2'-fluoro-6'methylene carbocyclic nucleosides and nucleotides and methods of treating poxvirus infections with same

Abstract

The present invention relates to 2'-fluoro-6'-methylene carbocyclic nucleosides and nucleotides, pharmaceutical compositions containing these nucleosides and nucleotides, and their use in the treatment or prophylaxis of poxvirus infections and secondary disease states and conditions thereof.

Description

[0001] 2'-FLUORO-6'METHYLENE CARBOCYCLIC NUCLEOSIDES AND NUCLEOTIDES AND METHODS OF TREATING POXVIRUS INFECTIONS WITH SAME

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to 2'-fluoro-6-methylene carbocyclic nucleosides and nucleotides, pharmaceutical compositions containing these nucleosides and nucleotides, and their use in the treatment or prophylaxis of poxvirus infections and secondary7disease states and conditions thereof.

[0004] BACKGROUND

[0005] On May 6, 2022, a new case of Monkeypox (Mpox) was detected in a UK resident who had traveled to Nigeria from late April to early May.1Having originally been identified in 1958, Mpox has generally been regionally endemic to tropical regions of central and west Africa.2, 3However, the recent spread of cases has been widely outside of this region. The US Department of Health and Human Services declared that the recent monkeypox virus (MPXV) outbreak as a public health emergency.4The most recent global data on the Mpox outbreak from the Centers for Disease Control (CDC, updated October7th, 2024) reveal a total of -102,000 confirmed cases. Approximately 98% of cases were reported in locations that have not historically been known to have Mpox.5This includes 32,063 cases in the United States with 60 deaths, 10,967 in Brazil with 16 deaths, 7,896 in Spain with 3 deaths, 3,816 in Germany, 3,892 in the UK, and 4,195 in France.6MPXV, a DNA virus, is the etiological agent of a zoonotic disease. Until now, two genetic clades of Mpox have been identified and distinguished as the Central African clade (Congo Basin clade, clade I) and the West African clade (clade II). Remarkably, these distinct genetic clades have been reproducible and maintained for more than 30 years among MPXV isolates from various regions of the world. Evidence from previous outbreaks suggests that clade I is more virulent. Previously, clade I has historically triggered small outbreaks, typically confined to a few households or communities in Central Africa. Fortunately, the ongoing outbreak is caused by the West African clade (clade II), which has a significantly lower fatality rate as compared to the Congo basin central African clade (clade I), which has around 10% fatality.7, 8MPXV, primarily endemic in West and Central Africa,9now has raised significant concerns due to its recent emergence in non-endemic areas outside of Africa. MPXV was considered a neglected zoonotic pathogen, but its global spread serves as a wake-up call for scientific communities worldwide.10 While the virus typically triggers mild symptoms in individuals with strong immune systems, it poses a severe threat when it infects those who are immunocompromised, children, the elderly, pregnant women, and individuals with underlying health conditions such as human immunodeficiency virus (HIV) / AIDS and diabetes. The currently published report of the World Health Organization (WHO) and CDC revealed that infection of MPXV is related to sexual transmission.11The data are worrisome, because they indicate that MPXV is spreading as a sexually transmitted disease (STD), then MPXV has the potential to create pandemic concerns similar to the infection of HIV and need special attention for antiviral development.12The infection of MPXV raises serious concerns for human health, particularly due to its potential association with sexually transmitted diseases (STDs), posing a global threat.

[0006] Currently, no specific FDA-approved treatment is available for human Mpox infection. All current therapies are smallpox-centric and are being applied or may be used for Mpox treatment. The antiviral tecovirimat (Tpoxx®, FIG. 1), cidofovir (CDV), brincidofovir (BCV, prodrug of cidofovir), and vaccinia immune globulin are currently available from the Strategic National Stockpile (SNS) as options for the treatment of Mpox infection.

[0007] However, there are no definitive data available for the application of these drugs to treat MPXV infections. Tecovirimat (Tpoxx) was approved by the US-FDA for the treatment of the human smallpox virus.13Use of tecovirimat for the treatment of Mpox infection in the United States is permitted only through an FDA-regulated Expanded Access Investigational New Drug (EA-IND) mechanism.14Presently, the National Institute of Allergy and Infectious Diseases (NIAID) is managing two clinical trials for tecovirimat, specifically to treat patients with Mpox.15Cidofovir (Vestide®) has demonstrated in-vitro and in-vivo antiviral activity against MPXV.16Unfortunately, cidofovir is associated with severe side effects of nephrotoxicity.17Furthermore, the use of cidofovir is restricted by its limited oral absorption and the necessity7for intravenous infusion during administration.18Brincidofovir, a prodrug of cidofovir, has demonstrated potent antiviral activity against Mpox.19However, a recent study indicated that patients who were treated with brincidofovir (200 mg) once a week, out of seven patients, three were confirmed with elevated alanine transaminase liver enzyme resulting in cessation of the full course of treatment.20The CDC has decided to administer JYNNEOS or Imvanex (MVA-BN) vaccine to individuals at an increased risk of encountering MPXV infection. JYNNEOS is a live vaccinia vaccine that was initially authorized for the management of smallpox virus infection.21However, due to the lack of effective antivirals, immunocompromised, cancer-treating patients and organ transplant patients are still at a very high risk of MPXV infection and new antivirals with safe and improved antiviral activity7are urgently needed. SUMMARY OF THE INVENTION

[0008] One aspect of the invention is directed to methods of treating or reducing the likelihood of an infection caused by a poxvirus in a subject in need thereof. The methods can comprise administering to the subject an amount of a compound effective to treat or reduce the likelihood of the infection.

[0009] In some versions, the compound has a structure of:

[0010]

[0011] wherein:

[0012] R1and Rlaare each independently H, an acyl group, a C1-C20 alkyl group, a C1-C20 ether group, an amino acid residue (D or L), a phosphate, diphosphate, triphosphate, phosphoester, phosphodiester, or phosphorami date group, or together R1and Rlaform a carbodiester, phosphodiester, or cyclic phosphoramidate group with the oxygen atoms to which they are bonded;

[0013] RLis C(RH)2, wherein each RHis independently H, F, Cl, Br, or I;

[0014] B is:

[0015]

[0016] A1and A6are each independently N, CH, or C;

[0017] A2, A4, A5, and A7are each independently N, NR, CR, or CR2;

[0018] A3is C, N, or CR;

[0019] each --- between adjacent atoms represents a bond that is present or absent;

[0020] RM1, RM2, RM3, and RM4are each independently RN, R°, =0, OR, =S, or SR; each RNand R° is independently -NHR2or R;

[0021] each R2is independently H, an acyl group, OR, SR, a C1-C20 alkyl group, a C1-C20 ether group, or an amino acid residue (D or L);

[0022] each R is independently H, F, Cl, Br, I, C1-C4 alkyl (preferably CH3), -C≡N, -C≡C-Ra,

[0023]

[0024] each Rais independently H or a C1-C4 alky l group; and

[0025] each X is independently H, C1-C4 alkyl (preferably, CHs), F, Cl, Br or I, or a pharmaceutically acceptable salt, stereoisomer, hydrate, or solvate thererof.

[0026] In some versions, the compound has a structure of:

[0027]

[0028] In some versions, the compound has a structure of:

[0029]

[0030] In some versions, Rlais H, a C2-C20 acyl group, or a C1-C20 alkyl group. In some versions, Rlais H. In some versions, Rlais a C2-C20 acyl group. In some versions, Rlais a C2- Ce acyl group. In some versions, Rlais a Ci-Ce alkyl group.

[0031] In some versions, A1is N or C; A2is N or CR; A3is C or N; A4is N, NR, or CR; A5is N or NR; RM1is RNor =0; RM2is RN, R°, or =0; A6is N, or CH; A7is N or NR; RM3is =0; and / or RM4is RNor =0. In some versions, A1is N or C; A2is N or CR; A3is C or N; A4is N, NR, or CR; A5is N or NR; RM1is RNor =0; RM2is RN, R°, or =0; A6is N, or CH; A7is N or NR; RM3is =0; and RM4is RNor =0.

[0032] In some versions, B is:

[0033]

[0034] In some versions, A1is N or C. In some versions, A1is N. In some versions, A1is C. In some versions, A1is CH.

[0035] In some versions, A2is N. In some versions, A2is NR, optionally, wherein the NR of A2is NH. In some versions, A2is CR, optionally, wherein the CR of A2is CH. In some versions, A2is CR2, optionally, wherein the CR2 of A2is CH2.

[0036] In some versions, A3is C. In some versions, A3is N. In some versions, A3is CR, optionally, wherein the CR of A3is CH.

[0037] In some versions, A4is N. In some versions, A4is NR, optionally, wherein the NR of A4is NH. In some versions, A4is CR, optionally, wherein the CR of A4is CH. In some versions, A4is CR2, optionally, wherein the CR2 of A4is CH2.

[0038] In some versions, A5is N. In some versions, A5is NR, optionally, wherein the NR of A5is NH. In some versions, A5is CR, optionally, wherein the CR of A5is CH. In some versions, A5is CR2, optionally, wherein the CR2 of A5is CH2. In some versions, A5is N or NR, optionally, wherein the NR of A5is NH.

[0039] In some versions RM1is RNor =0. In some versions RM1is RN. In some versions RM1is =0.

[0040] In some versions, RM2is RN, R°, or =0. In some versions, RM2is RN. In some versions, R is R°. In some versions, RM2is =0.

[0041] In some versions, A1is C; A2is CR, optionally, wherein the A2of CR is CH; A3, A4, and A5are each N; RM1is RN, and RM2is R°.

[0042] In some versions, B is:

[0043]

[0044] In some versions, A6is N or CR, optionally, wherein the CR of A6is CH. In some versions. A6is N. In some versions, A6is CR, optionally, wherein the CR of A2is CH. In some versions, A7is N. In some versions, A7is NR, optionally, wherein the NR of A7is NH. In some versions, A7is CR, optionally, wherein the CR of A7is CH. In some versions, A7is CR2, optionally, wherein the CR2 of A7is CH2. In some versions, A7is N or NR, optionally, wherein the NR of A7is NH.

[0045] In some versions, RM3is RN, R° or =0. In some versions, R315is RN. In some versions, R31' is R°. In some versions, RM3is =0.

[0046] In some versions RM4is RNor =0. In some versions RM4is RN. In some versions RM4is =0.

[0047] In some versions, A6is CR, optionally, wherein the CR of A6is CH; A7are each N; R31’ is =0; and RM4is RN.

[0048] In some versions B is:

[0049]

[0050]

[0051] In some versions, B is:

[0052]

[0053] In some versions, B is:

[0054]

[0055] In some versions, B is:

[0056]

[0057] In some versions, B is:

[0058]

[0059] In some versions, B is:

[0060]

[0061] In some versions, RNis -NHR2. In some versions, RNis NH2. In some versions, RNis R.

[0062] In some versions, R° is -NHR2. In some versions, R° is NH2. In some versions, R° is R.

[0063] In some versions, the R2on the -NHR2of RNis H, a C2-C20 acyl group, or a C1-C20 alkyl group. In some versions, the R2on the -NHR2of RNis H. In some versions, the R2on the -NHR2of RNis a C2-C20 acyl group. In some versions, the R2on the -NHR2of RNis a C2-C6 acyl group. In some versions, the R2on the -NHR2of RNis a Ci-Ce alkyl group. In some versions, the R2on the -NHR2of R° is H, a C2-C20 acyl group, or a C1-C20 alkyl group. In some versions, the R2on the -NHR2of R° is H. In some versions, the R2on the -NHR2of R° is a C2-C20 acyl group. In some versions, the R2on the -NHR2of R° is a C2-C6 acyl group. In some versions, the R2on the -NHR2of R° is a Ci-Ce alkyl group. In some versions, each R2is independently H, a C2-C20 acyl group, or a C1-C20 alkyl group. In some versions, each R2is H. In some versions, each R2is a C2-C20 acyl group. In some versions, each R2is a C2-C6 acyl group. In some versions, each R2is a Ci-Ce alkyl group.

[0064] In some versions, the R of RNis H, F, Cl, Br, I, or C1-C4 alkyl (preferably CH3). In some versions, the R of RNis H or F. In some versions, the R of RNis H. In some versions, the R of R° is H, F, Cl, Br, I, or C1-C4 alkyl (preferably CH3). In some versions, the R of R° is H or F. In some versions, the R of R° is H. In some versions, each R is independently H, F, Cl, Br, I, or C1-C4 alkyl (preferably CH3). In some versions, each R is H or F. In some versions, each R is H.

[0065] In some versions, R1is H, a C2-C20 acyl group, or a Ci-C 20 alkyl group. In some versions, R1is H. In some versions, R1is a C2-C20 acyl group. In some versions, R1is a C2-C6 acyl group. In some versions, R1is a Ci-Ce alkyl group.

[0066] In some versions, R1is an acyl, phosphate, phosphodiester, or phosphoramidate group. In some versions, R1is:

[0067]

[0068] wherein each R5is independently H, a C1-C20 alkyl group, or an alkoxy alkyl, aryl oxy alkyl, aryl, alkoxy, or alkoxy carbonyloxy group, each of which groups may be optionally substituted, with the proviso that at least one R5group is other than H, or the two R5groups together form a five- or six-membered heterocyclic group.

[0069] In some versions, R1is:

[0070]

[0071] In some versions, B' is a group according to the structure:

[0072]

[0073] wherein: i is 0, 1, 2 or 3; and R7is H, a C1-C20 alkyl group, or an acyl, alkoxyalkyl, aryloxyalkyl, or aryl group, each of which groups may be optionally substituted.

[0074] In some versions, B' is a group according to the structure:

[0075]

[0076] In some versions, R1in the compound is:

[0077]

[0078] and the compound is administered in a composition having a stereoisomeric excess of the compound with respect to a stereoisomer of the compound wherein R1in the stereoisomer is:

[0079]

[0080] The stereoisomeric excess in such versions can be at least 0.1%, at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%.

[0081] In some versions, R1is:

[0082]

[0083] and the compound is administered in a composition having a stereoisomeric excess of the compound with respect to a stereoisomer of the compound wherein R1in the stereoisomer is:

[0084]

[0085] The stereoisomeric excess in such versions can be at least 0.1%, at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%.

[0086] In some versions, R1and Rlatogether are:

[0087]

[0088] In some versions, R6is independently H, a C1-C20 alkyl group, or an alkoxy alkyl, aryloxyalkyl, aryl, alkoxy, or alkoxycarbonyloxy group, each of which groups may be optionally substituted. In some versions, R6is an optionally substituted aryl. In some versions, R6is an aryl. In some versions, R6is an optionally substituted phenyl. In some versions, R6is a phenyl.

[0089] In any version herein, R8can be a sidechain of an amino acid or an optionally substituted C1-C20 alky l group. In some versions, R8is a C1-C20 alkyl group. In some versions, R8is an optionally substituted Ci-Ce alkyl group. In some versions, R8is a Ci-Ce alky l group. In some versions, R8is an optionally substituted C1-C4 alkyl group. In some versions, R8is a C1-C4 alkyl group. In some versions, R8is methyl, ethyl, isopropyl, or isobutyl. In some versions, R8is methyl or isopropyl.

[0090] In any version herein, Rpcan be H, nitro, cyano, methoxy, or a C1-C4 alkyl group optionally substituted with 1-3 halogen substituents. In some versions, Rpis H or a C1-C4 alkyl group. In some versions, Rpis H or a C1-C3 alkyl group. In some versions, Rpis H.

