WO2014124430A1

WO2014124430A1 - Nucleotide and nucleoside therapeutic compositions and uses related thereto

Info

Publication number: WO2014124430A1
Application number: PCT/US2014/015763
Authority: WO
Inventors: Dennis C. Liotta; George R. Painter; Gregory R. BLUEMLING
Original assignee: Emory University
Priority date: 2013-02-11
Filing date: 2014-02-11
Publication date: 2014-08-14

Abstract

This disclosure relates to nucleotide and nucleoside therapeutic compositions and uses related thereto. In certain embodiments, the disclosure relates to halogenated nucleosides optionally conjugated to a phosphorus oxide or pharmaceutically acceptable salts thereof. In certain embodiments, the disclosure relates to conjugate compounds or pharmaceutically acceptable salts thereof comprising an amino acid ester or a sphingolipid or derivative linked by a phosphorus oxide to a nucleotide or nucleoside. In certain embodiments, the disclosure contemplates pharmaceutical compositions comprising these compounds for uses in treating infectious diseases, viral infections, and cancer.

Description

NUCLEOTIDE AND NUCLEOSIDE THERAPEUTICS COMPOSITIONS AND USES RELATED THERETO

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benfit of U.S. Provisional Application No. 61/763,333, filed on February 11, 2013 which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Nucleoside and nucleotide phosphates and phosphonates are clinically useful as antiviral agents. Two examples are tenofovir disoproxil fumarate for the treatment of human

immunodeficiency virusand adefovir dipivoxil for the treatment of hepatitis B virus infections. Administration of three or more antiretroviral agents in combination, e.g., Highly Active

Antiretroviral Therapy (HAART), has significantly reduced the morbidity and mortality associated with HIV infection. However, there is a growing need for new antiviral agents to address the critical issues of resistance and penetration into viral sanctuaries (commonly referred to as privileged compartments). Permeability into privileged compartments may be partially responsible for the current inability of chemotherapy to totally clear a patient of HIV infection and the emergence of resistance.

Anti-viral agents that are unphosphorylated nucleotides and nucleotide derivatives need to be phosphorylated to actively inhibit viral replication. Nucleoside analogues enter a cell via two types of broad-specificity transporters, concentrative nucleoside transporters (CNTs) and equilibrative nucleoside transporters (ENTs). Once inside, they utilize the host's nucleoside salvage pathway for sequential phosphorylation by deoxynucleoside kinases (dNKs), deoxynucleoside monophosphate kinases (dNMPKs) and nucleoside diphosphate kinase

(NDPK). However, intracellular activation of these compounds is often compromised by the high substrate specificity of the host's endogenous kinases. In vitro and in vivo studies have demonstrated that the first and/or second phosphorylation, catalyzed by dNKs and dNMPKs, often represent the rate-limiting steps in nucleoside analogue activation. Thus, there is a need to identifying improved antiviral nucleoside analogues with structural features that are sufficiently activated by cellular kinases.

McGuigan et al, J Med Chem, 2005, 48(10):3504-3515, report phenylmethoxyalaninyl phosphoramidate of abacavir as a prodrug leads to enhancement of antiviral potency. Painter et al, Antimicrob Agents Chemother, 2007, 51(10):3505-3509, report promoting the oral availability of tenoforir with a hexadecyloxypropyl prodrug ester, designated CMX157.

Sphingolipids play roles in cell-cell and cell-substratum interactions, and help regulate growth and differentiation by a variety of mechanisms, such as inhibition of growth factor receptor kinases and effects on numerous cellular signal transduction systems. U.S. patent 6,610,835 discloses sphingosine analogues. It also discloses methods of treating infections and cancer. Pruett et al, J. Lipid Res. 2008, 49(8), 1621-1639, report on sphingosine and

derivatives.Bushnev et al, ARKIVOC, 2010, (viii):263-277, report an asymmetric synthetic method for preparing sphingolipid derivatives. Dougherty et al, Org. Lett. 2006, 8(4), 649-652, report the synthesis of 1-deoxysphingosine derivatives. Wiseman et al, Org. Lett. 2005, 7(15), 3155-3157, report 1 -deoxy-5-hydroxysphingo lipids in anticancer and stereoselective syntheses of 2-amino-3,5-diols.

References cited herein are not an admission of prior art.

