quenching. Incorporation of 32 P] into high molecular weight products was assayed by filter binding.

[0260] Example 47. Human DNA polymerase inhibition assay: Human DNA polymerases alpha (catalog number 1075), beta (catalog number 1077), and gamma (catalog number 1076) were purchased from CHIMERx (Madison, WI). Inhibition of beta and gamma DNA polymerase activities was assayed in microtiter plates in 50 mM Tris-HCl (pH 8.7), KCl (10 mM for beta, 100 mM for gamma), 10 mM MgCl2, 0.4 mg / mL BSA, 1 mM DTT, 15% glycerol, 0.05 mM of dCTP, dTTP, and dATP, 10 μCi 32 ​Assayed in a 50 μL reaction mixture containing 32 P]-α-dGTP (800 Ci / mmol), 20 μg activated calf thymus DNA, and the test compound at the indicated concentrations. In each assay, the enzyme reaction was allowed to proceed for 30 minutes at 37 °C, followed by transfer to a glass fiber filter plate and subsequent precipitation with 10% trichloroacetic acid (TCA). The plate was then washed with 5% TCA, followed by one wash with 95% ethanol. When the filter was dry, radioactivity incorporation was measured using a liquid scintillation counter (Microbeta).

[0261] Example 48. HIV-infected PBMC assay: Fresh human peripheral blood mononuclear cells (PBMC) were obtained from a commercial source (Biological Specialty) and determined to be seronegative for HIV and HBV. Depending on the volume of donor blood received, the leukophoresed blood cells were washed several times with PBS. After washing, the leukophoresed blood was diluted 1:1 with Dulbecco's phosphate-buffered saline (PBS) and layered on a 15 mL Ficoll-Hypaque density gradient in a 50 mL conical centrifuge tube. These tubes were centrifuged at 600 g for 30 minutes. The banded PBMC were gently aspirated from the resulting interface and washed three times with PBS. After the final wash, the cell number was determined by trypan blue dye exclusion, and the cells were 6Resuspended in RPMI 1640 with 15% fetal bovine serum (FBS), 2 mmol / L L-glutamine, 2 μg / mL PHA-P, 100 U / mL penicillin and 100 μg / mL streptomycin at cells / mL and incubated at 37 °C for 48 - 72 hours. After incubation, PBMC were centrifuged and resuspended in tissue culture medium. The cultures were maintained until use by semi-volume culture exchange with fresh IL-2-containing tissue culture medium every 3 days. The assay was initiated with PBMC 72 hours after PHA-P stimulation.

[0262] To minimize the effects of donor variability, the PBMC used in the assay were a mixture of cells derived from three donors. Immediately prior to use, target cells were resuspended in fresh tissue culture medium at 1×10 6 cells / mL and seeded at 50 μL / well into the inner wells of a 96-well round-bottom microtiter plate. Then, 100 μL of 2-fold concentrated compound-containing medium was transferred to the 96-well plate containing cells in 50 μL of medium. AZT was used as an internal assay standard.

[0263] After adding the test compound to the wells, 50 μL of a predetermined dilution of the HIV virus (prepared from 4-fold the final desired in-well concentration) was added and mixed well. For infection, 50 - 150 TCID 50 of each virus was added (final MOI approximately 0.002). PBMC were exposed to the virus in triplicate and cultured in a 96-well microtiter plate in the presence or absence of various concentrations of the test substance as described above. Seven days after culturing, HIV-1 replication was quantified in the tissue culture supernatant by measurement of reverse transcriptase (RT) activity. Wells with cells and virus only served as virus controls. Another plate was prepared identically without virus for the drug cytotoxicity test.