[0091] In any version, herein, R" can be a C1-C20 alkyd group or a phenyl or heteroary 1 group, each of which groups may be optionally substituted. In some versions, R" is a C1-C20 alky l group. In some versions, R" is an optionally substituted Ci-Ce alkyl group. In some versions, R" is a Ci-C6alkyl group. In some versions, R" is an optionally substituted C1-C4 alkyl group. In some versions, R" is a C1-C4 alkyd group. In some versions, R" is methyl, ethyl, isopropyl, or isobutyl. In some versions, R" is methyl or isopropyl.

[0092] In some versions, the compound is:

[0093]

[0094] or a pharmaceutically acceptable salt thereof.

[0095] In some versions, the compound is:

[0096]

[0097] or a pharmaceutically acceptable salt thereof, and the compound is administered in a composition having a stereoisomeric excess of the compound with respect to a stereoisomer of the compound having the structure of:

[0098]

[0099] or a pharmaceutically acceptable salt thereof. The stereoisomeric excess in such versions can be at least 0.1%, at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%. In some versions, the compound is:

[0100]

[0101] or a pharmaceutically acceptable salt thereof, and is administered in a composition having a stereoisomeric excess of the compound with respect to a stereoisomer of the compound having the structure of:

[0102]

[0103] or a pharmaceutically acceptable salt thereof. The stereoisomeric excess in such versions can be at least 0.1%, at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%.

[0104] In some versions, the subject has been exposed to a second subject having an infection caused by the poxvirus. In some versions, the subject is suspected of having an infection caused by the poxvirus. In some versions, the subject has an infection caused by the poxvirus.

[0105] In some versions, the poxvirus is an orthopox virus. In some versions, the poxvirus is a monkeypox virus.

[0106] Another aspect of the invention is directed to a compound of the invention for use in a method of treating or reducing the likelihood of an infection caused by a poxvirus in a subject in need thereof. The method can comprise administering to the subject an amount of a compound effective to treat or reduce the likelihood of the infection. Any consideration pertaining to the methods outlined herein (including compounds, compositions, viruses, and / or subjects) can apply to such uses.

[0107] Another aspect of the invention is directed to a composition of the invention for use in a method of treating or reducing the likelihood of an infection caused by a poxvirus in a subj ect in need thereof. The method can comprise administering to the subject an amount of a composition effective to treat or reduce the likelihood of the infection. Any consideration pertaining to the methods outlined herein (including compounds, compositions viruses, and / or subjects) can apply to such uses.

[0108] Another aspect of the invention is directed to the use of a compound of the invention in the manufacture of a medicament for treating or reducing the likelihood of an infection caused by a poxvirus in a subject in need thereof. Any consideration pertaining to the methods outlined herein (including compounds, compositions viruses, and / or subjects) can apply to such uses.

[0109] The objects and advantages of the invention will appear more fully from the following detailed description of the preferred embodiment of the invention made in conjunction with the accompanying drawings.

[0110] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1. Chemical structures and brand names of the antiviral drugs against MPXV.

[0111] FIG. 2. Structures of FMCA (12) and its phosphoramidate prodrug FMCAP (17). FIG. 3. Structural modification of entecavir to FMCAP (17).

[0112] FIG. 4. Scheme 1: Synthesis of phosphoramidate prodrugs of 2'-flouro-6'-methylene carbocyclic purine analogs (17-20). Reagents and Conditions: (a) Ref.34(b) i) (HCHO)4, i-PrNH·TFA, diisopropylamine, THF; ii) NaBH₄, CeCl₃·7H₂O, Methanol; (c) Ref.33(d) Appropriate Boc-protected purine, DIAD, TPP, THF; (e) 2 M solution of TFA in DCM; (f) Phosphorochloridate reagent 16, NMI, THF.

[0113] FIG. 5. Scheme 2: Synthesis of phosphoramidate prodrugs of 2'-flouro-6'-methylene carbocyclic guanosine analog (FMCGP, 22). Reagents and Conditions: (a) 2 M solution of TFA in DCM; (b) HCOOH, 50 °C; (c) Phosphorochloridate reagent 16, NMI, THF; 0 °C-rt; (d) 2 M solution of TFA in water.

[0114] FIG. 6. Scheme 3: Separation of both chiral pure Sp (23) and / ?p (24) isomers of FMCAP (17) via chiral chromatography.

[0115] FIG. 7. Scheme 4: Synthesis of cytosine analog (FMCC, 29) and its phosphoramidate prodrug (34) via intermediate 3. Reagents and Conditions: (a) DIAD, TPP, THF; (b) 7 N ammonia solution in methanol; (c) 2,4,6-trisisopropylbenzeneslfonyl chloride, DMAP, EtsN, CH3CN; (d) 2 M solution of TFA in DCM; (e) TBDPSC1, imidazole, DMF; (f) 3,4-dihydro-2H-pyran, p-TSA, DCM; (g) 1 M solution of TBAF in THF, THF; (h) phosphorami date reagent 16, 2 M solution of ‘BuMgCI in THF, THF; (i) 2 M solution of TFA in DCM.

[0116] FIG. 8. Inhibition of VACV-GFP in Huh-7 cells / 96-well format by 17 (FMCAP) and its chiral pure p (23) and p (24) isomer of FMCAP.

[0117] FIG. 9. Racemic FMCAP (17) inhibits VACV-GFP propagation at nM concentration. Reconstruction of imaging fields captured on a CX7 Celllnsight HCS microscope 48 h postinfection with an indicated concentration of 17. In bottom panels, nuclei are in black, VACV-GFP is white.

[0118] DETAILED DESCRIPTION OF THE INVENTION

[0119] The following definitions are used to describe the invention. If a term is not specifically defined herein, the meaning given to the term is that which one of ordinary skill would apply to the term within the context of the term’s use.

[0120] The term ‘’compound”, as used herein, unless otherwise indicated, refers to any specific chemical or compound disclosed herein but may include, within context, tautomers, anomers, and where applicable, stereoisomers, optical isomers (enantiomers), or diastereomers (two chiral centers) thereof of these compounds, as well as pharmaceutically acceptable salts thereof, solvates, and / or polymorphs thereof. It is noted that in the event that a carbon range is provided for a compound, that range signifies that each and every carbon individually is considered part of the range. For example, a C1-C20 group describes a group with a single carbon, two carbon atoms, three carbon atoms, four carbon atoms, etc. up to twenty carbons.

[0121] The term “patient” or “subject” is used throughout the specification to describe an animal, preferably a domesticated animal or a human, more preferably a human to whom treatment, including prophylactic treatment, with the compositions according to the present invention is provided. For treatment of those infections, conditions, or disease states which are specific for a specific animal such as a human subj ect, the term “subj ecf ’ refers to that specific animal.

[0122] The term “pharmaceutically acceptable salt” is used throughout the specification to describe, where applicable, a salt form of one or more of the compounds described herein. The salts can increase the solubility of the compound in the gastric juices of the patient’s gastrointestinal tract in order to promote dissolution and the bioavailability of the compounds. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids, where applicable. Suitable salts include those derived from alkali metals such as potassium and, sodium, alkaline earth metals such as calcium, magnesium and ammonium salts, among numerous other acids well known in the pharmaceutical art. Sodium and potassium salts are particularly preferred as neutralization salts of the phosphates according to the present invention.

[0123] The term ‘'pharmaceutically acceptable derivative” is used throughout the specification to describe any pharmaceutically acceptable prodrug form (such as an ester, ether or amide or other prodrug group) which, upon administration to a patient, provides directly or indirectly the present compound or an active metabolite of the present compound. Any specific compound explicitly described herein by formula or structure can be provided in a form of a pharmaceutically acceptable derivative thereof and used as the compound in the methods described herein.

[0124] “Treat,” treating,” “treatment,” and the like means decreasing, suppressing, attenuating, diminishing, or arresting the underlying cause of a disease, disorder, or condition or any symptom associated therewith, or stabilizing the development or progression of a disease, disorder, condition, and / or any symptom associated therewith.

[0125] “Poxvirus” refers to a virus from the family poxviridae. Exemplary poxviruses include viruses from genera of poxviruses that infect humans, including Orthopoxvirus, Parapoxvirus, Yatapoxvirus, Molluscipoxvirus. Poxviruses from the genus Orthopoxvirus are referred to herein as “orthopox” viruses. Exemplary orthopox viruses include smallpox virus (variola), vaccinia virus, cowpox virus, and monkeypox (Mpox) virus. Exemplary poxviruses from the genus Parapoxvirus include orf virus, pseudocowpox, and bovine papular stomatitis virus. Exemplary poxviruses from the genus Yatapoxvirus include tanapox virus, and yaba monkey tumor virus. Exemplary poxviruses from the genus Molluscipoxvirus include molluscum contagiosum virus (MCV). The compounds of the invention can be used to treat or reduce the likelihood of an infection caused by any poxvirus from any of the aforementioned types.

[0126] “Exposed” as used herewith respect to a subject being exposed to a second subject refers to being in direct contact with the second subj ect or being within a distance of the second subject and for a time sufficient for transmission of a poxvirus infection to occur.

[0127] The term “alkyl” shall mean within its context a C1-C20 a linear, branched, or cyclic fully saturated hydrocarbon radical, which may be optionally substituted. It is noted that in the event that a carbon range is provided, that range signifies that each and every carbon is considered part of the range. For example, a C1-C20 group describes a group with a single carbon, two carbon atoms, three carbon atoms, four carbon atoms, etc. Exemplary' carbon ranges for the alkyls disclosed herein include C1-C20, C1-C12, Ci-Cs, C1-C5, C1-C4, C1-C3, or C1-C2. C1-C4 and C1-C3 alkyl groups are preferably linear or branched. Exemplary alkyls include methyl, ethyl, propyl, isopropyl, butyl, and isobutyl.

[0128] The term “ether” shall mean an optionally substituted Cito C20 ether group, formed from an oxygen and an alkyl group, or alternatively, may also contain at least one oxygen within the alkyl or alkylene chain.

[0129] The term “aromatic” or “aryl” shall mean within its context a substituted or unsubstituted monovalent carbocyclic aromatic radical having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl, anthracene, phenanthrene). Other examples include optionally substituted heterocyclic aromatic ring groups (“heteroaromatic” or “heteroaryl”) having one or more nitrogen, oxygen, or sulfur atoms in the ring, and preferably include five or six-membered heteroaryl groups, such as imidazole, furyl, pyrrole, furanyl, thiene, thiazole, pyridine, pyrazine, triazole, oxazole, among others, but can also include fused ring heteroaryl groups such as indole groups, among others. The preferred aryl group in compounds according to the present invention is a phenyl or a substituted phenyl group.

[0130] The term “heterocycle” shall mean an optionally substituted moiety which is cyclic and contains at least one atom other than a carbon atom, such as a nitrogen, sulfur, oxygen or other atom, which ring may be saturated and / or unsaturated.

[0131] The term “optionally substituted” is used synonymously with “substituted or unsubstituted.”

[0132] The term “unsubstituted” shall mean substituted only with hydrogen atoms. The term “substituted” shall mean, within the chemical context of the compound defined, a substituent selected from: optionally substituted hydrocarbyl (which may be substituted with an optionally substituted alkyl group or a fluoro group), preferably an alkyl (generally, no greater than about 3 carbon units in length), including CF3; an optionally substituted aryl; halogen (F, Cl, Br, I); thiol; hydroxyl; carboxyl; optionally substituted C1-C3 alkoxy; alkoxycarbonyl; CN; nitro; or an optionally substituted amine (e.g. an alkyleneamine or a C1-C3 monoalkyl or dialkyl amine). The optionally substituted moieties may be substituted with 3 or more unsubstituted substituents, preferably no more than 3 unsubstituted substituents and preferably with 1 or 2 unsubstituted substituents.

[0133] “ — ” between adjacent atoms indicates a bond that is present or absent depending on the valency of the adjacent atoms in a given specified structural context. The bond may comprise localized electrons between the adjacent atoms or delocalized electrons depending on the given specified structural context.

[0134] The term “acyl” is used throughout the specification to describe a group represented by the structure:

[0135]

[0136] wherein RAis a Ci to C20 alkyl group which may be optionally substituted. Preferred acyl groups are those where RAis a Cito C12 alkyl group. An acyl group in combination with a hydroxyl group results in an ester, and an acyl group in combination with an exocyclic amine group results in an amide. Acyl groups according to the present invention also include, for example, those acyl groups derived from benzoic acid and related acids, 3-chlorobenzoic acid, succinic, capric and caproic, lauric, myristic, palmitic, stearic and oleic groups, among numerous others and may include such related groups as sulfone groups such as mesylate groups. All groups may be appropriately substituted within context as otherwise described herein. One of ordinary skill in the art will recognize the acyl groups which will have utility in the present invention, either to synthesize the target pharmaceutical compounds or as prodrug of the nucleosides according to the present invention.

[0137] The term “amino acid’' or “amino acid residue” shall mean, within context, a radical of a D- or L-amino acid which is covalently bound through a carboxylic acid moiety of the amino acid to a nitrogen or oxygen on the remainder of the compound, thus forming, respectively, an amide or ester group linking the amino acid to the remainder of the compound. Amino acids may also be used to provide phosphoramidate groups in the compounds according to the present invention as otherwise described herein. Representative amino acids include both natural and unnatural amino acids, preferably including, for example, alanine, P-alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, glutamine, glycine, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, proline, serine, threonine, valine, tryptophan or tyrosine, among others. The chemical structures of the side groups of these amino acids are well known in the art.

[0138] The term “phosphate ester” or “phosphodiester” (which term includes phosphotri ester groups and phosphoramidate groups in context) is used throughout the specification to describe mono-phosphate groups at the 5' position of the carboy clic sugar synthon which are mono- or diesterified (or amidated and optionally esterified in the case of a phosphoramidate) such that the phosphate group is negatively charged or is rendered neutral, i.e., has a neutral charge. In some cases, the phosphodiesters or cyclic phosphoramidate groups are phosphate groups bound to both the 5’ and 3’ positions of the carboylic sugar synthon. Phosphate esters, phosphodi esters, phosphoramidate, and cyclic phosphoramidate groups for use in the present invention include those represented by the structures:

[0139]

[0140] each R5and R6is independently selected from H, a Ci to C20 alkyl group, alkoxyalkyl, aryloxyalkyl, such as phenoxymethyl, optionally substituted aryl (especially an optionally substituted phenyl group) and alkoxy, among others, including alkoxycarbonyloxy groups (e.g., (isopropoxy carbonyl)oxy] -methoxy) each of which groups may be optionally substituted (e.g., a phenyl or other group may be optionally substituted as otherwise described herein or preferably with from one to three, Ci-Ce alkyl groups, halogen, preferably F, Cl or Br, nitro, cyano, or C2-C6 carboxy ester groups) with the proviso that at least one R5group is other than H, or the two R5groups together form a five- or six-membered heterocyclic group;

[0141] B' is a group having the structure:

[0142]

[0143] or a group obtained from an amino acid (a natural or unnatural amino acid such as, for example, alanine, P-alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, glutamine, glycine, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, proline, serine, threonine, valine, tryptophan or tyrosine, among others) to preferably provide a group according to the structure:

[0144]

[0145] wherein i is 0, 1, 2 or 3 (preferably 0), R7is a Cito C20 alkyl, a C2to C2oacyl, alkoxyalkyl, aryloxyalkyl, such as phenoxymethyl, aryl, or alkoxy, among others, each of which may be optionally’ substituted; R8is sidechain of an amino acid, preferably a sidechain of an amino acid selected from the group consisting of alanine, 0-alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamic acid, glutamine, glycine, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, proline, serine, threonine, valine, tryptophan or tyrosine (preferably R8is derived from alanine, leucine, isoleucine or threonine, more preferably alanine-R8is methyl); and R" is a Ci to C20 alkyl or a phenyl or heteroaryl group, each of which groups is optionally substituted. “Nucleoside” in the phosphate esters, phosphodiesters, phosphoramidate, and cyclic phosphoramidate structures above refer to:

[0146]

[0147] and the connectivities of the phosphate esters, phosphodi esters, phosphoramidate, and cyclic phosphoramidate structures to the nucleoside occur through RXO, RlaO, or the combination of the two, in either orientation.