SUMMARY OF THE INVENTION

This disclosure relates to nucleotide and nucleoside therapeutic compositions and uses related thereto. In certain embodiments, the disclosure relates to halogenated nucleosides optionally conjugated to a phosphorus oxide or salts thereof. In certain embodiments, the disclosure relates to conjugate compounds or salts thereof comprising an amino acid ester or a sphingolipid or derivative linked by a phosphorus oxide to a nucleotide or nucleoside. In certain embodiments, the disclosure contemplates pharmaceutical compositions comprising these compounds for uses in treating infectious diseases, viral infections, and cancer. In certain embodiments, the disclosure relates to phosphorus oxide prodrugs of 2'- fluoronucleosides for the treatment of positive-sense and negative-sense RNA viral infections through targeting of the virally encoded RNA-dependent RNA polymerase (RdRp). This disclosure also provides the general use of sphingolipids to deliver nucleoside analogs for the treatment of infectious disease and cancer.

In certain embodiments, the disclosure relates to conjugate compounds or salt thereof comprising a sphingolipid or derivative linked by a phosphorus oxide to a nucleotide or nucleoside. In certain embodiments, the phosphorus oxide is a phosphate, phosphonate, polyphosphate, or polyphosphonate, wherein the phosphate, phosphonate or a phosphate in the polyphosphate or polyphosphonate is optionally a phosphorothioate orphosphoroamidate. In certain embodiments, the sphingolipid is covalently bonded to the phosphorus oxide through an amino group or a hydroxy 1 group.

In certain embodiments, the nucleotide or nucleoside is a heterocycle comprising two or more nitrogen heteroatoms substituted with an oxo, amino, or carbamoyl, wherein the substituted heterocyclyl is optionally substituted with one or more, the same or different alkyl, halogen, or cycloalkyl. In certain embodiments, the nucleotide or nucleoside comprises pyrimidine-2,4- dione, 4-aminopyrimidin-2-one, purin-6-amine, 2-amino-purin-6-one, 5-methylpyrimidine-2,4- dione, 3-methylpyrimidine-2,4-dione, 4-amino-5-methylpyrimidin-2-one, 4- (methylamino)pyrimidin-2-one, 4-(dimethylamino)pyrimidin-2-one, 2-methyl-purin-6-amine, N- methyl-purin-6-amine, N,N-dimethyl-purin-6-amine, pyrrolo[2,3-d]pyrimidin-4-amine; N- cyclopropyl-pyrrolo[2,3-d]pyrimidin-4-amine, purin-2-amine, purine-2,6-diamine, purin-6-one, N⁶-cyclopropyl-purine-2,6-diamine, 2-fluoro-purin-6-amine, 5-(trifluoromethyl)pyrimidine-2,4- dione, 4-amino-5-fluoropyrimidin-2-one, 5-ethylpyrimidine-2,4-dione, 5-(2- bromovinyl)pyrimidine-2,4-dione, 5-fluoropyrimidine-2,4-dione, 5-iodopyrimidine-2,4-dione, l,2,4-triazole-3-carboxamide, or 3-oxo-3,4-dihydropyrazine-2-carboxamide.

In certain embodiments, the sphingolipid is saturated or unsaturated 2-aminoalkyl or 2- aminooctadecane optionally substituted with one or more substituents. In certain embodiments, the sphingolipid derivative is saturated or unsaturated 2-aminooctadecane-3-ol optionally substituted with one or more substituents. In certain embodiments, the sphingolipid derivative is saturated or unsaturated 2-aminooctadecane-3,5-diol optionally substituted with one or more substituents. In certain embodiments, the disclosure contemplates pharmaceutical compositions comprising any of the compounds disclosed herein and a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition is in the form of a pill, capsule, tablet, or saline buffer comprising a saccharide. In certain embodiments, the composition may contain a second active agent such as a pain reliever, anti-inflammatory agent, non-steroidal antiinflammatory agent, anti-viral agent, anti-biotic, or anti-cancer agent.

In certain embodiments, the disclosure relates to methods of treating or preventing an infection comprising administering an effective amount of a compound disclosed herein to a subject in need thereof. Typically, the subject is diagnosed with or at risk of an infection from a virus, bacteria, fungi, protozoa, or parasite.

In certain embodiments, the disclosure relates the methods of treating a viral infection comprising administering an effective amount of a pharmaceutical composition disclosed herein to a subject in need thereof. In certain embodiments, the subject is a mammal, for example a human. In certain embodiments, the subject is diagnosed with a chronic viral infection. In certain embodiments, administration is under conditions such that the viral infection is no longer detected. In certain embodiments, the subject is diagnosed with a RNA virus, DNA virus, or retroviruses. In certain embodiments, the subject is diagnosed with a virus that is a double stranded DNA virus, sense single stranded DNA virus, double stranded RNA virus, sense single stranded RNA virus, antisense single stranded RNA virus, sense single stranded RNA retrovirus or a double stranded DNA retrovirus.