[0264] Reverse transcriptase activity assay - Reverse transcriptase activity was measured in the cell-free supernatant using a standard radioactive incorporation polymerization assay. Tritiated thymidine triphosphate (TTP; New England Nuclear) was purchased at 1 Ci / mL and 1 μL was used per enzyme reaction. The rAdT stock solution was prepared by mixing 0.5 mg / mL poly rA and 1.7 U / mL oligo dT in distilled water and stored at -20 °C. The RT reaction buffer was freshly prepared daily and consisted of 125 μL of 1 mol / L EGTA, 125 μL of dH2O, 125 μL of 20% Triton X-100, 50 μL of 1 mol / L Tris (pH 7.4), 50 μL of 1 mol / L DTT, and 40 μL of 1 mol / L MgCl2. For each reaction, 1 μL of TTP, 4 μL of dH2O, 2.5 μL of rAdT, and 2.5 μL of the reaction buffer were mixed. 10 microliters of this reaction mixture was placed in a round-bottom microtiter plate, 15 μL of the virus-containing supernatant was added, and mixed. The plate was incubated at 37 °C in a humidified incubator for 90 minutes. After incubation, 10 μL of the reaction volume was spotted onto a DEAE filter mat in an appropriate plate format and washed 5 times (5 minutes each) in 5% sodium phosphate buffer, 2 times (1 minute each) in distilled water, and 2 times (1 minute each) in 70% ethanol, and then air-dried. The dried filter mat was placed in a plastic sleeve and 4 mL of Opti-Fluor O was added to the sleeve. Incorporated radioactivity was quantified using a Wallac 1450 Micorobeta Trilux liquid scintillation counter.

[0265] Example 49. HBV: HepG2.2.15 cells (100 μL) in RPMI 1640 medium with 10% fetal bovine serum were seeded at 1 × 10 4In addition to adding to all wells of a 96-well plate at the density of cells, the plate was incubated at 37 °C in an environment of 5% CO2 for 24 hours. After incubation, six 10-fold serial dilutions of the test compound prepared in RPMI1640 medium with 10% fetal bovine serum were added in triplicate to individual wells of the plate. Six wells of the plate received only the medium as a virus-only control. The plate was incubated at 37 °C in an environment of 5% CO2 for 6 days. The medium was replaced on the third day with the medium containing each compound at the indicated concentration. 100 microliters of the supernatant was recovered from each well for analysis by qPCR of viral DNA, and cytotoxicity was evaluated on the sixth day by XTT staining of the cell culture monolayer.

[0266] 10 microliters of the cell culture supernatant collected on the sixth day was diluted in qPCR dilution buffer (40 μg / mL fragmented salmon sperm DNA) and boiled for 15 minutes. Quantitative real-time PCR was performed in a 386-well plate using an Applied Biosystems 7900HT Sequence Detection System and SDS 2.4 software that supports it. The boiled DNA of each 5-microliter (5 μL) sample and 10-fold serial dilutions of the quantitative DNA standard were subjected to real-time Q-PCR with Platinum Quantitative PCR SuperMix-UDG (Invitrogen) and specific DNA oligonucleotide primers (IDT, Coralville, ID) HBV-AD38-qF1 (5’-CCG TCT GTG CCT TCT CAT CTG-3’), HBV-AD38-qR1 (5’-AGT CCA AGA GTY CTC TTA TRY AAG ACC TT-3’), and HBV-AD38-qP1 (5’-FAM CCG TGT GCA / ZEN / CTT CGC TTC ACC TCT GC-3’BHQ1) at a final concentration of 0.2 μM for each primer and a total reaction volume of 15 μL. The copy number of HBV DNA in each sample was interpolated from the standard curve by SDS.24 software, and the data was imported into an Excel spreadsheet for analysis.

[0267] The 50% cytotoxic concentration of the test substance is induced by measuring the reduction of the tetrazolium dye XTT in the treated tissue culture plates. XTT is metabolized to soluble formazan products by the mitochondrial enzyme NADPH oxidase in metabolically active cells. The XTT solution was prepared daily as a 1 mg / mL stock in PBS. The phenazine methosulfate (PMS) stock solution was prepared at 0.15 mg / mL in PBS and stored at -20 °C in the dark. The XTT / PMS solution was prepared immediately before use by adding 40 μL of PMS per 1 mL of the XTT solution. 50 microliters of XTT / PMS was added to each well of the plate and the plate was incubated at 37 °C for 2 - 4 hours. An incubation of 2 - 4 hours was empirically determined to be within the linear response range of XTT dye reduction for the number of cells indicated in each assay. An adhesive plate sealer was used instead of the lid, and the sealed plate was inverted several times to mix the soluble formazan products. The plate was read at 450 nm (650 nm reference wavelength) using a Molecular Devices SpectraMax Plus 384 spectrophotometer. The data was collected by Softmax 4.6 software and imported into an Excel spreadsheet for analysis.