[0148] Preferred monophosphate esters for use in prodrug forms according to the present invention are those where one R5is a Ci to C20 alkyl group, more preferably a Ci to C3 alkyl group, all of which groups may be optionally substituted.

[0149] Other compounds which are preferred are as otherwise set forth herein, especially, where R1is a phosphoramidate group as otherwise described herein. A preferred phosphoramidate is:

[0150]

[0151] wherein R8, Rp, and R" are as defined elsewhere herein.

[0152] In some versions, R1and Rlatogether form a cyclic phosphoramidate structure, wherein R1and Rlatogether are:

[0153]

[0154] wherein R8and R" are as defined elsewhere herein. In some versions, R8is a C1-C6alkyl group. In some versions, R8is an optionally substituted C1-C4 alkyl group. In some versions, R8is a C1-C4 alkyl group. In some versions, R8is methyl, ethyl, isopropyl, or isobutyl. In some versions, R8is methyl or isopropyl. In some versions, R" is a C1-C6alkyl group. In some versions, R" is an optionally substituted C1-C4 alkyl group. In some versions, R" is a C1-C4 alkyl group. In some versions, R" is methyl, ethyl, isopropyl, or isobutyl. In some versions, R8is methyl, ethyl, or isopropyl. In some versions, R8is methyl or isopropyl, and R" is methyl, ethyl, or isopropyl. R8is methyl, and R" is methyl, ethyl, or isopropyl. In some versions, R8and R" are both isopropyl.

[0155] The term “independently'’ is used herein to indicate that the variable, which is independently applied, varies independently from application to application.

[0156] The term “effective amount” shall mean an amount or concentration of a compound according to the present invention which is effective within the context of its administration or use, which may be inhibitory, prophylactic and / or therapeutic. Within context, all active compounds which are used in the present invention are used in effective amounts. The present compound also relates to combinations of compounds which contain effective amounts of each of the compounds used, whether that combination is additive or synergistic in effect, provided that the overall effect of the combination of compounds is to inhibit the growth, reduce the likelihood of, or treat viral infections in patients as otherwise described herein.

[0157] The term “D-configuration” as used in the context of the present invention refers to the configuration of the compounds according to the present invention which mimics the natural configuration of sugar moieties as opposed to the unnatural occurring nucleosides or “L” configuration. The term “0” or “ anomer” is used to describe nucleoside analogs according to the present invention in which the nucleoside base is configured (disposed) above the plane of the carbocyclic moiety in the compound.

[0158] The term “stereoisomeric excess” refers to a greater abundance of a first defined stereoisomer of a compound of the invention with respect to a second defined stereoisomer of the compound of the invention. Stereoisomeric excess in a given composition can be expressed as a percent stereoisomeric excess (%se) according to the following formula:

[0159]

[0160] where is the mole fraction of the first defined stereoisomer of the total moles of the first and second defined stereoisomers, and F2 is the mole fraction of the second defined stereoisomer of the total moles of the first and second defined stereoisomers. The first and second stereoisomers can have 1 or more (e.g., 2, 3, 4, etc.) chiral centers. The first and second stereoisomers can be defined with respect to each other with respect to any one or more of such chiral centers, such that any stereoisomeric configuration at the non-defined chiral centers are permitted. In some versions, the first and second defined stereoisomers are epimers. In some versions, the first and second defined stereoisomers are enantiomers. The term “stereoisomeric excess” does not imply the presence of the second defined stereoisomer in the composition, such that in a limiting case the first defined stereoisomer can have a stereoisomeric excess of 100%. The stereoisomeric excess of the first defined stereoisomer over the second defined stereoisomer for any two pairs of stereoisomers provided herein can be at least 0.1%, at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100%.

[0161] The compounds of the invention can be coadministered with at least one other agent. The terms “coadminister” and “coadministration” are used synonymously to describe the administration of at least one of the compounds according to the present invention in combination with at least one other agent, preferably at least one additional anti-viral agent, including other nucleoside anti-viral agents which are specifically disclosed herein in amounts or at concentrations which would be considered to be effective amounts at or about the same time. While it is preferred that coadministered agents be administered at the same time, agents may be administered at times such that effective concentrations of both (or more) agents appear in the patient at the same time for at least a brief period of time. Alternatively, in certain aspects of the present invention, it may be possible to have each coadministered agent exhibit its inhibitory effect at different times in the patient, with the ultimate result being the inhibition of the virus and the treatment of the infections. Of course, when more than one viral or other infection or other condition is present, the present compounds may be combined with agents to treat that other infection or condition as required. In certain preferred compositions and methods, the compounds of the invention are coformulated and / or coadministered with at least one additional antiviral agent. Exemplary antiviral agents include brincidofovir, cidofovir, tecovirimat, acyclovir, famciclovir, ganciclovir, valaciclovir, vidarabine, ribavirin, zoster- immune globulin (ZIG), lamivudine, adefovir dipivoxil, entecavir, telbivudine, clevudine, tenofovir Hepsera (adefovir dipivoxil), lamivudine, entecavir, telbivudine, tenofovir, emtricitabine, clevudine, valtoricitabine, amdoxovir, pradefovir, racivir, BAM 205, nitazoxanide, UT 231-B, Bay 41-4109, EHT899, zadaxin (thymosin alpha-1), NM 283, ribavirin, VX-950 (telaprevir), SCH 50304, TMC435, VX-500, BX-813, SCH503034, R1626, ITMN-191 (R7227), R7128, PF-868554, TT033, CGH-759, GI 5005, MK-7009, SIRNA-034, MK-0608, A-837093, GS 9190, ACH-1095, GSK625433, TG4040 (MVA-HCV), A-831, F351, NS5A, NS4B, ANA598, A-689, GNI-104, IDX102, ADX184, GL59728, GL60667, PSI-7851, TLR9 Agonist, PHX1766, SP-30, and mixtures thereof.

[0162] The present invention also relates to pharmaceutical compositions comprising an effective amount of a compound as described above, optionally in combination with a pharmaceutically acceptable carrier, additive or excipient. In alternative embodiments, pharmaceutical compositions may also contain one or more additional antiviral agents as otherwise described herein in combination with an additive, carrier or excipient.

[0163] Pharmaceutical compositions based upon the nucleoside compounds according to the present invention comprise one or more of the above-described compounds in an amount effective to treat or reduce the likelihood of a poxvirus infection, optionally in combination with a pharmaceutically acceptable additive, carrier or excipient. One of ordinary skill in the art will recognize that the effective amount will vary with the infection or condition to be treated, its severity, the treatment regimen to be employed, the pharmacokinetics of the agent used, as well as the patient or subject (animal or human) to be treated.

[0164] The compound according to the present invention is preferably formulated in admixture with a pharmaceutically acceptable carrier. In general, it is preferable to administer the pharmaceutical composition in orally-administrable form, but certain formulations may be administered via a parenteral, intravenous, intramuscular, transdermal, buccal, subcutaneous, suppository, or other route. Intravenous and intramuscular formulations are preferably administered in sterile saline. In certain instances, transdermal administration may be preferred. Of course, one of ordinary skill in the art may modify the formulations within the teachings of the specification to provide numerous formulations for a particular route of administration without rendering the compositions of the present invention unstable or compromising their therapeutic activity. In particular, the modification of the present compounds to render them more soluble in water or other vehicle, for example, may be easily accomplished by minor modifications (salt formulation, esterification, etc.) which are well within the ordinary skill in the art. It is also well within the practitioner’s skill to modify the route of administration and dosage regimen of a particular compound in order to manage the pharmacokinetics of the present compounds for maximum beneficial effect in patients. In certain pharmaceutical dosage forms, the pro-drug form of the compounds, especially including acylated (acetylated or other) and ether (alkyl and related) derivatives, phosphate esters, and various salt forms of the present compounds, are preferred. One of ordinary skill in the art will recognize how to readily modify the present compounds to prodrug forms to facilitate delivery of active compounds to a targeted site within the host organism or patient. The practitioner also will take advantage of favorable pharmacokinetic parameters of the pro-drug forms, where applicable, in delivering the present compounds to a targeted site within the host organism or patient to maximize the intended effect of the compound.

[0165] The amount of compound included within active formulations according to the present invention is an amount effective for treating the poxvirus infection or reducing the likelihood of poxvirus infection. In general, an effective amount of the present compound in pharmaceutical dosage form usually ranges from about 0.05 mg / kg to about 100 mg / kg per day or more, more preferably, slightly less than about 1 mg / kg to about 25 mg / kg per day of the patient or considerably more, depending upon the compound used, the condition or infection treated and the route of administration. The compound according to the present invention is preferably administered in amounts ranging from about 0.5 mg / kg to about 25 mg / kg per day of the patient, depending upon the pharmacokinetics of the agent in the patient. This dosage range generally produces effective blood level concentrations of active compound which may range from about 0.05 to about 100 micrograms / cc of blood in the patient.

[0166] Administration of the active compound may range from continuous (intravenous drip) to several oral administrations per day (for example, Q. I. D.) or transdermal administration and may include oral, topical, parenteral, intramuscular, intravenous, sub-cutaneous, transdermal (which may include a penetration enhancement agent), buccal, and suppository administration, among other routes of administration. Enteric coated oral tablets may also be used to enhance bioavailability of the compounds from an oral route of administration. The most effective dosage form will depend upon the bioavailability / pharmacokinetics of the particular agent chosen as well as the severity of disease in the patient. Oral dosage forms are particularly preferred, because of ease of administration and prospective favorable patient compliance.

[0167] To prepare the pharmaceutical compositions according to the present invention, an effective amount of one or more of the compounds according to the present invention is preferably intimately admixed with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques to produce a dose. A carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral. In preparing pharmaceutical compositions in oral dosage form, any of the usual pharmaceutical media may be used. Thus, for liquid oral preparations such as suspensions, elixirs and solutions, suitable carriers and additives including water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like may be used. For solid oral preparations such as powders, tablets, capsules, and for solid preparations such as suppositories, suitable carriers and additives including starches, sugar carriers, such as dextrose, mannitol, lactose and related carriers, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like may be used. If desired, the tablets or capsules may be enteric-coated or sustained release by standard techniques. The use of these dosage forms may significantly enhance the bioavailability of the compounds in the patient.

[0168] For parenteral formulations, the carrier will usually comprise sterile water or aqueous sodium chloride solution, though other ingredients, including those which aid dispersion, also may be included. Of course, where sterile water is to be used and maintained as sterile, the compositions and carriers must also be sterilized. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed.

[0169] Liposomal suspensions (including liposomes targeted to viral antigens) may also be prepared by conventional methods to produce pharmaceutically acceptable carriers. This may be appropriate for the delivery of free nucleosides, acyl / alkyl nucleosides or phosphate ester pro-drug forms of the nucleoside compounds according to the present invention.

[0170] In various embodiments according to the present invention, the compounds are administered in oral dosage form in amounts ranging from about 5 pg, about 250 pg, about 1 mg, or about 5 mg up to about 500 mg or more at least once a day, preferably, up to four times a day. In some embodiments according to the present invention the compounds are administered in oral dosage form in amounts ranging from about 5 mg up to about 500 mg or more at least once a day, preferably, up to four times a day. The present compounds are preferably administered orally, but may be administered parenterally, topically or in suppository form.

[0171] The compounds according to the present invention, may advantageously be employed prophylactically to prevent or reduce the likelihood of a poxvirus infection or to prevent or reduce the likelihood of the occurrence of clinical symptoms associated with the poxvirus infection, or to prevent or reduce the likelihood of the spread of a poxvirus infection to another person. Thus, the present invention also encompasses methods for the prophylactic treatment of poxvirus infection. In this aspect, the present compositions may be used to prevent, reduce the likelihood of and / or delay the onset of a viral infection or a virus related disease state or condition or the spread of infection to other people. This prophylactic method comprises administering to a patient in need of such treatment or who is at risk for the development of a poxvirus infection, including a virus related disease state or condition or an infected patient who wishes to prevent or reduce the likelihood of a viral infection from spreading to another person, an amount of a compound according to the present invention alone or in combination with another anti-viral effective for alleviating, preventing, or delaying the onset of the poxvirus infection. In the prophylactic treatment according to the present invention, it is preferred that the antiviral compound utilized should be as low in toxicity and preferably nontoxic to the patient. It is particularly preferred in this aspect of the present invention that the compound which is used should be maximally effective against the virus and should exhibit a minimum of toxicity to the patient. In the case of compounds of the present invention for the prophylactic treatment of viral infections, these compounds may be administered within the same dosage range for therapeutic treatment (e.g., from about 5 u, about 250 ug, about 1 mg, or about 5 mg up to about 500 mg or more from one to four times per day for an oral dosage form) as a prophylactic agent to prevent the proliferation of the viral infection or alternatively, to prolong the onset of or reduce the likelihood of a patient contracting a virus infection which manifests itself in clinical symptoms.

[0172] The following documents are incorporated by reference in their entireties: US Patent 8,816,074, US Patent 8,946,244, US Patent 9,334,273, US Patent 10,533,008, US Patent 10,995,093, and US Patent 11,945,833.

[0173] The elements and method steps described herein can be used in any combination whether explicitly described or not.

[0174] All combinations of method steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.

[0175] As used herein, the singular forms “a,” "an." and “the” include plural referents unless the content clearly dictates otherwise.

[0176] Numerical ranges as used herein are intended to include every number and subset of numbers contained within that range, whether specifically disclosed or not. Further, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 2 to 8, from 3 to 7, from 5 to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

[0177] All patents, patent publications, and peer- reviewed publications (i.e., “references”) cited herein are expressly incorporated by reference to the same extent as if each individual reference were specifically and individually indicated as being incorporated by reference. In case of conflict between the present disclosure and the incorporated references, the present disclosure controls.

[0178] It is understood that the invention is not confined to the particular construction and arrangement of parts herein illustrated and described, but embraces such modified forms thereof as come within the scope of the claims.

[0179] EXAMPLES SUMMARY

[0180] The US Department of Health and Human Services declared that the recent monkeypox virus (MPXV) outbreak is a public health emergency. The data of Centers for Disease Control (CDC) and World Health Organization (WHO) for Monkeypox (Mpox) infections are worrisome because it indicates that MPXV is spreading as a sexually transmitted disease (STD) and has the potential to create pandemic concerns, which requires special attention for antiviral development. Here, a phosphoramidate prodrug of 2'-fluoro-6'-methylene-carbocyclic adenosine (FMCAP) and its purine and pyrimidine analogs are reported. FMCAP (17) and its chiral prodrugs. S'p (23) and Ap (24) analogs have demonstrated antiviral activity against vaccinia virus (VACV, an orthopox virus closely related virus to MPXV) and Monkeypox Virus-Luciferase (MPXV-Luc). Chiral pure Ap (24) isomer of FMCAP expressed an EC50 value of 85 nM against vaccinia virus compared to cidofovir (EC 50 = 6.7 pM) and brincidofovir (EC50 = 0.21 pM) and was 71-fold more potent than cidofovir and 2.1 -fold more potent than brincidofovir. Interestingly, against MPX-Luc, compound 24 expressed (EC50 = 0.43 pM) potent antiviral activity and reveals potential for preclinical development.