In certain embodiments, the subject is diagnosed with influenza A virus including subtype H1N1, influenza B virus, influenza C virus, rotavirus A, rotavirus B, rotavirus

Qrotavirus D,rotavirus E, SARS coronavirus, human adenovirus types (HAdV-1 to 55), human papillomavirus (HPV)Types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59,parvovirus B19, molluscum contagiosum virus, JC virus (JCV), BK virus, Merkel cell polyomavirus, coxsackie A virus, norovirus, Rubella virus, lymphocytic choriomeningitis virus (LCMV), yellow fever virus, measles virus, mumps virus, respiratory syncytial virus, rinderpest virus, California encephalitis virus, hantavirus, rabies virus, ebola virus, marburg virus, herpes simplex virus- 1 (HSV-1), herpes simplex virus-2 (HSV-2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, roseolovirus, or Kaposi's sarcoma- associated herpesvirus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E or human immunodeficiency virus (HIV).

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

In certain embodiments, pharmaceutical compositions disclosed herein are administered in combination with a second antiviral agent such as abacavir, acyclovir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla, boceprevir, cidofovir,

combivir,darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, interferon type III, interferon type II, interferon type I, lamivudine, lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfmavir, nevirapine, nexavir, oseltamivir, peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin , raltegravir, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, stavudine, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir,

tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine zalcitabine, zanamivir, or zidovudine and combinations thereof.

In certain embodiments, the disclosure relates to methods of treating a cancer comprising administering an effective amount of a pharmaceutical composition disclosed herein to subject in need thereof. In certain embodiments, the cancer is selected from bladder cancer, lung cancer, breast cancer, melanoma, colon and rectal cancer, non-hodgkin lymphoma, endometrial cancer, pancreatic cancer, kidney cancer, prostate cancer, leukemia, thyroid cancer, and brain cancer.

In certain embodiments, the compositions are administered in combination with a second anti-cancer agent such as temozolamide, bevacizumab, procarbazine, lomustine, vincristine, gefitinib, erlotinib, docetaxel, cis-platin, 5-fluorouracil, gemcitabine, tegafur, raltitrexed, methotrexate, cytosine arabinoside, hydroxyurea, adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin, vinblastine, vindesine, vinorelbine, taxol, taxotere, etoposide, teniposide, amsacrine, topotecan,

camptothecin, bortezomib, anagrelide, tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene, fulvestrant, bicalutamide, flutamide, nilutamide, cyproterone, goserelin, leuprorelin, buserelin, megestrol, anastrozole, letrozole, vorazole, exemestane, finasteride, marimastat, trastuzumab, cetuximab, dasatinib, imatinib, combretastatin, thalidomide, and/or lenalidomide or combinations thereof.

In certain embodiment, the disclosure relates to uses of compounds disclosed herein in the production or manufacture of a medicament for the treatment or prevention of an infectious disease, viral infection, or cancer.

In certain embodiments, the disclosure relates to derivatives of compounds disclosed herein or any of the formula.

Additional advantages of the disclosure will be set forth in part in the description which follows. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure, as claimed.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 illustrates certain embodiments of the disclosure. The base may be a naturally occurring base, a modified, for example a methylated base, or any active derivatives thereof, such as those found in currently approved nucleoside bases found in antiviral or anticancer agents. In certain embodiments, R may be HOH₂C- as well as their mono-, di-, and triphosphates. In certain embodiments, Ri is hydrogen, methyl, fluoromethyl, hydroxymethyl, difluoromethyl, trifluoromethyl, or C2 units (acetylenyl, ethyl, vinyl, cyano, etc.).

Figure 2 illustrates bases for embodiments provided in Figure 1 or any of the

embodiments disclosed herein.

Figure 3 illustrates the unraveling of McGuigan prodrugs in vivo. The metabolic unraveling of these prodrugs begins with an esterase-catalyzed cleavage of the carboxylic ester, followed by several chemical rearrangement steps resulting in an amino acid phosphoramidate. The final cleavage is carried out by one of several endogenous phosphoramidases, one of which has been identified to be the histidine triad nucleotide binding protein 1 (hF Tl)

Figure 4 illustrates embodiments of cyclobutyl nucleosides. The base may be any base or derivative thereof disclosed herein. R may be hydrogen, fluoro, hydroxy or azide, and n may be 1 or 2.