[0268] Example 50. Dengue virus RNA-dependent RNA polymerase reaction conditions The RNA polymerase assay was performed in 1.5 ml tubes at 30 °C using 100 μl of reaction mix. The final reaction conditions were 50 mM Hepes (pH 7.0), 2 mM DTT, 1 mM MnCl2, 10 mM KCl, 100 nM UTR-Poly A (self-annealing primer), 10 μM UTP, 26 nM RdRp enzyme. Reaction mixes with different compounds (inhibitors) were incubated at 30 °C for 1 hour. To evaluate the amount of pyrophosphate generated during the polymerase reaction, 30 μl of the polymerase reaction mix was mixed with the luciferase-coupled enzyme reaction mix (70 μl). The final reaction conditions for the luciferase reaction were 5 mM MgCl2, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 200 μU ATP sulfurylase, 5 μM APS, 10 nM luciferase, 100 μM D-luciferin. For detection of the light signal, the white plate containing the reaction sample (100 μl) was immediately transferred to a luminometer Veritas (Turner Biosystems, CA).

[0269] Example 51. Procedures for cell incubation and analysis Huh-7 cells were seeded at 0.5×10 in 1 mL of complete medium in a 12-well tissue culture-treated plate 6Cells were seeded in wells. The cells were allowed to adhere overnight at 37°C / 5% CO2. A 40 μM stock solution of the test article was prepared in 100% DMSO. From the 40 μM stock solution, a 20 μM solution of the test article in 25 ml of complete DMEM medium was prepared. For compound treatment, the medium was aspirated from the wells and 1 mL of the 20 μM solution was added to the appropriate wells in complete DMEM medium. Another plate of "no" compound added cells was also prepared. The plates were incubated at 37°C / 5% CO2 at the following time points: 1, 3, 6, and 24 hours. After incubation at the desired time points, the cells were washed twice with 1 mL of DPBS. The cells were extracted by adding 500 μl of 70% methanol / 30% water with internal standard added to each well treated with the test article. Untreated blank plates were extracted with 500 μL of 70% methanol / 30% water per well. The samples were centrifuged at 16,000 rpm for 10 minutes at 4°C. The samples were analyzed by LC-MS / MS using an ABSCIEX 5500 QTRAP LC-MS / MS system equipped with a Hypercarb (PGC) column.

[0270] Example 52: Procedure for Rodent Pharmacokinetic Experiments DBA-1J mice (6 - 8 weeks old, female) were acclimated for 2 or more days after receipt. The body weight of the mice was measured on the day before dosing, and the dosing volume was calculated. The mice were dosed with the drug by forced oral administration at 30 mg / kg, 100 mg / kg, and 300 mg / kg. The mice were sacrificed at 8 time points: 0.5, 1, 2, 3, 4, 8, and 24 hours (3 mice per time point for the test drug). The mice were euthanized and their organs were collected (see below). To collect blood, the mice were euthanized with CO2 at the appropriate time points listed above. Blood was obtained by cardiac puncture (0.3 ml) at each time point. After blood collection, the organs were removed from the mice (see below). The Li-heparin tubes containing blood were gently inverted 2 or 3 times and mixed well to process the blood. Then, the tubes were placed in a rack in ice water until centrifugation was possible (less than 1 hour). As soon as possible, the blood was centrifuged in a refrigerated centrifuge at approximately 2000 × g for 10 minutes to obtain plasma. Then, using a 200 μL pipette, the plasma was transferred to labeled 1.5 ml Eppendorf tubes in ice water. Then, the plasma was frozen in a freezer or on dry ice. The samples were stored at -80 °C before analysis. Organs were recovered from the euthanized mice. Organs (lungs, liver, kidneys, spleen, and heart) were removed, placed in tubes, and immediately frozen in liquid nitrogen. Then, the tubes were transferred to dry ice. The samples were stored in cryogenic tissue vials. The samples were analyzed by LC-MS / MS using an ABSCIEX 5500 QTRAP LC-MS / MS system equipped with a Hypercarb (PGC) column.