[0181] INTRODUCTION

[0182] To tackle the above-explained associated problem with current antivirals to treat Mpox infection, a library of synthesized carbocyclic nucleosides / nucleotide analogs have been screened against Modified Vaccinia virus Ankara-expressing GFP protein (MVA-GFP, a closely related virus to Mpox). As the MVA virus cannot infect human cells, infections were tested in baby hamster kidney 21 cells (BHK-21). In these efforts, a nucleotide analog (FMCAP) was identified a potent compound against MPXV. FMCAP is phosphoramidate prodrug of 2'-Fluoro-6'-methylene-carbocyclic adenosine (FMCA, FIG.2).

[0183] The antiviral activity of FMCAP against orthopox viruses was validated as it was subsequently shown to have potent activity against vaccinia virus (VACV; a closely related virus to MPXV) in the nanomolar range compared to cidofovir and brincidofovir’s micromolar range activity. Noteworthy to mention that FMCAP was found >70-fold more potent than cidofovir and ~2.5-fold more potent than brincidofovir and is almost as potent as tecovirimat. FMCAP offers a direct-acting antiviral (DAA) preclinical candidate against Mpox, which maybe used against MPXV infection. Earlier, in search of a better anti-HBV nucleoside agent, 2'-fluoro-6'-methylene carbocyclic adenosine (FMCA, 12) and its phosphoramidate prodrugs (FMCAP, 17, FIG. 2) were reported to be active against drug-resistant HBV strains. FMCA expressed antiviral activities against HBV with two mutations in reverse transcriptase (rt) that cause drug resistance to adefovir and lamivudine (L180M / M204V) in vitro.2Furthermore, when tested against three-mutation lamivudine / entecavir-resistant HBV (L180M / S202G / M204V), FMCAP (17) exhibited superior in vitro activity7compared to lamivudine or entecavir.23- 24Unexpectedly, it is shown herein that FMCAP is a potent preclinical candidate against the MPXV. This is highly surprising, given the massive phylogenetic differences between HBV and MPXV, as well as the respective polymerase targets (HBV reverse transcriptase, vs. the unrelated three-protein DNA polymerase complex comprising the F8, A22, and E4 proteins.2’

[0184] RESULTS AND DISCUSSION FMCAP (17) is a derived analog of the drug entecavir. Entecavir is specifically classified as a 2'-deoxy-6'-methylene carbocyclic nucleoside. Structurally, it differs from natural nucleosides in that the oxygen atom is replaced by a methylene group. This modification imparts unique properties to entecavir, making it a potent antiviral agent, particularly against the hepatitis B virus (HBV).26Whereas in the case of FMCAP (17), the 2'-position of carbocyclic moiety is substituted by a fluorine atom, and the base is replaced byadenine in place of guanine (FIG. 3). Additionally, FMCAP 5'-hydroxy is capped with the phosphoramidate moiety, which increases the lipophilicity and results in better cellular absorption.

[0185] It has been proven that inserting a fluorine atom at the 2'-position of a nucleoside strengthens the glycosidic bond against nucleoside hydrolase / phosphorylase enzymes and increases metabolic stability27In a previous report, 2'-fluoro-5-methyl- / EL-arabinofuranosyluracil (L-FMAU or clevudine) was found to be effective against HBV.28Another fluorine-containing drug, clofarabine,29also shows significant biological activity. Additionally, the insertion of 2'-fluorine has been proposed to contribute to interactions with target polymerases,27which turns out to be beneficial for antiviral activity.23-30Additionally, it is proven that the alteration in the sugar or base moiety enhances nucleoside stability and affinity for the viral DNA polymerase, thereby selectively inhibiting the replication of viruses more effectively. By practicing alteration either in base or sugar moieties of nucleosides / nucleotides, several clinical candidates have emerged as antiviral and anticancer agents.31Due to the tedious and challenging synthesis, the 2'-fluoro-6'-methylene-carbocyclic nucleoside analogs have not been well explored and pose potential for antiviral drug development. After the exciting antiviral potency of racemic FMCAP against MPXV, the complete structure-activity relationship (SAR) of this class of molecules against poxviruses and herpesviruses was explored. In search of anti-MPXV lead compounds that are superior to cidofovir and brincidofovir, a series of purine-derived phosphoramidate prodrugs of N-methyl FMCA (13), FMCG (21), and its N-6-methyl prodrug of FMCG (15) were synthesized. Additionally, pyrimidine-derived analogs of 2'-fluoro-6'-methylene-carbocyclic cytosine (FMCC, 29) and its prodrug FMCCP (34) were synthesized and tested against the MVA-GFP (a closely related virus to Mpox) in BHK-21 cells.

[0186] Chemistry

[0187] Earlier, the synthesis of FMCAP (17) via Vince lactam and carbocyclic ketone (1) was reported.12, 33To synthesize the purine and pyrimidine analogs, first, synthesis of ketone (1) was accomplished from D-ribose in 9 steps.34Furthermore, compound 1 was converted to crucial 2'-fluoro-containing intermediate 3 via our earlier reported procedure (Scheme 1 (FIG.

[0188] 4)).33Compound 3 was coupled with the appropriate N,N'-diBoc-protected purine base under Mitsunobu conditions. Synthesis of appropriate Boc-protected purine bases (4-7) were carried out by the reported protocol of Dey, S. et al.35N,N'-diBoc-protected base (4-7) coupled with key intermediate 2-fluoro-l -hydroxy carbocyclic ring (3) in the presence of diisopropyl azodicarboxylate (DIAD) and triphenylphosphine (TPP) in THF under Mitsunobu condition to produce coupled product (8-11).

[0189] The tert-butyl and Boc protecting groups of compounds 8, 9, 10, and 11 were removed by using 2 M solution of TFA in DCM at room temperature (rt) to afford final nucleoside FMCA (12), 6-A-methyl FMCA (13), 6-chloro-FMCG (14), and 6-V-methyl FMCG (15) in 75-80% yields Scheme 1 (FIG. 4). To improve the antiviral potency and cellular uptake, 5'-hydroxy of compounds 12-15 were capped with phosphoramidate moiety and targeted phosphoramidate prodrugs 17-20 were synthesized. Additionally, phosphoramidate prodrugs help bypass the first, rate-limiting step of mono phosphorylation and assist in the generation of the active triphosphate nucleotide substrate of the viral polymerase.36To bypass the first rate-limiting step of mono-phosphorylation, phosphoramidate prodrugs of compounds 12, 13, 14, and 15 were synthesized by condensing nucleosides with phosphorochloridate intermediate 16. First, phosphorochloridate reagent 16 was prepared by coupling phenyl phosphoryl chloride with L-alanine isopropyl ester in DCM at -78 °C in 65% yield.33After that, nucleosides 12, 13, 14, and 15 were condensed with 16 in the presence of N-methyl imidazole (NMI) in THF at room temperature (rt) to produce targeted racemic (Rp / < S’p) phosphoramidate prodrugs, 17 (FMCAP), 18 (6-N-methyl-FMCAP), 19 (6-chloro-FMCGP) and 20 (6-N-methyl-FMCGP).37Noteworthy to mention that compound 20 is a double prodrug and it is 6-N-methyl and 5 '-phosphoramidate prodrug of guanosine analog.

[0190] The next target was the synthesis of phosphoramidate prodrug of guanosine analog (FMCGP, 22) (Scheme 2 (FIG. 5)). To accomplish the synthesis FMCGP, initially, analog of guanosine 21 was synthesized. As discussed in Scheme 1 (FIG. 4), intermediate 3 was condensed with 2-N,N'-diBoc-6-chloro adenine (6) in the presence of DIAD and TPP in THF under Mitsunobu condition to afford coupled compound 10 in 56% yield. To construct guanosine analog 21, intermediate 10 served as key compound and Boc and tert-butyl groups of 10 were deprotected with formic acid to afford 21. Compound 10 was treated with formic acid at 50 °C to produce final deprotected guanosine analog 21. To synthesize phosphoramidate prodrug 22 (FMCGP), initially regular practiced route 1 was adopted, and coupling of nucleoside 21 with phosphoramidate reagent (16) was tried with various protocols33, 38, 39, but in each attempt, only reactant 21 was observed as such without formation of coupled product 22. Then, it was thought that THF is not a good solvent for the coupling of 2'-fluoro-6'-methylene-carbocyclic guanosine analog (21) with phosphoramidate reagent 16.

[0191] Consequently, for coupling various solvents such as DMF, DCM, toluene, etc. were tried with an increased equivalent of NMI, however, each effort was unsuccessful in furnishing the final phosphoramidate prodrug 22. Therefore, it was concluded that either due to the low solubility of guanosine or the conformational constraint of compound 21, it is not coupling with phosphoramidate coupling reagent. Therefore, route 2 was adopted to construct the final phosphoramidate prodrug of guanosine (22). Compound 10 was treated with the 2 M solution of TFA in DCM at rt to give intermediate 14. Compound 10 was stirred at rt in 2 M solution of TFA in DCM, which provides approximately 60% of intermediate 14 and 40% of the final guanosine nucleoside analog (21) by converting 6-chloro to 6-hydroxy of intermediate 14.

[0192] Finally, intermediate 14 was coupled with phosphoramidate reagent 16 in the presence of NMI in THF to yield phosphoramidate prodrug of 6-chloro guanosine analog (19). Conversion of 6-chloro to 6-hydroxy of 19 was carried out by treatment of 2 M aqueous solution of TFA which finally yielded targeted prodrug FMCGP (22) in good yield.

[0193] After identifying potent anti-Mpox activity of racemic (Rp / Sp) FMCAP (17), it was of great interest to separate chiral pureSp and / ?p isomer of FMCAP (17). The racemic Sp / Rp, FMCAP (17) was separated into chiral pure Sp (23) and Rp (24) isomer of FMCAP via chiral chromatography (Scheme 3 (FIG. 6)). To determine the specific conformations of both 5' and Rp isomers, initially, X-ray crystallographic studies were attempted, but it was not possible to obtain crystals. Therefore, the conformation of both Sp and / p isomers was determined by the published studies of sofosbuvir by Bruce Ross et al. via phosphorus NMR.39The reported literature revealed that31P-NMR of chiral pure phosphoramidate prodrug of. S'p isomer demonstrates a high field ppm level value of phosphorus in comparison of Rp isomer.38In31P-NMR analysis, chiral pure S'p isomer of FMCAP (23) exhibited a value of 3.42 ppm, and 7?p (24) expressed 2.89 ppm of phosphorus.

[0194] The next goal was to synthesize pyrimidine analogs of 2'-fluoro-6'-methylene carbocyclic cytosine (FMCC, 29) and its phosphoramidate prodrug 34. In this effort, 3-N-benzoyl-protected uracil base (25) was first coupled with key intermediate 3 (Scheme 4 (FIG.

[0195] 7)) under Mitsunobu coupling conditions with DIAD and TPP in THF to give coupled product 26 in 41% yield. Selective A-benzoyl deprotection of 26 was performed with 7 N methanolic ammonia solution. Compound 26 was treated with 7 N solution of ammonia in methanol at rt to render compound 27 in 52 % yield. To synthesize FMCC (29), uracil intermediate 27 was converted into the cytosine moiety. Compound 27 was treated with 2,4,6-triisopropylbenzenesulfonyl chloride in the presence of 4-(dimethylamino)pyridine (DMAP) and triethylamine (EbN) in acetonitrile to obtain cytosine intermediate 28 in 74% yield. Finally, after deprotection of t-butyl protecting groups of 27 by 2 N solution of TFA in DCM furnished cytosine analog (FMCC, 29) in 81% yield.

[0196] At the first attempt to synthesize phosphoramidate prodrug FMCCP (34), a direct coupling of phosphorochloridate intermediate 16 was tried in the presence of either NMI orlBuMgCl (2 M solution in THF); however, in both cases the reaction was unsuccessful. Then, it was predicted that the lower solubility of 29 may restrict its coupling with the phosphoramidate reagent. Therefore, to increase the solubility of compound 29, selective 3'-hydroxy of protection of 29 was performed with dihydropyran (DHP). First, the protection of 5'-hydroxy of 29 was carried out with tert-butyl diphenyl silyl chloride (TBDPSC1). 29 was treated with the TBDPSC1 in the presence of imidazole in DMF to produce 5'-hydroxy protected intermediate 30 in 69% yield. After that, the 3 '-hydroxy of 30 was protected with 3,4-dihydro-2H-pyran (DHP) in the presence of para toluene sulfonic acid o-TSA) in DCM to give 3' and 5'-protected compound 31 in 76% yield. Selective deprotection of TBDPS of 31 was carried out with fefra-butylammonium fluoride (TBAF). Compound 31 was stirred with 1 M solution of TBAF in THF to afford 32 in 82% yield. Eventually, coupling of 32 with phosphoramidate reagent 16 was performed in the presence of 1 M solution 'BuMgO in THF to obtain phosphorami date product 33 in 54%. Finally, 3 '-deprotection of THP of 33 was performed with a 2 M solution of TFA in DCM to produce phosphoramidate prodrug of FMCCP (34) in 48% yield.

[0197] Antiviral Activity

[0198] To evaluate the anti-MPXV activity of synthesized nucleoside analogs as well as their prodrugs, initially, nucleos(t)ides were tested against Modified Vaccinia virus Ankara-expressing GFP protein (MVA-GFP) in BHK-21 cells. First all compounds were screened at 20 pM and those with >50% inhibition in >3 independent experiments (each in at least duplicates) were chosen for further antiviral evaluation (Table 1). Compounds 12, 17, 21, and 29 showed >50% inhibition and were selected for dose-dependent evaluation. Furthermore, compounds that have demonstrated >50% inhibition (Table 1) were tested in vitro against vaccinia virus-green fluorescent protein (VACV-GFP, a closely related virus to MPXV) in Huh7-C3 cells at the range 20 pM to 9.1 nM using cidofovir and brincidofovir as controls.40Huh7-C3 cells were seeded in 96-well plates at a concentration of 1 x 104cells / well, then treated with compounds and subsequently infected with VACV-GFP at 0.06 multiplicity of infection (MOI). Total fluorescence intensity per well was recorded and used to calculate ECso values, which were defined as the concentration where compound reduces 50% fluorescence intensity.

[0199] Table 1. Preliminary screening results of hit nucleos(t)ide analogs against MVA-GFP in BHK-21 cells (NI = no inhibition; ND = not determined).