Figure 5 illustrates a scheme for the synthesis of cyclobutyl nucleosides, a) Zn-Cu couple, Et₂0, reflux; b) NH₄C1, EtOH; c) i: LDA, THF, -78°C ii: electrophile; d) nucleophile, ammonium salt; e) L-Selectride, THF; f) i: PPI1₃, 4-nitrobenzoic acid, DIAD, THF ii: K₂CO₃, MeOH; g) brosyl chloride, Et₃N, DCM; h) DBU, purine or pyrimidine, DMSO; i) pTSA, EtOH, R may be hydrogen, fluoro, hydroxy or azide, and n may be 1 or 2.

Figure 6 illustrates embodiments of mono- and diphosphate structural types.

Figure 7 illustrates schemes for the synthesis of conjugates.

Figure 8 illustrates the preparation of embodiments of the disclosure.

Figure 9 illustrates certain embodiments of the disclosure.

Figure 10 is the X-ray crystal structure for compound 32 of Example 6. DETAILED DESCRIPTION OF THE INVENTION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

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

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.

As used herein, the term "phosphorus oxide" refers to any variety of chemical moieties that contain a phosphorus-oxygen (P-0 or P=0) bond. When used as linking groups herein, the joined molecules may bond to oxygen or directly to the phosphorus atoms. The term is intended to include, but are not limited to phosphates, in which the phosphorus is typically bonded to four oxygens and phosphonates, in which the phosphorus is typically bonded to one carbon and three oxygens. A "polyphosphate" generally refers to phosphates linked together by at least one phosphorus-oxygen-phosphorus (P-O-P) bond. A "polyphosphonate" refers to a polyphosphate that contains at least one phosphorus-carbon (C-P-O-P) bond. In addition to containing phosphorus-oxygen bond, phosphorus oxides may contain a phosphorus-thiol (P-S or P=S) bond and/or a phosphorus-amine (P-N) bond, respectively referred to as phosphorothioate or phosphoroamidate. In phosphorus oxides, the oxygen atom may form a double or single bond to the phosphorus or combinations, and the oxygen may further bond with other atoms such as carbon or may exist as an anion which is counter balanced with a cation, e.g., metal or quaternary amine.

As used herein, "alkyl" means a noncyclic, linear or branched, unsaturated or saturated hydrocarbon such as those containing from 1 to 22 carbon atoms. Unsaturated alkyl groups contain from 2 to 22 carbon atoms.The term "lower alkyl" or "Ci_₄alkyl" refers to an alkyl group that contains from 1 to 4 carbon atoms. The term "higher alkyl" refers to an alkyl group that contains from 8 to 22 carbon atoms. Representative saturated, linear alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-septyl, n-octyl, n-nonyl, and the like; while saturated, branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.

Unsaturated alkyls contain at least one double or triple bond between adjacent carbon atoms (referred to as an "alkenyl" or "alkynyl", respectively). Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3 -methyl- 1-butenyl, 2-methyl-2-butenyl, 2,3- dimethyl-2-butenyl, and the like; while representative straight chain and branched alkynyls include acetylenyl, propynyl, 1- butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3- methyl- 1-butynyl, and the like.

Non-aromatic mono or polycyclic alkyls are referred to herein as "carbocycles" or

"carbocyclyl" groups that contain 3 to 30 carbon atoms Representative saturated carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated carbocycles include cyclopentenyl and cyclohexenyl, and the like.

"Heterocarbocycles" or heterocarbocyclyl" groups are carbocycles which contain from 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur which may be saturated or unsaturated (but not aromatic), monocyclic or polycyclic, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. Heterocarbocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,

tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl,

tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

"Aryl" means an aromatic carbocyclic monocyclic or polycyclic ring that contains 6 to 32 carbon atoms, such as phenyl or naphthyl. Polycyclic ring systems may, but are not required to, contain one or more non-aromatic rings, as long as one of the rings is aromatic.

As used herein, "heteroaryl" refers an aromatic heterocarbocycle having 1 to 4

heteroatoms selected from nitrogen, oxygen and sulfur, and containing at least 1 carbon atom, including both mono- and polycyclic ring systems. Polycyclic ring systems may, but are not required to, contain one or more non-aromatic rings, as long as one of the rings is aromatic. Representative heteroaryls are furyl, benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl, isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl, isooxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl. It is contemplated that the use of the term "heteroaryl" includes N-alkylated derivatives such as a 1- methylimidazol-5-yl substituent.