[0271] Pharmacokinetic parameters: · T max after oral dosing is 0.25 - 0.5 hours. · C max is 3.0, 7.7, and 11.7 ng / ml after oral dosing at 30, 100, and 300 mg / kg; · Bioavailability (relative to intraperitoneal delivery) is 65% at 30 mg / kg and 39 - 46% at 100 and 300 mg / kg oral dosing; · EIDD-1931 plasma T 1 / 2It is 2.2 hours after intravenous administration and 4.1 - 4.7 hours after oral administration. · After oral administration of 300 mg / kg, the plasma level at 24 hours is approximately 0.4 μM; after administration of 100 mg / kg it is approximately 0.1 μM.

[0272] Example 53: Procedure for a mouse model of chikungunya infection 100 pfu of CHIK virus was injected into the plantar pads of C57BL - 6J mice. The test groups consisted of an uninfected and untreated group, an infected and untreated group, an infected group receiving a high dose of 35 mg / kg intraperitoneal EIDD - 01931, and an infected group receiving a low dose of 25 mg / kg intraperitoneal EIDD - 01931. The two test groups receiving EIDD - 01931 received the compound 12 hours before exposure and then daily for 7 days. The plantar pads were evaluated daily for 7 days for inflammation (foot thickness). Arthritis (histology) induced by CHIK virus was evaluated at the ankle joint using PCR after 7 days.

[0273] Example 54: N(4)-Hydroxycytidine for the prevention and treatment of alphavirus infection An activity test in the vero cell cytopathic effect (CPE) model of infection showed that the ribonucleoside analog N(4)-hydroxycytidine (EIDD - 01931) was active against Ross River, EEE, WEE, VEE, and CHIK viruses, with EC 50 values of 2.45 μM, 1.08 μM, 1.36 μM, 1.00 μM, and 1.28 μM, respectively. The cytotoxicity profile of the compound was acceptable and the selectivity index ranged from a low value of 8 in CEM cells to a high value of 232 in Huh7 (liver) cells.

[0274] Example 55: If high-titer VEE virus can reach the brain within hours of aerosol exposure, a direct-acting antiviral drug would be desirable if therapeutic levels of the drug could be rapidly achieved in the brain. Pilot pharmacokinetic studies were conducted in male Sprague-Dawley rats dosed with 5 and 50 mg / kg of EIDD-01931 by forced oral administration to determine the pharmacokinetic parameters and the tissue distribution profile of the compound in major organ systems including the brain. EIDD-01931 is orally available, dose-proportional, and has a calculated bioavailability (%F) of 28%. Organ samples (brain, lung, spleen, kidney, and liver) were recovered from the 50 mg / kg dose group at 2.5 and 24 hours post-dose. EIDD-01931 was well-distributed in all tissues tested; of particular note, based on estimates from cell data, it was readily distributed in brain tissue at therapeutic drug levels. Once in the brain, EIDD-01931 was rapidly metabolized to its active 5'-triphosphate form, giving brain levels of 526 and 135 ng / g at 2.5 and 24 hours, respectively. Even after 24 hours, the levels of EIDD-01931 and its 5'-triphosphate in the brain were significant, suggesting that once-daily oral dosing may be sufficient for treatment.

[0275] Alternatively, drug delivery by aerosol (intranasal spray) administration may rapidly achieve therapeutic drug levels in the nasal mucosa and brain. EIDD-01931 has an acceptable toxicology profile after 6 days of once-daily intraperitoneal (IP) injection in mice, and the NOEL (no-observed-effect level) is 33 mg / kg; weight loss was observed at the highest dose tested (100 mg / kg), but reversed upon discontinuation of dosing.

[0276] Example 56: Several derivatives of EIDD-01931 showed antiviral activity in screening against various viruses. The activity data are shown in the following table.