[0200]

[0201]

[0202] The cytotoxicity of compounds was evaluated in Huh-7 cells in CellTiter 96 NonRadioactive Cell Proliferation assay system (Promega) using the 2,3-Bis-(2-Methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, disodium salt (XTT) method.41Doseresponse curves (EC50) and cell viability (CC50) were determined using GraphPad software (San Diego, California). In dose-dependent screening, the antiviral results of select compounds were encouraging: 17 (FMCAP), a prodrug of FMCA (12) exhibited an EC50 value of 0.094 pM (Table 2), however, 12 (FMCA), 21 (FMCG), and 29 (FMCC) demonstrated lower EC50 values of 20.8, 225, and 12.0 pM respectively without any cytotoxicity up to > lOOpM. However, the phosphoramidate prodrug 34 (FMCCP) of 29 (FMCC) expressed an improved antiviral potency EC50 value of 3.9 pM, which is almost 4-times more potent than FMCC (29), demonstrating that the prodrug approach is beneficial. Noteworthy to mention that compound 17 (FMCAP) was the racemic mixture of both Sp and?p isomers. After witnessing these outstanding antiviral results of 17 (FMCAP), the core focus turned to investigating the antiviral efficacy of the chiral pure Sp and Rp isomers of 17 (FMCAP) against vaccinia virusgreen fluorescent protein (VACV-GFP) and Monkeypox Virus-Luciferase (MPXV-Luc). A repeated scalable synthesis of 17 was performed and both chiral pure Sp (23) and Sp (24) isomers were separated via chiral chromatography.

[0203] Table 2: Antiviral activity of select synthesized compounds against vaccinia virus- green fluorescent protein (VACV- GFP) in Huh7-C3 Cells.

[0204]

[0205]

[0206] The antiviral screening of pure Sp and Rp isomer of FMCAP exhibited promising results against VACV- GFP. Racemic 17 (FMCAP) exhibited an EC50 value of 0.094 pM with SI > 1064 (Table 2; experiments conducted in at least 3 technical and 2 biological replicates each, FIGS.8 and 9); however, chiral pure. S'p (23) and / p (24) isomers exhibited EC50 values of 0.1280 (SI > 781) and 0.085 pM (>1176) respectively, compared to drug cidofovir42(EC50 = 6.7 pM; SI > 14.9) and its prodrug brincidofovir42(EC50 = 0.21 pM, SI > 476).

[0207] This finding was exciting, as the Rp (24) isomer of FMCAP exhibited enhanced antiviral potential than Sp (23) isomer. Rp isomer of FMCAP was found 78 times more potent than cidofovir, ~ 2.5 times more potent than brincidofovir and almost equally potent as tecovirimat against VAVC-GFP. Notably, cidofovir is associated with severe effects of nephrotoxicity17and its lipid conjugate prodrug brincidofovir is also not safe and demonstrated elevated liver enzyme during the treatment of Mpox-infected patients.20The prodrug 17 (FMCAP) was 221 times more potent (Table 2) than 12 (FMCA), which validates the hypothesis that the first step of mono-phosphoration was a significant roadblock for the antiviral activity of 12 (FMCA, parental nucleoside). After the interesting antiviral data of FMCAP (17) against VACV, cytosine phosphoramidate 34, (FMCCP,) 17 (FMCAP), and its chiral pure. S’p (23) and Rp (24) isomers were evaluated against MPXP-Luc in Huh7 cells (Table 3)

[0208] Table 3: Antiviral activity of select phosphoramidate analogs of synthesized compounds against Monkeypox Virus-Luciferase (MPXV-Luc) in Huh7-C3 Cells.

[0209]

[0210] Against MPXV-Luc, FMCAP (17) exhibited EC 50 of 0.46 pM with SI >214. However, its chiral pure < Sp and / p analogs have demonstrated EC50 value of 0.51 and 0.43, respectively with a good SI. Our finding indicates that Rp (24) isomer of FMCAP is an excellent candidate for preclinical development against MPXV and other poxviruses. Compound 17 (FMCAP) has previously demonstrated potent activity against wild-type and drug-resistant hepatitis B virus (HBV) and is a preclinical candidate against the drug-resistant HBV.24, 32, 33However, the findings herein indicated that 17 (FMCAP) and its chiral pure Sp (23) / Rp (24) have potent activity against MPXV and hold potential for preclinical development as a broad-spectrum direct-acting antiviral (DAA) against poxviruses.

[0211] Anti-orthopox virus activity of known drugs that have been approved for the treatment of HBV (entecavir, FTC, besifovir, telbivudine), HIV (ddl, AZT, Abacavir), HCV (sofosbuvir) or HSV (acyclovir, penciclovir) infections were also examined (Table 4). None of these significantly inhibited MVA-GFP in BHK-21 cells. This result highlights the enormous differences in nucleos(t)ide susceptibility of DNA synthesis by MPXV vs. other viruses, and demonstrates that inhibition of MPXV by FMCAP is a novel unexpected finding, despite the reported inhibitory of the compound against the highly divergent HBV virus.

[0212] Table 4. Lack of inhibitory activity of known HIV, HBV, HCV antiviral drugs tested against MVA-GFP in BHK-21 cells.

[0213]

[0214] Conclusions

[0215] Here, the results show that the 2'-fluoro-6'-methylene carbocyclic adenosine phosphoramidate prodrug (17, FMCAP) has excellent antiviral activity' against VACV-GFP and MPXV -Luc and has potential for further preclinical development against MPXV. Both chiral pure isomers of FMCAP 5p (23) and Rp (24) were identified more potent in comparison to drug cidofovir and brincidofovir. Against VACV-GFP, chiral pure Rp (24) isomer of FMCAP exhibited 78 times more potency than cidofovir and ~ 2.5 times more potent than brincidofovir. Additionally, compound 17 was found to be almost as potent as tecovirimat. Furthermore, extended SAR studies of 2'-fluoro-6'-methylene carbocyclic purine and pyrimidines analogs were performed, and various new purine and pyrimidine derivations have been synthesized. Considering the associated toxicities of cidofovir and brincidofovir, additional, FMCAP (17) and its chiral pure Sp and Rp isomers are excellent candidate for the preclinical and clinical development against poxviruses.

[0216] EXPERIMENTAL SECTION

[0217] General Analytical Methods

[0218] Reagents and anhydrous solvents were purchased from commercial sources and used without further purification. Moisture-sensitive reactions were performed using oven-dried glassware under a nitrogen or argon atmosphere. Reactions were monitored by thin-layer chromatography plates (TLC silica gel GF 250 microns) that were visualized using a Spectroline UV lamp (254 nm) and developed with 15% solution of sulfuric acid in methanol. Column chromatography was performed on silica gel 60A, 40-63pM (230 X 400 mesh, Sorbent Technologies). Preparative normal phase chromatography was performed on a CombiFlash Rf 150 (Teledyne Isco) with pre-packed RediSep Rf silica gel cartridges or on RediSep® gold C18 reverse phase columns. Melting points were recorded on a Mel-temp II laboratory device and are uncorrected. Nuclear magnetic spectra were recorded on Varian Inova 500 spectrometer at 500 MHz for 'H NMR, 202 MHz for31P NMR, 125 MHz for13C NMR and 470 MHz for19F NMR with tetramethylsilane as an internal standard. CFCh (trichloro-fluoro methane was used as the internal standard (reference) for19F-NMR. Chemical shifts (5) are quoted as s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (double doublet) and dt (double triplet). Optical rotations were measured on a JASCO DIP-370 digital polarimeter. High-resolution mass spectroscopy (HRMS) spectra were measured on Bruker Ultra-high resolution QTOF MS Impact II spectrometer. Samples were infused at 3 pL / min, and spectra were obtained in the positive or negative ionization mode with a typical resolution of 20,000 or greater. Purity of all tested compounds are >95%, as determined by their elemental analysis (Table- SI) or by HPLC / UV. Elemental analysis were performed by the Atlantic Microlab Inc. Norcross, GA. HPLC / UV were determined with a Waters HPLC coupled to a photodiode array. 5 / zL of sample 0.5 mg / mL in methanol, or in acetonitrile or in mixture of DMSO / MeOH (0.5: 10) were injected, using an XBrigde C18, 3.5 zzm. (4.6 X 150) mm column at 25 °C with flow rate 0.8 mL / min or with UPLC BEH Cl 8, 1.7 z.zm (100 X 2.1) mm at 50 °C with a flow7rate 0.55 mL / min. The mobile phases were a mixture of A = 10 mM ammonium acetate in water and B = acetonitrile (ACN), and A = 0.05% formic acid (FA) in water and B = 0.05% in acetonitrile (ACN). Purity is given as % of absorbance at Max plot. Optical purity of chiral intermediates and final compound were determined by the chiral HPLC. Chiral HPLC / UV were determined with a Waters HPLC coupled to a photodiode array. 10 pL of samples 0.5 mg / mL in methanol, were injected, using an CHIRALCEL OX-H, 5pmm (4.6 X 250mm) column at 30 °C with flow rate 3.0 mL / min.

[0219] General Procedure for the Synthesis of Compounds 8-11

[0220] To a stirred solution of tri phenylphosphine (2.5 eq), in THF (50 mL) at -10 °C, DIAD was added (2.5 eq) dropwise, reaction mixture was stirred at this temperature for 30 minutes, and then a solution of appropriated Boc protected purine base (1.5 eq) in THF (20 mL) was added. This mixture was stirred for 30 min at 0 °C. Then reaction mixture was again cooled to -20 °C and compound 3 (1 eq.) in THF (10 mL) was added dropwise. The reaction temperature was raised to room temperature and stirred for 1.5 h. The reaction was quenched with methanol and solvent was removed under reduced pressure, the crude residue was purified by silica gel column chromatography (5% EtOAc / hexane) to give couple compounds 8-11 as white foam.

[0221] 9-(( / / ?,3 / ?,- / / ?)-3-tert-butoxy-4-(tert-butoxymethyl)-2-fluoro-5-methylenecyclopentyl)- / V, X-diboc-9H-purin-6-amine (8). Yield (3.2 g, 74 %). [a]24D = -51.47 (c 1.0, CHCh); ’H NMR (500 MHz, CDCh) 5 8.91 (s, 1H), 8.24 (s, 1H), 5.97 (d, J = 30.5 Hz, 1H), 5.32 (s, 1H), 4.90 (dd, J= 9.0, 52.5 Hz, 1H), 4.49-4.77 (m, 1H), 4.33 (d, J = 14.0 Hz, 1H), 3.62-3.60 (m, 1H), 3.54-3.50 (m, 1H), 2.85 (bs, 1H), 1.47 (s, 18H), 1.28 (s, 9H), 1.27 (s, 9H); ^C H} NMR (125 MHz, CDCh) 8 153.9, 152.0, 150.4, 150.2, 150.0, 146.4, 145.3, 128.1, 111.7, 109.9, 83.7, 75.7, 73.2, 62.6, 49.8, 28.2, 27.8, 27.5;19F-NMR (470 MHz, CDCh) 8 -191.1 (ddd, J= 17.5, 35.0 & 49 Mz, 1F); HRMS (El) Calcd for (C30H46FN5O6+H)* 592.3505, found 592.3592.

[0222] r<?r / -butyl(9-((l R,2R,4R)-3-(tert-biitoxy)-4-(r<? / ‘ / -butoxymethyl)-2-fluoro-5-methylenecyclo-pentyl)-9H-purin-6-yl)(methyl)carbamate (9). Mp 215-218 °C; [a]24D = -46.0 (c 0.5, MeOH); 'H NMR (500 MHz, CDCh) 8 8.80 (s, 1H), 8.18 (s, 1H), 5.97 (d, J = 31.0 Hz, 1H), 5.33 (s, 1H), 4.97 (dt, J= 6.0 & 56.5 Hz, 1H), 4.97 (s, 1H), 4.32 (d, J= 14.0 Hz, 1H), 3.61-3.51 (m, 2H), 3.56 (s, 3H), 2.85 (bs, 1H), 1.52 (s, 9H), 1.28, (s, 18H);13C{1H} NMR (125 MHz, CDCh): 6 154.0, 153.6, 153.1, 151.7, 146.5, 143.5, 126.1, 111.6, 81.9, 73.5, 73.2, 62.7, 49.8, 35.0, 28.2, 27.5, 21.9;19F NMR (470 MHz, CDCh) 8 -191.22 (ddd, J= 32.0, 45.5 & 46.0 Hz, IF); HRMS (El) Calcd for (C26H4oFN504+H)+506.3137, found 506.3138. di-tert-butylacetyl(9-((lR,2R,4R)-3-(tert-butoxy)-4-(tert-butoxymethyl)-2-fluoro-5-methylenecyclopentyl)-6-chloro-9H-purin-2-yl)carbamate (10).JH NMR (500 MHz, CDCI3) 88.25 (s, lH), 7.19 (s, 1H), 5.77 (d, J = 28.0 Hz, 1H), 5.26 (s, 1H), 4.78 (s, 1H), 4.73 (d, J= 49.5 Hz, 1H), 4.47 (d, J= 13.5 Hz, 1H), 3.53-3.43 (m, 2H), 2.73 (bs, 1H), 1.38 (s, 18H), 1.18 (s, 9H), 1.17 (s, 9H);13CfH} NMR (125 MHz, CDCh): 5 153.2, 152.0, 151.0, 150.6, 150.0, 145.7, 129.3 95.9 (d,.7 = 197.0 Hz), 83.6, 75.7, 73.3 28.2, 27.9, 27.5;19F NMR (470 MHz, CDCh) 8 -191.75 (ddd, J = 14.0, 28.0 & 42.0 Hz, IF); HRMS (El) Calcd for (C3OH45C1N506+H)+626.3115, found 626.3118.

[0223] te / -butyl (2-(bis(fer / -butoxycarbonyl)amino)-9-((lR,2R,4R)-3-( / er / -butoxy)-4-(terZ-butoxymethyl)-2-fluoro-5-methylenecyclopentyl)-91Z-purin-6-yl)(methyl)carbamate (11).1H NMR (500 MHz, CDCI3) 88.17 (s, 1H), 7.29 (s, 1H), 5.88 (d, J= 31.0 Hz, 1H), 5.28 (s, 1H), 4.82 (d, J= 51.5 Hz, 1H), 4.77 (s, 1H), 4.30 (d, J= 13.5 Hz, 1H), 3.60-3.53 (m, 1H), 3.50 (s, 3H), 2.83 (bs, 1H), 1.49 (s, 9H), 1.47 (s, 18H), 1.27 (s, 18H); ^C H} NMR (125 MHz, CDCh): 6 154.5, 154.1, 153.4, 151.8, 151.1, 146.4, 144.0 124.3, 111.5, 97.3, 83.1, 81.9, 75.6, 73.4, 62.6, 58.3, 49.7, 34.8, 28.2, 27.9, 27.5, 21.9, 21.7;19FNMR (470 MHz, CDCh) 8 -191.33 (ddd, J = 10.5, 31.5 & 45.5 Hz, IF); HRMS (El) Calcd for (C36H57FN6O8+H)+721.4295, found 721.4300.

[0224] General Procedure for the Synthesis of Compounds 12-15

[0225] Compound 8-11 (1.0 eq.) was dissolved in DCM (30 mL). Added trifluoroacetic acid (6 mL) to this solution and mixture was stirred at room temperature for 16 h. TFA with excess solvent was removed under reduced pressure and residue was co-evaporated three times with methanol to remove residual trifluoroacetic acid and neutralized with aqueous ammonia solution, concentrated under reduced pressure. The obtained crude was purified by column chromatography on silica gel (6%methanol / DCM) to give final nucleosides 12-15 as white solid in good yields.