As used herein, "heterocycle" or "heterocyclyl" refers to mono- and polycyclic ring systems having 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur, and containing at least 1 carbon atom. The mono- and polycyclic ring systems may be aromatic, non-aromatic or mixtures of aromatic and non-aromatic rings. Heterocycle includes heterocarbocycles, heteroaryls, and the like.

"Alkylthio" refers to an alkyl group as defined above attached through a sulfur bridge. An example of an alkylthio is methylthio, (i.e., -S-CH₃).

"Alkoxy" refers to an alkyl group as defined above attached through an oxygen bridge. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n- butoxy, s-butoxy, t-butoxy, n- pentoxy, and s-pentoxy. Preferred alkoxy groups are methoxy, ethoxy, n-propoxy, i- propoxy, n-butoxy, s-butoxy, and t-butoxy.

"Alkylamino" refers an alkyl group as defined above attached through an amino bridge. An example of an alkylamino is methylamino, (i.e., -NH-CH₃).

"Alkanoyl" refers to an alkyl as defined above attached through a carbonyl bride (i.e., - (C=0)alkyl).

"Alkylsulfonyl" refers to an alkyl as defined above attached through a sulfonyl bridge

(i.e., -S(=0)₂alkyl) such as mesyl and the like, and "Arylsulfonyl" refers to an aryl attached through a sulfonyl bridge (i.e., - S(=0)₂aryl).

"Alkylsulfinyl" refers to an alkyl as defined above attached through a sulfinyl bridge (i.e. -S(=0)alkyl).

The term "substituted" refers to a molecule wherein at least one hydrogen atom is replaced with a substituent. When substituted, one or more of the groups are "substituents." The molecule may be multiply substituted. In the case of an oxo substituent ("=0"), two hydrogen atoms are replaced. Example substituents within this context may include halogen, hydroxy, alkyl, alkoxy, nitro, cyano, oxo, carbocyclyl, carbocycloalkyl, heterocarbocyclyl,

heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -NRaRb, -NRaC(=0)Rb, - NRaC(=0)NRaNRb, -NRaC(=0)ORb, - NRaS0₂Rb, -C(=0)Ra, -C(=0)ORa, -C(=0)NRaRb, - OC(=0)NRaRb, -ORa, -SRa, -SORa, - S(=0)₂Ra, -OS(=0)₂Ra and -S(=0)₂ORa. Ra and Rb in this context may be the same or different and independently hydrogen, halogen hydroxyl, alkyl, alkoxy, alkyl, amino, alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl.

The term "optionally substituted," as used herein, means that substitution is optional and therefore it is possible for the designated atom to be unsubstituted.

As used herein, "salts" refer to derivatives of the disclosed compounds where the parent compound is modified making acid or base salts thereof, including pharmaceutically acceptable salts thereof. Examples of salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkylamines, or dialkylamines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. In typical embodiments, the salts are conventional nontoxic pharmaceutically acceptable salts including the quaternary ammonium salts of the parent compound formed, and non-toxic inorganic or organic acids. Preferred salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2- acetoxybenzoic, fumaric,

toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.

"Subject" refers any animal, preferably a human patient, livestock, rodent, monkey or domestic pet.

The term "prodrug" refers to an agent that is converted into a biologically active form in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent compound. They may, for instance, be bioavailable by oral administration whereas the parent compound is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. A prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis.

As used herein, the term "derivative" refers to a structurally similar compound that retains sufficient functional attributes of the identified analogue. The derivative may be structurally similar because it is lacking one or more atoms, substituted with one or more substituents, a salt, in different hydration/oxidation states, e.g., substituting a single or double bond, substituting a hydroxy group for a ketone, or because one or more atoms within the molecule are switched, such as, but not limited to, replacing an oxygen atom with a sulfur or nitrogen atom or replacing an amino group with a hydroxyl group or vice versa. Replacing a carbon with nitrogen in an aromatic ring is a contemplated derivative. The derivative may be a prodrug. Derivatives may be prepared by any variety of synthetic methods or appropriate adaptations presented in the chemical literature or as in synthetic or organic chemistry text books, such as those provide in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Wiley, 6th Edition (2007) Michael B. Smith or Domino Reactions in Organic Synthesis, Wiley (2006) Lutz F. Tietze hereby incorporated by reference.