[0277]

Table 1

[0278]

Table 2

[0279]

Table 3

[0280]

Table 4

[0281]

Table 5

[0282] Example 57: Compounds Screened in the CHIKV CPE Assay

Chemical Structure

[0283] Example 58: Compounds Screened in the CHIKV CPE Assay

[0284]

Table 6

[0285] Example 59: Compounds Screened in the CHIKV CPE Assay

Chemical Structure

Chemical Structure

[0286] Example 60:

[0287]

Table 7

[0288] Example 61: Compounds screened by CHIKV CPE assay [Chemical formula] [Chemical formula]

[0289] Example 62:

[0290] [Table 8]

Claims

1. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound having the structure of Formula IA below, or a pharmaceutically acceptable salt thereof. 【Chemical 1】 (In the formula, R ７ is H; X is CH2; Y is H; R １ is carboxy, formyl or esteryl, R １ is one or more of the same or different R ２０ or R １ O- is a carboxylic acid ester; R ４ is hydroxy; R ５ is hydrogen; R ２０ is deuterium, alkyl, alkenyl, alkynyl, halogen, cyano, hydroxy, amino, amido, formyl, carboxy, carbamoyl, alkoxy, alkylamino, (alkyl) ２ Amino, (C ３ ~C ６ ) carbocyclyl or aryl, where R ２０ represents one or more of the same or different R ２１ may be optionally replaced by; and R ２１ is halogen, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methoxycarbonyl, ethoxycarbonyl, (C ３ ~C ６ ) carbocyclyl or aryl; Here, the lipid is C ６～２２ (including aryl substituted with alkyl, alkoxy, polyethylene glycol, or alkyl groups)

2. R １ The composition of claim 1 , wherein O− is a carboxylic acid ester.

3. R １ The composition of claim 1 , wherein is an esteryl.

4. R １ is R ２０ and R ２０ The composition of claim 1 , wherein is alkyl.

5. R １ The composition of claim 1 , wherein is formyl.

6. R １ is R ２０ and R ２０ The composition of claim 6 , wherein is alkyl.

7. R １ The composition of claim 1 , wherein is carboxy.

8. R １ is R ２０ and R ２０ The composition of claim 7 , wherein is alkyl.

9. R ２０ is R ２１ and R ２１ The composition of any one of claims 1 to 8, wherein is aryl.

10. A pharmaceutical composition for treating or preventing an infection caused by an Orthomyxoviridae virus, a Paramyxoviridae virus, an RSV virus, an influenza virus, a Filoviridae virus, or an Ebola virus, comprising a pharmaceutically acceptable excipient and a compound having the structure of formula IA below or a pharmaceutically acceptable salt thereof: 【Chemistry 2】 (In the formula, R ７ is H; X is CH2; Y is H; R １ is carboxy, formyl or esteryl, R １ is one or more of the same or different R ２０ or R １ O- is a carboxylic acid ester; R ４ is hydroxy; R ５ is hydrogen; R ２０ is deuterium, alkyl, alkenyl, alkynyl, halogen, cyano, hydroxy, amino, amido, formyl, carboxy, carbamoyl, alkoxy, alkylamino, (alkyl) ２ Amino, (C ３ ~C ６ ) carbocyclyl or aryl, where R ２０ represents one or more of the same or different R ２１ may be optionally replaced by; and R ２１ is halogen, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio, methoxycarbonyl, ethoxycarbonyl, (C ３ ~C ６ ) carbocyclyl or aryl; Here, the lipid is C ６～２２ (including aryl substituted with alkyl, alkoxy, polyethylene glycol, or alkyl groups)

11. R １ The composition of claim 10, wherein O- is a carboxylic acid ester.

12. R １ The composition of claim 11 , wherein is an esteryl.

13. R １ is R ２０ and R ２０ The composition of claim 13 , wherein is alkyl.

14. R １ The composition of claim 10 , wherein is formyl.

15. R １ is R ２０ and R ２０ The composition of claim 14 , wherein is alkyl.

16. R １ The composition of claim 10, wherein is carboxy.

17. R １ is R ２０ and R ２０ The composition of claim 16 , wherein is alkyl.

18. R ２０ is R ２１ and R ２１ The composition of any one of claims 10 to 17, wherein is aryl.

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