[0226] (+)-9-[(17?, 2'R, 3'R, 47?)-2'-Fluoro-3'-hydroxy-4'-(hydroxymethyl)-5'-methylene-cydopentan-l'-yl]adenine (FMCA, 12). Yield 1.2 g, 77%; mp 215-218 °C;

[0227] [a]24D= +152.10 (c 0.5, MeOH); ’H NMR (500 MHz, CD3OD) 8 8.26 (s, 1H), 8.10 (d, 1H), 5.90 (d, J = 26.0 Hz, 1H), 5.46 (s, 1H), 4.96 (dt, J= 2.5 & 52.5 Hz, 1H), 4.95 (s, 1H), 4.44 (dd, J= 13.5 Hz, 1H), 3.88-3.82 (m, 2H), 2.81 (bs, 1H);13CfH} NMR (125 MHz, CD3OD): 8 156.0, 152.5, 149.9, 146.0, 141.1 (d, J= 5.3 Hz), 117.9, 111.7, 95.9 (d, J= 184.0 Hz), 72.9 (d, J= 23.6 Hz ), 61.7, 57.5 (d, J =17.4 Hz), 51.0;19F NMR (470 MHz, CD3OD) 8 -192.93 (ddd, J= 14.0, 28.0 & 56.0 Hz, IF); HRMS (El) Calcd for (Ci2Hi4FN5O2+H)+280.1204, found 280.1216. (+)-9-[(l'7?, 2'7?, 3'7?, 4'7?)-2'-Fluoro-3'-hydroxy-4'-(hydroxymethyl)-5'-methylene-cyclopentan-l'-yl]-6-A-methyladenine (13). Mp 215-218 °C; [OC]24D = +129.10 (c 0.5, MeOH); ’H NMR (500 MHz, CD3OD) 5 8.30 (s, 1H), 8.03 (s, 1H), 5.88 (d, J = 26.0 Hz, 1H), 5.44 (s, 1H), 4.94 (d, J = 52.5 Hz, 1H), 4.92 (s, 1H), 4.43 (d, J = 13.5 Hz, 1H), 3.90-3.82 (m, 2H), 3.15 (s, 3H), 2.80 (bs, 1H); ^CfH} NMR (125 MHz, CD3OD): 5 155.4, 152.5, 146.1, 140.5, 118.4, 111.7, 95.9 (d, J= 197.0 Hz), 73.0, 72.8, 61.7, 57.6, 57.4, 51.1;19F NMR (470 MHz, CD3OD) 5 -195.84 (ddd, J = 17.5, 28.0 & 42.0 Hz, IF); HRMS (El) Calcd for [CuHieFNsCh+H] 294.1361, found 294.1366.

[0228] (17?,27?,37?,57?)-3-(2-amino-6-chloro-9H-purin-9-yl)-2-fluoro-5-(hydroxymethyl)-4-methylenecyclopentan-l-ol (14). Yield (45 mg, 75%); 'HNMR (500 MHz, CD3OD) 58.01 (d, J= 2.5 Hz, 1H), 5.82 (d, J= 27.4 Hz, 1H), 5.43 (s, 1H), 4.98-4.91(m, 2H), 4.42 (dt, J= 2.4 & 13.5 Hz, 1H), 3.89-3.83 (m, 1H), 3.82-3.75 (m, 1H), 2.79 (t, J= 7.5 Hz, 1H);nC{’H} NMR (125 MHz, CD3OD) 5 160.5, 154.5, 150.3, 146.0, 142.8, 122.8, 111.7, 95.8 (J= 185.1), 73.1, 62.0, 57.5, 51.2;19F NMR (470 MHz, CD3OD) 5 -195.6 to -195.8 (m, IF); HRMS-ESI ( / z):

[0229] [M + H]+calculated for [Ci2Hi4ClFN5O2]+314.0815; found 314.0809.

[0230] (+)-9-[(l'7?, 2'7?, 3'7?, 4'7?)-2'-Fluoro-3'-hydroxy-4'-(hydroxymethyl)-5'-methylene-cyclopentan-r-yl|-2-amino-6- / V-niethyladenine (15). Mp 206-208 °C; [a]24D = + 139.10 (c 0.5, MeOH); ’H-NMR (500 MHz, CD3OD) 5 7.65 (s, 1H), 5.70 (d,.7= 28.0 Hz, 1H), 5.40 (s, 1H), 4.92 (s, 1H), 4.88 (d, J= 39.5 Hz, 1H), 4.40 (d, J=13.5 Hz, 1H), 3.87-3.75 (m, 2H), 3.08 (s, 3H), 2.78 (bs, 1H); ^C H} NMR (125 MHz, CD3OD): 5160.7, 155.8, 146.4, 137.4, 112.4, 111.1, 96.0 (d, J= 184.0 Hz), 73.0, 72.8, 62.0, 57.2, 57.1;19F NMR (470 MHz, CD3OD) 5 -195.74 (ddd, J = 19.5, 28.0 & 42.0 Hz, IF); HRMS (El) Calcd for [Ci3Hi7FN6O2+H]+309.1470, found 309.1475.

[0231] General Procedure for the Synthesis of Compounds 17-20

[0232] Phenyl di chlorophosphate (1.0 mol eq.) and the L-alanine isopropyl ester hydrochloride salt (1.0 mol eq.) was taken in anhydrous dichloromethane and cooled to -78 °C. Added tri ethylamine (2.0 mol eq.) dropwise at -78 °C and stirred for Ih. After 1 h the reaction mixture was slowly allowed to warm to rt and stirred for addition 2 h. The solvent was removed under reduced pressure and crude residue was re-suspended in anhydrous ether and filtered through a celite bed under nitrogen. Filtrate was concentrated to produce compound 16, which was used as such for the next step without further purification.

[0233] A+M ethyl imidazole. NMI (4 mmol eq.) was added to a stirring suspension of compounds 12-15 (1 mmol eq.) in dry THF under argon atmosphere at 0 °C. The phosphorochloridate 16 (1.5 mmol eq.) was added dropwise by dissolving in THF. The reaction mixture was warm to rt and continued stir for 16 h. Then volatiles were evaporated under reduced pressure and crude was purified by silica gel column chromatography (2% methanol / DCM) to give the phosphoramidate prodrugs 17-20 as off white solid.

[0234] {[(Z / ?,37?,- / ?)-3-(6-aniino-9H-purin-9-yl)-4-fluoro-5-hydroxy-2-methylenecyclo pentyl)methoxy](phenoxyphosphoryl amino} propionic Acid Isopropyl Ester (17). Yield (0.55 g, 61%); ‘H NMR (500 MHz, CDCh) 8 d 8.36 (s, 1H), 7.84 (d, 7= 24.5 Hz, 1H), 7.28-7.10 (m, 5H), 5.88 (d, J= 30.0 Hz, 1H), 5.80 (bs, 2H), 5.18 (d, J = 9.0 Hz 1H), 4.96-4.76 (m, 3H), 4.39-4.34 (m, 2H), 4.17- 4.04 (m, 2H), 3.90-3.88 (m, 2H), 3.00 (bs, 1H), 1.31 (d, J= 6.5 Hz, 3H), 1.16 (dd, 7=6.0, & 14.0 Hz, 6H):13C{1H} NMR (125 MHz, CDCh): 6 187.7, 173.3, 155.4, 153.1, 150.5, 144.5, 142.4, 140.9, 129.8, 125.2, 120.3, 118.7, 112.3, 95.9, 73.7, 50.5, 49.6, 21.6, 20.8;19F NMR (470 MHz, CDCh) 8 -192.81 (ddd, J= 17.5, 31.5 & 53.0 Hz, IF);31P NMR (CDCh, 202 MHz): 8 2.84, 2.37; HRMS (El) Calcd for ^HnFNeOeP+Hf 549.2021, found 549.2026.

[0235] Isopropyl ((((! / ?, 3 / ?, 4 / ?)-3-fluoro-2-hydroxy-4-(6-(methylamino)-9 / / -purin-9-yl )-5-methylenecyclopentyl)methoxy)(phenoxy)phosphoryl)-Z-alaninate (18). ’H NMR (500 MHz, CDCh): 8 8.33 (s, 1H), 7.72 (s, 1H), 7.25-7.19 (m, 2H), 7.16-7.12 (m, 2H), 7.13-6.98 (m, 1H), 6.20 (s, 1H), 5.89 (d, J= 31.5 Hz, 1H), 5.17 (s, 1 H), 4.96-4.90 (m, 2H), 4.80 (s, 1H), 4.73 (s, 1H), 4.39 (d, J = 15.0 Hz, 1H), 4.35-4.25 (m, 1H), 4.16-4.10 (m, 2H), 3.95-3.90 (m, 1H), 3.12 (s, 3H), 3.01 (s, 1H), 1.29 (d, J= 5.5 Hz, 3H), 1.5-1.14 (m, 6H);13C{XH} NMR (125 MHz, CDCls) 8: 173.2 (d,7= 19.1Hz), 155.5, 153.1, 150.6, 145.0 (7= 9.5 Hz), 140.2, 129.8, 125.1, 120.2, 119.0, 112.3, 73.6, 69.4 (d, J= 19.1 Hz), 67.0, 50.5, 49.7, 21.7, 20.9;19F NMR (470 MHz, CDCh) 8: -192.60 to -192.99 (m, IF);31PNMR (202 MHz, CDCh) 8: 2.75, 2.46; HRMS (El) Calcd for IC25H33FN6O6P+HJ+563.2178, found 563.2187.

[0236] Isopropyl ((((17?, 37?, 47?, 57?)-3-(2-amino-6-chloro-9 / / -purin-9-yl)-4-fluoro-5-hydroxy-2-methylenecyclopentyl)methoxy)(phenoxy)phosphoryl)-Z-alaninate (19).

[0237] Yield (35 mg, 47%); 'H NMR (500 MHz, CDCh) 87.75 (d, 7= 15.5 Hz, 1H), 7.34 (t, 7= 7.8 Hz, 2H), 7.24-7.14 (m, 3H), 5.77-5.61 (m, 1H), 5.31 (bs, 1H), 5.28-5.17 (m, 2H), 5.07-4.92 (m, 2H), 4.87 (d, 7= 14.8 Hz, 1H), 4.59-4.48 (m, 1H), 4.46-4.37 (m, 1H), 4.37-4.16 (m, 1H), 4.06-3.90 (m, 1H), 3.83-3.62 (m, 2H), 3.11-2.98 (m, 1H), 1.39 (d, 7= 7.1 Hz, 3H), 1.25-1.20 (m, 6H);13C{1H} NMR (125 MHz, CDCh) 8 173.4, 159.2, 154.2, 151.5, 150.6, 144.0, 142.6, 129.9, 125.3, 124.7, 120.3, 120.2, 113.0, 96.2, 94.7, 74.1, 69.7, 57.7, 50.6, 49.7, 49.4, 29.8, 21.7, 20.9;19F NMR (470 MHz, CDCh) 8 -193.8 to -194.5 (m, IF);31P NMR (202 MHz, CDCh) 83.69, 2.87; HRMS-ESI (w / z): [M + H]+calculated for ^HsoOeNeFClPf 583.1632; found 583.1624. Isopropyl ((((17?, 37?, 47?)-3-(2-amino-6-(methylamino)-977-purin-9-yl)-4-fluoro-5-hydroxy-2-methylenecyclopentyl)methoxy)(phenoxy)phosphoryl)-Z-alaninate (20). 'H NMR (500 MHz, CDC13) 5: 7.43 (s, 1H), 7.28-7.24 (m, 2H), 7.15 (t, J = 7.5 Hz, 2H), 7.12-7.05 (m, 1H), 6.23 (brs, 1H), 5.66 (d, 32.0 Hz, 1H), 5.23 (s, 1H), 5.13 (brs, 1H), 5.02-4.88 (m, 3H), 4.86 (s, 1H), 4.75 (s, 1H), 4.42-4.37 (m, 1H), 4.28 (brs, 1H), 4.20-4.05 (m, 1H), 3.98-3.85 (m, 1H), 3.62 (s, 1H), 3.11-2.98 (m, 4H), 1.29 (s, 3H), 1.18-1.13 (m, 6H);13C{*H} NMR (125 MHz, CDCh) 5: 173.3 (t, J = 6.15 Hz), 155.9, 150.6, 144.8, 129.8, 125.1, 120.2, 113.4, 112.2, 110.0, 95.0, 73.4, 73.3, 69.4 (d, J= 12.3 Hz), 67.3, 57.5, 53.4, 50.5, 49.8, 29.7, 21.6, 20.9;19F NMR (470 MHz, CDCh) 5: -193.3;31P NMR (202 MHz, CDCh) 5: 2.75, 2.34; HRMS (El) Calcd for [C25H33FN7O6P+H]+578.2287, found 578.2296.

[0238] (+)-9-[(17?, 27?, 37?, 47?)-2-Fluoro-3-hydroxy-4-(hydroxymethyl)-5-methylenecyclo-pentan-l-yl]-guanosine (21). Compound 10 (0.5 g, 0.6 mmol) was dissolved in 10 ml of formic acid and heated at 50 °C for 6 hours. After completely removing the volatile, the residue was further treated with aqueous methanolic ammonium hydroxide solution at room temperature for 1 hour. The reaction mixture was concentrated in reduced pressure and the obtained crude was purified by column chromatography on silica gel (MeOH / DCM) to give guanosine analogue 21 (150 mg, 63%) as white solid. Mp: 215-218 °C; [a]25D+ 44.69° (c 0.64, H2O): UV (H2O)max253.0 nm (e 13300, pH 2), 251.0 nm (s 13600, pH 7), 260.0 nm (e 11400, pH 11); 'H NMR (500 MHz, DMSO-hJ 8 10.70 (s, 1H, NH), 7.46 (d, J= 2.5 Hz, 1H), 6.59 (s, 1H), 5.65 (d, J= 3.0 Hz, 1H), 5.50 (d, J= 28.5 Hz, 1H), 5.31 (s, 1H), 5.01 (s, 1H), 4.85 (d, 1H), 4.80 (s, 1H), 4.25 (d, J= 12.5 Hz, 1H), 3.66-3.64 (m, 1H), 3.54 (s, 1H), 2.61 (s, 1H);13C{1H} NMR (125 MHz, DMSO-tC) 8 157.2, 154.3, 152.1, 147.3, 136.9, 116.0, 111.5, 97.2, 96.5 (d, J = 183.1 Hz) 72.6 (d, J= 22.8 Hz), 62.3, 57.3 (d, J = 16.6 Hz);19F NMR (470 MHz, DMSO-rfc) 8 -192.93 (m, IF); Anal. Calcd. For Ci2Hi4FN5O.2.5H2O C, 42.35; H, 5.63; N, 20.58; Found C, 42.25; H, 5.51; N, 20.59.

[0239] Isopropyl ((((17?,37?,47?,57?)-3-(2-amino-6-oxo-l,6-dihydro-977-purin-9-yl)-4-fluoro-5-hydroxy-2-methylenecyclopentyl)methoxy)(phenoxy)phosphoryl)-L-alaninate (22). Yield (13 mg, 53%);JH NMR (500 MHz, CD3OD) 8 7.61 (d, J= 18.6 Hz, 1H), 7.38 (t, J = 7.8 Hz, 2H), 7.27 (t, J= 9.0 Hz, 2H), 7.22 (d, J= 7.2 Hz, 1H), 5.74 (d, J= 32.4 Hz, 1H), 5.43 (d, J= 13.0 Hz, 1H), 5.04-4.93 (m, 4H), 4.45-4.22 (m, 3H), 4.00-3.91 (m, 1H), 3.04-2.94 (m,1H), 1.36 (dd, J = 7.1 & 13.9 Hz, 3H), 1.27-1.22 (m, 6H); ^C H} NMR (125 MHz, CD3OD) 8 161.9, 161.6, 158.1, 154.2, 151.0, 145.1, 129.4, 124.8, 120.2, 120.1, 118.1, 115.8, 112.3, 100.0, 68.9, 68.8, 50.6, 30.8, 20.7, 20.5, 19.0;19F NMR (470 MHz, CD3OD) 8 -195.1 to -195.4 (m, IF);31P NMR (202 MHz, CD3OD) 8 4.38, 4.06; HRMS-ESI (w / z): [M + H| calculated for [C24H3iO7N6FP]+565.1970; found 565.1947. Isopropyl((7? )-(((! / ?, 37?, 47?)-3-(6-amino-917-purin-9-yl)-4-fluoro-5-hydroxy-2-methylenecyclopentyl)methoxy)(phenoxy)phosphoryl)-£-alaninate (23).JH NMR (500 MHz, CDCh) 5 8.36 (s, 1H), 7.84-7.82 (m, 1H), 7.32 (t, J = 7.5 Hz, 2H), 7.22 (d, J= 8.0 Hz, 2H), 7.16 (t, J= 7.0 Hz, 1H), 5.99-5.84 (m, 3H), 5.25 (s, 1H), 5.03-4.87 (s, 2H), 4.82 (s, 1H), 4.49-4.38 (m, 2H), 4.23 (q, J= 10.4 Hz, 1H), 4.07-3.96 (m, 2H), 3.08-3.03 (m, 1H), 1.38 (d,.7= 6.6 Hz, 3H), 1.25-1.20 (m, 6H);13C{1H} NMR (125 MHz, CDCh) 8 173.3, 155.6, 153.2, 150.7, 141.0, 129.8, 125.2, 120.4, 112.3, 100.0, 96.4. 94.9. 74.0, 69.5, 57.8, 50.6, 49.7, 31.7, 29.8, 22.7, 21.8, 21.7, 21.1, 14.2;19F NMR (470 MHz, CDCh) 6 -192.6 to -192.8 (m, IF);31P NMR (202 MHz, CDCh) 63.42; HRMS (El) Calcd for [C24H3oFN606P+H]+549.2021, found 549.2024.

[0240] Isopropyl((A)-(((17?,37?,47?)-3-(6-amino-9 / 7-purin-9-yl)-4-fluoro-5-hydroxy-2-methylenecyclopentyl)methoxy)(phenoxy)phosphoryl)-£-alaninate (24). 'H NMR (500 MHz, CDCh) 6 8.37 (s, 1H), 7.87 (d, J= 3.0 Hz, 1H), 7.34 (t, J= 8.0 Hz, 2H), 7.25-7.22 (m, 2H), 7.17 (t, J = 7.5 Hz, 1H), 5.95 (d, J= 29.9 Hz, 1H), 5.68 (brs, 2H), 5.23 (s, 1H), 5.07-4.90 (m, 2H), 4.83 (s, 1H), 4.52-4.41 (m, 2H), 4.20 (q, J= 10.4 Hz, 1H), 4.03-3.91 (m, 3H), 3.10-3.05 (brs, 1H), 1.39 (d, J= 6.6 Hz, 3H), 1.25 (d, J= 6.2 Hz, 6H);13C{1H} NMR (125 MHz, CDCh) 8 173.3, 155.5, 153.2, 150.6, 144.6, 141.0, 129.9, 125.2, 120.2, 118.8, 112.3, 100.0, 96.4, 94.6, 74.0, 69.5, 57.9, 50.6, 49.8, 21.7, 20.0;19F NMR (470 MHz, CDCh) 8 -192.8 to -193.1 (m, IF);31P NMR (202 MHz, CDCh) 82.89; HRMS (El) Calcd for IC. H.oFNeOeP+HI 549.2021, found 549.2027.

[0241] 3-Benzoyl-l-((lR,2R,3R,4R)-3-( / er / -butoxy)-4-(ter / -butoxymethyl)-2-fluoro-5-methylenecyclopentyl)pyrimidine-2,4(lH,3H)-dione (26). To a stirred mixture of N3-benzoyluracil 25 (236 mg, 1.09 mmol) and TPP (449 mg, 1.82 mmol) in anhydrous THF (15 mL) was added dropwise diisopropyl azodicarboxylate (DIAD, 0.36 mL, 1.82 mmol) at 0 °C under nitrogen atmosphere. After complete addition of DIAD, reaction mixture was cooled to -15 °C, added intermediate 3 (200 mg, 1.73 mmol) by dissolving in THF at this temperature. The mixture was stirred at -10 °C for 2.5 h and then quenched with methanol; the solvent was removed under vacuum. The residue was purified by flash silica gel column chromatography (17% EtOAc / hexane) to give 26. (121 mg, 41% yield) as a gummy solid. ’H NMR (500 MHz, CDCh): 87.95 (d, J= 7.5 Hz, 2H), 7.66 (t, J= 7.5 Hz, 1H), 7.51 (t, J= 7.5 Hz, 2H), 7.45 (d, J= 8.0 Hz, 1H), 5.81-5.73 (m, 2H), 5.40 (s, 1H), 5.09 (s, 1H), 4.87-4.74 (m, 1H), 4.21 (dt, J = 14.5 & 2.6 Hz, 1H), 3.55-3.50 (m, 1H), 3.44 (td, J= 8.9 & 2.2 Hz, 1H), 2.73 (brs, 1H), 1.22 (s, 9H), 1.20 (s, 9H); ^C H} NMR (125 MHz, CDCh) 8: 162.2, 154.5, 153.6, 153.0, 135.3, 132.0, 130.6, 129.3, 128.8, 128.2, 110.2, 73.0, 70.8, 48.0, 28.4, 27.5, 22.0, 21.4;19F NMR (470 MHz, CDCh) 5: -193.69 to -193.90 (m, IF); HRMS-ESI (m / z): [M + Na]+calculated for [C26H33FN2O5Na]+495.2271; found 495.2282.

[0242] l-((lR,2R,3R,4R)-3-(r<’ / r-butoxy)-4-(r<?r / -butoxymethyl)-2-fluoro-5-methylenecyclo-pentyl)pyrimidine-2,4(lH,3H)-dione (27). Compound 26 (120 mg, 0.25 mmol) was dissolved in 7 N ammonia solution in methanol (20 mL) and stirred for 24 h at rt. The solvent was evaporated under vacuum and the residue was purified by column chromatography (4% Methanol / DCM) to give 27 as a white solid (62 mg 52% yield).1H NMR (500 MHz, CDCh) 5 8.41 (bs, 1H), 7.32 (dd, J = 1.8 & 8.2 Hz, 1H), 5.78 (dd, J= 3.0 & 30.0 Hz, 1H), 5.67 (dd, J = 2.2 & 8.2 Hz, 1H), 5.35-5.33 (m, 1H), 4.98 (t, J= 2.5 Hz, 1H), 4.78 (dd, J= 4.6 & 53 Hz, 1H), 4.11 (d, J= 14.6 Hz, 1H), 3.52-3.48 (m, 1H), 3.41 (td, J = 2.0) & 9.2 Hz, 1H), 2.75-2.69 (m, 1H), 1.26 (s, 9H), 1.23 (s, 9H); ^C H} NMR (125 MHz, CDCh): 5 163.5, 151.6, 145.4, 144.0, 112.1, 101.6, 97.8, 95.3, 75.6, 72.9, 62.4, 59.1, 49.8, 28.2, 27.4;19FNMR (470 MHz, CDCh) 5 -193.6 (ddd, J= 14.0, 31.5 & 49.0 Hz, IF); HRMS (El) Calcd for (Ci9H29FN2O +H)+369.2190, found 369.2187.

[0243] 4-amino-l-((lR,2R,3R,4R)-3-( r / -butoxy)-4-(ter / -butoxymethyI)-2-fluoro-5-methylenecyclopentyl)pyrimidin-2(lH)-one (28). To a stir solution of compound 27 (1.5 g, 4.08 mmol) in anhydrous acetonitrile (30 mL), 2,4,6-triisopropyl benzenesulfonyl chloride (2.46 g, 8.16 mmol), 4-(dimethylamino)pyridine(DMAP) (0.49 g, 4.08 mmol) and triethylamine (2.28 ml, 16.3 mmol) were added at 0 °C. The mixture was stirred at ambient temperature for 12 h. After that, 28% solution of ammonium hydroxide (15 mL) was added and stirred at room rt for 6h. The reaction mixture was concentrated under reduced pressure and crude was purified by column chromatography (5% MeOH / DCM) to give the compound 28 as a white solid (Yield 1.1 g, 74%). Mp 189-192 °C; [a]24D= +68.34 (c 1.0, CHCh); 'H NMR (500 MHz, CDCh) 5 7.37 (dd, J= 1.8 & 7.5 Hz, 1H), 6.05 (d, J= 7.3 Hz, 1H), 5.95-5.85 (m, 1H), 5.30 (s, 1H), 4.96 (s, 1H), 4.75 (dd, J = 3.7 & 53.2 Hz, 1H), 4.15 (d, J = 14.5 Hz, 1H), 3.50-3.45 (m, 3H), 3.40-3.34 (m, 1H), 2.74-2.68 (m, 1H), 1.22 (s, 9H), 1.20 (s, 9H); ^C H} NMR (125 MHz, CDCh): 5 164.7, 156.3, 146.5, 145.7, 111.7, 98.0, 96.5, 94.4, 75.6, 73.2, 62.8, 60.1, 50.3, 28.3, 27.6;19F NMR (470 MHz, CDCh) 8 -193.5 to -193.68 (m, IF); HRMS (El) Calcd for (CI9H30FN3O3+H)+368.2344, found 368.2348.

[0244] 4-amino-l-((lR,2R,3R,4R)-2-fluoro-3-hydroxy-4-(hydroxymethyl)-5-methylenecyclopeiityl)pyrimidin-2(lH)-one (29). Compound 28 (0.3 g, 0.82 mmol) was dissolved in DCM (20 mL). Added 2.5 ml of trifluoroacetic acid (TFA) and stirred for 24 h at rt. The solvent and TFA were evaporated under vacuum and the obtained residue was dissolved in methanol and neutralized with 28% aqueous ammonia solution. The mixture was concentrated under reduced pressure and purified by column chromatography (5% Methanol / DCM) to give the 29 as a white solid. Yield (170 mg, 81%). Mp 192-193 °C; [CC]24D = +65.34 (c 1.0, MeOH); 'H NMR (500 MHz, CD3OD) 57.63 (d, J= 8.0 Hz, 1H), 6.00 (d, J = 7.5 Hz, 1H), 5.94 (d, J = 29.0 Hz, 1H), 5.49 (s, 1H), 5.09 (s, 1H), 4.88 (d, J = 38.5 Hz, 1H), 4.31 (d, J = 13.5 Hz, 1H), 3.80-3.68 (m, 2H), 2.75 (bs, 1H); ^C H} NMR (125 MHz, CD3OD): 5 167.2, 157.8, 150.5, 149.4, 115.8, 100.1 (d, J= 187.0 Hz), 97.8, 76.9, 65.9, 63.6, 55.5;19F NMR (470 MHz, CD3OD) 5 -197.4 (m, IF); HRMS (El) Calcd for (C11H14FN3O3+H) 256.1097, found 256.1095.

[0245] 4-ainiiio-l-((l / ?,3 / ?,4 / ?,5 / ?)-3-((( / e / -biityldiphenylsilyl)oxy)methyl)-5-fluoro-4-hydroxy-2-methylenecyclopentyl)pyrimidin-2(H / )-one (30). To a stirred solution of 4-amino-l-((lR,27?,3J?,4R)-2-fluoro-3-hydroxy-4-(hydroxymethyl)-5-methylenecyclopentyl)pyrimidin-2(lF / )-one (FMCC, 29) (90 mg, 0.35 mmol) in dry7DMF (1.5 mL), imidazole (72 mg, 1.05 mmol) and TBDPS-C1 (77 pL, 0.29 mmol) was added. The mixture was stirred at rt for 6 h. After that, reaction mixture quenched with 20 mL of water and extracted into EtOAc (3 X 10 mL). The combined organic layer was washed with brine (20 mL), finally with water (10 mL) and dried over anhydrous Na2SO4. filtered, and concentrated under reduced pressure. The obtained crude was purified by flash silica gel column chromatography (8% MeOH / DCM) to give compound 30 as a colorless thick liquid. Yield (120 mg, 69%). 'H NMR (500 MHz, CDCh) 57.66 (t, J= 7.5 Hz, 4H), 7.45-7.35 (m, 6H), 7.25 (s, 1H), 7.19-7.09 (m, 1H), 6.02 (d, J = 31.2 Hz, 1H), 5.67 (d, J= 9.2Hz, 1H), 5.14 (s, 1H), 5.03-4.86 (m, 1H), 4.80 (s, 1H), 4.40 (d, J= 16.4 Hz, 1H), 3.75 (q, J= 8.3 Hz, 2H), 2.84 -2.79 (m, 1H), 1.06 (s, 9H); ^C H} NMR (125 MHz, CDCh) 5 168.5, 165.3, 157.4, 145.9, 145.4, 135.8, 133.3, 129.9, 127.9, 111.8, 100.0, 94.7, 64.5, 53.9, 51.8, 31.8, 29.3, 27.0, 19.3;19F NMR (470 MHz, CDCh) 5 -195.22 (d, J = -103.4 Hz, IF); HRMS-ESI ( / z): [M + H]+calculated for [C27H33FN3O3SI]+494.2270; found 494.2257.

[0246] 4-amiiio-l-((l / ?,3 / ?,4 / ?,5 / ?)-3-((( / e / -butyldiphenylsilyl)oxy)methyl)-5-fluoro-2-methylene-4-((tetrahydro-2 / / -pyran-2-yl)oxy)cyclopentyl)pyrimidin-2(177)-one (31). To a stirred solution of compound 30 (90 mg, 0.18 mmol, 1.0 eq) in dry DCM (2 mL), 4-dihydro-277- yran (50 pL, 0.54 mmol) and catalytic amount of -TS A was added. The reaction mixture was stirred at rt for 6 h. After that, volatiles were removed under reduced pressure and obtained residue was purified by flash silica gel column chromatography (8% MeOH / DCM) to give 31 as a white fluffy solid. Yield (80 mg, 76%). Mp: 82-85 °C; 'H NMR (500 MHz, CDCh) 8 7.73-7.65 (m, 4H), 7.47-7.36 (m, 6H), 7.19 (t, J= 7.4 Hz, 1H), 5.99 (dd, J= 7.0 & 34.2 Hz, 1H), 5.77-5.71 (m, 1H), 5.25-5.10 (m, 1H), 5.02-4.70 (m, 2H), 4.52-4.36 (m, 1H), 3.90-3.63 (m, 3H), 3.55-3.44 (m, 1H), 3.02-2.80 (m, 1H), 1.85-1.45 (m, 7H), 1.09-1.05 (m, 9H); ^C H} NMR (125 MHz, CDCh) 8 164.8, 156.4, 145.6, 135.7, 135.6, 133.4, 133.2, 130.0, 129.9, 127.9, 112.1, 100.0, 98.5, 97.5, 96.6, 95.1, 94.3, 76.4, 64.5, 62.9, 62.3, 60.4, 50.6, 31.0, 30.5, 29.8, 26.9, 25.3, 19.3, 18.9;19F NMR (470 MHz, CDCh) 5 -195.9 to -196.5 (m, IF); HRMS-ESI (m / zy. [M + Na]+calculated for [C32H4oFN304SiNa]+600.2670; found 600.2643.

[0247] 4-aiiiino-l-((l / ?,2 / ?,3 / ?,4 / ?)-2-niioro-4-(hydroxyinethyl)-5-methylene-3-((tetraliydro-2 / / -pyran-2-yl)oxy)cyclopentyl)pyriini(lin-2( l / / )-one (32). To a stirred solution of compound 31 (100 mg, 0.17 mmol) in dry THF (1.5 mL) at 0 °C, 1 M solution of TBAF in THF was added (0.2 mL, 0.2 mmol) and the mixture was stirred at rt for 4 h. The mixture volatiles were removed under reduced pressure and the residue was purified by silica gel column chromatography (9% MeOH / DCM) to give 32 as a white fluffy solid. Mp 96-98 °C; yield (48 mg, 82%); ‘H NMR (500 MHz, CD3OD) 8 7.46 (ddd, J= 1.5, 7.5, & 13.9 Hz, 1H), 6.04-5.87 (m, 2H), 5.48-5.40 (m, 1H), 5.18-5.04 (m, 1H), 5.00-4.96 (m, 1H), 4.92-4.88 (m, 1H), 4.84-4.81 (m, 1H), 4.34 (dd, J= 13.9 & 50.9 Hz, 1H), 3.98-3.90 (m, 1H), 3.85-3.76 (m, 1H), 3.74-3.54 (m, 2H), 3.35-3.24 (m, 1H), 2.95-2.81 (m, 1H), 1.91-1.52 (m, 7H), 1.50-1.41 (m, 1H), 1.05 (t, J = 7.4 Hz, 1H); ^CfH} NMR (125 MHz, CD3OD) 8 166.2, 157.8, 145.9, 145.0, 110.8, 98.7, 97.5, 96.4, 94.9, 94.4, 93.5, 78.5, 76.9, 62.6, 62.4, 62.2, 60.3, 58.1, 53.5, 50.6, 50.3, 30.6, 25.1, 23.5, 19.4, 19.2, 12.7;19F NMR (470 MHz, CD3OD) 6 -197.3 to -198.1 (m, IF): HRMS-ESI (m / z): [M + H]' calculated for ICisH Y hOfi 340.1667; found 340.1653.

[0248] Isopropyl ((((l / ?,3 / ?,4 / ?,5 / ?)-3-(4-amiiio-2-oxopyrimidiii-l(2 / / )-yl)-4-fluoro-2-methylene-5-((tetrahydro-2 / / -pyran-2-yl)oxy)cyclopentyl)methoxy)(phenoxy)phosphoryl)-L-alaninate (33). To a stirred solution of compound 32 (60 mg, 0.17 mmol) in dry THF (2 mL) at -78 °C, IM 'BuMgCl solution in THF (0.37 mL, 0.38 mmol) was added dropwise. After that, the mixture was warmed to 0 °C and stirred at the same temperature for 30 min. Again, the mixture was cooled to -78 °C and a solution of compound 16 (115 mg, 0.38 mmol) was added by dissolving in dry THF (1.5 mL) and stirring at rt for 16 h. The reaction mixture was quenched with MeOH (2 mL), and volatiles were removed under reduced pressure. The obtained residue was diluted with DCM (20 mL), washed with water (2 X 15 mL), and finally with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude was purified by silica gel column chromatography (6% MeOH / DCM) to give compound 33 as a white fluffy solid. Mp 58-63 °C; Yield (58 mg, 54%); 'H NMR (500 MHz, CDCh) 87.35-7.28 (m, 2H), 7.18 (dd, J= 5.6 & 35.8 Hz, 4H), 6.05 (dd, J= 18.1 & 33.5 Hz, 1H), 5.72 (q, J = 6.0 & 6.8 Hz, 1H), 5.32 (d, J = 17.1 Hz, 1H), 5.14- 4.92 (m, 2H), 4.87-4.63 (m, 1H), 4.41-3.75 (m, 5H), 3.57-3.43 (m, 1H), 3.13-2.93 (m, 1H), 2.89-2.57 (m, 1H), 1.82-1.44 (m, 5H), 1.41-1.32 (m, 3H), 1.26-1.17 (m, 6H); ^C H} NMR (125 MHz, CDCh) 8 173.0, 165.3, 156.7, 150.9, 145.2, 144.6, 129.8, 125.0, 120.3, 120.2, 120.1, 112.6, 98.5, 98.0, 94.4, 69.3, 66.9, 62.5, 53.5, 50.4, 48.1, 30.6, 29.8, 25.2, 21.8, 21.7, 19.4, 19.1;19F NMR (470 MHz, CDCls) 5 -195.6 to -195.9 (m, 1F);31P NMR (202 MHz, CDCh) 5 3.21-2.82; HRMS-ESI (wUz): [M + Na]+calculated for [C2sH38FN4O8PNa]+631.2309; found 631.2296.

[0249] Isopropyl ((((17?,37?,47?,57?)-3-(4-amino-2-oxopyrimidin-l(27 / )-yl)-4-fluoro-5-hydroxy-2-methylenecyclopentyl)methoxy)(phenoxy)phosphoryl)-L-aIaninate (34). To a stirred solution of compound 33 (30 mg, 0.04 mmol) in DCM (1 mL) at 0 °C, 20% TFA in DCM (0.2 mL) was added and the mixture was stirred at same temperature for 2 h. After that, the reaction mixture was concentrated under reduced pressure and obtained residue was redissolved in DCM (20 mL), washed with 10% NaHCCL (5 mL), and finally with brine (10 mL), dried over Na2SO4 and filtered. The organic layer was concentrated under reduced pressure. The crude was purified by silica gel column chromatography (8% MeOH / DCM) to give 34 as an off-white solid. Yield (12 mg, 48%); 'HNMR (500 MHz, CD3OD) 57.46-7.37 (m, 3H), 7.30-7.21 (m, 3H), 6.01 (d, J= 30.9 Hz, 1H), 5.88 (t, J= 6.3 Hz, 1H), 5.49 (s, 1H), 5.06-4.98 (m, 2H), 4.92 (d, J=4.9 Hz, 1H), 4.82 (d, =4.1 Hz, 1H), 4.35-4.18 (m, 3H), 4.00-3.91 (m, 1H), 3.01-2.91 (m, 1H), 1.38 (d, J= 7.5 Hz, 3H), 1.27 (q, J= 5.9 Hz, 6H);13C{1H} NMR (125 MHz, CD3OD) 5 173.2, 166.1 150.1, 145.1, 144.9, 129.5, 124.8, 120.1, 120.0, 112.4, 97.0, 95.5, 94.4, 73.0, 72.8, 68.9, 66.4, 59.7, 59.6, 50.3, 49.7, 20.6, 19.1;19F NMR (470 MHz, CD3OD) 5 -196.8 to -197.05 (m, IF);31P NMR (202 MHz, CD3OD) 5 4.36 & 4.03; HRMS-ESI (m z): [M + Na]+calculated for [C23H3oFN407PNa]+547.1734; found 547.1714.

[0250] Cells and Viruses

[0251] Human hepatoma-derived Huh-7-C3 cells were used for all cell culture-based assays. Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) with 4.5 g / L-glucose, L-glutamine, and sodium pyruvate (DMEM, IX, Coming, Manassas, VA) supplemented with 10% heat-inactivated fetal bovine serum (Benchmark FBS; Gemini Bio Products, West Sacramento, CA), penicillin-streptomycin (5,000 lU / mL), and amphotericin B (250 pg / mL). Vaccinia strain VacWR-p7.5eGFP (VACV-GFP) was obtained from the Viral Vector Core at the University of Iowa. Virus seed stocks were amplified in Huh7-C3 cells in infection medium comprising DMEM with 2.5% FBS and 1% penicillin-streptomycin.

[0252] Compounds and Formulations

[0253] All compounds were prepared as 10 mM stocks in DMSO and stored at -80 °C. The control compounds, cidofovir (CDV; BEI Resources, Manassas, VA), brincidofovir (BCV, AdooQ Bioscience, Irvine, CA), and tecovirimat (TPOXX; Millipore Sigma, Burlington, MA), are commercially available. Stock compounds were diluted in DMSO and / or complete tissue culture media prior to being added to cells.

[0254] Antiviral Efficacy Assay

[0255] Huh-7-C3 cells were seeded at a density of IxlO4cells per well in 96- well plates and allowed to incubate overnight. On the second day, the cells were treated with compounds subsequently infected with VACV-GFP at 0.06 MOI. At 48hpi, plates were read on a BioTek Cytation 5 where bright field and fluorescence images were taken at 4x magnification. Total fluorescence intensity per well was recorded and used to calculate ECso values, which were defined as the concentration where there is a 50% reduction in fluorescence intensity. Doseresponse curves were generated using Prism software (Graphpad, San Diego, CA, USA). The therapeutic index was defined as the CC50 divided by the EC50.

[0256] Evaluation of Cytotoxicity

[0257] The cytotoxic effects of test compounds for Huh-7-C3 cells were determined by CellTiter 96 Non-Radioactive Cell Proliferation assay system (Promega) using the 2,3-Bis-(2-Methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, disodium salt (XTT) method under the same conditions as the antiviral assay but in the absence of VACV-GFP. Huh-7-C3 cells were seeded at a density of IxlO4cells per well in 96-well plates and allowed to incubate overnight. On the second day, different concentrations of test compounds were added to Huh-7-C3 cells. Following 2 days of incubation at 37 °C in a CO2 incubator, the XTT reagent was added, and the cells were incubated for 3 hours at 37°C. Subsequently, the absorbance of the samples was measured using a microplate reader (BioTek). The cytotoxic concentration (CC50) was determined based on the viability of mock-infected cells.

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Claims

CLAIMSWhat is claimed is:

1. A method of treating or reducing the likelihood of an infection caused by a poxvirus in a subject in need thereof, the method comprising administering to the subject an amount of a compound effective to treat or reduce the likelihood of the infection, wherein the compound has a structure of:wherein:R1and Rlaare each independently H, an acyl group, a C1-C20 alkyl group, a C1-C20 ether group, an amino acid residue (D or L), a phosphate, diphosphate, triphosphate, phosphoester, phosphodiester, or phosphoramidate group, or together R' and Rlaform a carbodiester, phosphodiester, or cyclic phosphoramidate group with the oxygen atoms to which they are bonded;RLis C(RH)2, wherein each RHis independently H, F, Cl, Br, or I;B is:A1and A6are each independently N, CH, or C;A2, A4, A5, and A7are each independently N, NR, CR, or CR2;A3is C, N, or CR;each --- between adjacent atoms represents a bond that is present or absent;RM1, RM2, RM3, and RM4are each independently RN, R°, =0, OR, =S, or SR;each RNand R° is independently -NHR2or R;each R2is independently H, an acyl group, OR, SR, a C1-C20 alkyl group, a C1-C20 ether group, or an amino acid residue (D or L);each R is independently H, F, Cl, Br, I, C1-C4 alkyd (preferably CH?), -C≡N, -C≡C-Ra,each Rais independently H or a C1-C4 alkyl group; andeach X is independently H, C1-C4 alkyl (preferably, CH?), F, Cl, Br or I,or a pharmaceutically acceptable salt, stereoisomer, hydrate, or solvate thererof.

2. The method of claim 1, wherein the compound has a structure of:

3. The method of any prior claim, wherein:A1is N or C;A2is N or CR;A3is C orN;A4is N, NR, or CR;A5is N or NR;RM1is RNor =0;RM2is RN, R°, or =0;A6is N, or CH;A7is N or NR;RM3is =0; and / orRM4is RNor =0.

4. The method of any prior claim, wherein B is:

5. The method of any prior claim, wherein B is:

6. The method of any prior claim, wherein each RNis -NHR2.

7. The method of any prior claim, wherein each RNis NH2.

8. The method of any prior claim, wherein each R° is H.

9. The method of any prior claim, wherein each R is H.

10. The method of any prior claim, wherein Rlais H.

11. The method of any prior claim, wherein:R1is:each R5and R6is independently H, a C1-C20 alkyl group, or an alkoxyalkyl, aryloxyalkyl, aryl, alkoxy, or alkoxycarbonyloxy group, each of which groups may be optionally substituted, with the proviso that at least one R5group is other than H, or the two R5groups together form a five- or six-membered heterocyclic group;B' is a group according to the structure:i is 0, 1, 2 or 3;R7is H, a C1-C20 alkyl group or an acyl, alkoxyalkyl, aryloxyalkyl, or aryl group, each of which groups may be optionally substituted;R8is a sidechain of an amino acid or an optionally substituted C1-C20 alkyl group; and each R" is independently a C1-C20 alkyd group or a phenyl or heteroaryl group, each of which groups may be optionally substituted.

12. The method of any prior claim, wherein:R1is:wherein:R6is H, a C1-C20 alkyl group, or an alkoxyalkyl, aryloxyalkyl, aryl, alkoxy, or alkoxycarbonyloxy group, each of which groups may be optionally substituted;R8is a sidechain of an amino acid or an optionally substituted Ci- C20 alkyl group; andR" is a C1-C20 alkyl group or a phenyl or heteroaryl group, each of which groups may be optionally substituted; andthe compound is administered in a composition having an stereoisomeric excess of the compound with respect to a stereoisomer of the compound wherein R1in the stereoisomer is:

13. The method of any one of claims 1-11, wherein:R1is:R8is a sidechain of an amino acid or an optionally substituted C1-C20 alkyl group;R" is an optionally substituted C1-C20 alkyl group; andRpis H, nitro, cyano, methoxy, or a C1-C4 alkyl group optionally substituted with 1-3 halogen substituents.

14. The method of claim 13, wherein:R1is:the compound is administered in a composition having an stereoisomeric excess of the compound with respect to a stereoisomer of the compound wherein R1in the stereoisomer is:

15. The method of any one of claims 13-14, wherein Rpis H or a C1-C4 alkyl group.

16. The method of any one of claims 13-15, wherein Rpis H.

17. The method of any one of claims 1-9, wherein:R1and Rlatogether are:R8is a sidechain of an amino acid or an optionally substituted C1-C20 alkyl group; and R" is a C1-C20 alkyl group or a phenyl or heteroaryl group, each of which groups may be optionally substituted.

18. The method of any one of claims 11-17, wherein R8is a C1-C4 alkyl group.

19. The method of any one of claims 11-18, wherein R" is a C1-C4 alkyl group.

20. The method of claim 1, wherein the compound is:or a pharmaceutically acceptable salt thereof.

21. The method of claim 1, wherein the compound is:or a pharmaceutically acceptable salt thereof, and the compound is administered in a composition having a stereoisomeric excess of the compound with respect to a stereoisomer of the compound having the structure of:or a pharmaceutically acceptable salt thereof.

22. The method of any prior claim, wherein the subject has been exposed to a second subject having an infection caused by the poxvirus.

23. The method of any prior claim, wherein the subject is suspected of having an infection caused by the poxvirus.

24. The method of any prior claim, wherein the subj ect has an infection caused by the poxvirus.

25. The method of any prior claim, wherein the poxvirus is an orthopox virus.

26. The method of any prior claim, wherein the poxvirus is a monkeypox virus.

27. The method of claim 1, wherein the subj ect has a monkeypox virus infection, the compound is administered in amount effective to treat the monkeypox virus infection, and the compound is:or a pharmaceutically acceptable salt thereof.

28. The method of claim 27, wherein the compound is:or a pharmaceutically acceptable salt thereof, and is administered in a composition having a stereoisomeric excess of the compound with respect to a stereoisomer of the compound having the structure of:or a pharmaceutically acceptable salt thereof.

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