As used herein, the terms "prevent" and "preventing" include the full or partial inhibition of the recurrence, spread or onset of a referenced pathological condition or disease. It is not intended that the present disclosure be limited to complete prevention. In some embodiments, the onset is delayed, or the severity of the disease is reduced.

As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e.g., patient) is cured and the disease is eradicated. Rather, embodiments, of the present disclosure also contemplate treatment that merely reduces symptoms, and/or delays disease progression.

As used herein, the term "combination with" when used to describe administration with an additional treatment means that the agent may be administered prior to, together with, or after the additional treatment, or a combination thereof.

Nucleoside Analogues as Antiviral Agents

Nucleoside analogs utilize the host's nucleoside salvage pathway for sequential phosphorylation by deoxynucleoside kinases (dNKs), deoxynucleoside monophosphate kinases (dNMPKs) and nucleoside diphosphate kinase (NDPK). However, intracellular activation of these compounds is often compromised by the high substrate specificity of the host's

endogenous kinases. In vitro and in vivo studies have demonstrated that the first and/or second phosphorylation, catalyzed by dNKs and dNMPKs, often represent the rate-limiting steps in nucleoside analog activation. These significant blockades in the phosphorylation cascade of a given nucleoside analog will result in the lack of any observable activity in cellular assays. To circumvent these blockades, several kinase bypass strategies have been developed. For example, McGuigan phosphoramidates are chemical conjudgates used for kinase bypass. See Serpi et al, J Med Chem, 2012, 55(10):4629-4639. The metabolismof these prodrugs begins with an esterase- catalyzed cleavage of the carboxylic ester, followed by several chemical rearrangement steps resulting in an amino acid phosphoramidate. The final cleavage is carried out by one of several endogenous phosphoramidases, one of which has been identified to be the histidine triad nucleotide binding protein 1 (hINTI).

An alternative prodrug strategy to circumvent these blockades is to utilize sphingoid bases to mask nucleotide analog phosphates. Sphingoid bases have the potential for delivering nucleotide analog phosphates to critical tissues such as the brain. The design concept driving the use of sphingoid bases to form nucleoside-lipid conjugates is based on observations that the sphingoid base analogs are: (a) well absorbed after oral administration, (b) resistant to oxidative catabolism in enterocytes, and (c) achieve high concentrations in the brain. Based on data for intestinal uptake of traditional phospholipid drug conjugates in mice and our data for sphingoid base oral absorption in rats, our sphingoid base conjugates should be well absorbed and resist first pass metabolism. After absorption, sphingoid bases, including sphingosine-1 -phosphate, are transported in blood via both lipoproteins and free plasma proteins like albumin. Active epithelial cell uptake of sphingoid base phosphates has been demonstrated to occur via the ABC transporter, CFTR, but passive protein transport and endocytotic uptake are also possible; it is believed that extracellularly delivered drug conjugates would be processed similarly by target cells in the central nervous system (CNS) and the gut-associated lymphoid tissue (GALT). The rat sphingolipid PK studies mentioned above resulted in 24 hour tissue concentrations exceeding plasma Cmax concentrations by 10 to 300+ fold, with lung and brain levels being particularly high and without evidence of toxicity. This approach has significant potential for conjugate delivery of high drug concentrations to critical tissues.

Compounds

In certain embodiments, the disclosure relates to halogenated nucleosides conjugated to a phosphorus moiety or pharmaceutically acceptable salts thereof.

In certain embodiments, the disclosure relates to compounds of the following formula:

Formula I or pharmaceutically acceptable salts thereof wherein,

X is O or CH₂ or CD₂;

R¹ is a phosphonate, or polyphosphonate,

wherein the phosphonate is optionally a phosphorothioate or phosphoroamidate, and wherein the phosphonate, phosphorothiolate, or phosphoroamidate is optionally substituted with one or more, the same or different R⁵, and

wherein the phosphonate, phosphorothiolate, or phosphoroamidate optionally forms a phosphorus containing heterocyclic ring, and

wherein the phosphorothiolate, or phosphoroamidate optionally forms a phosphorus containing heterocyclic ring with the R² carbon; R² is hydrogen, hydroxy, alkoxy, azide, or halogen;

R³ is hydrogen, hydroxy, halogen, cyano, or Ci_₂₂ alkyl optionally substituted with one or more, the same or different, R⁵;

R⁴ is a heterocyclyl comprising two or more nitrogen heteroatoms substituted with an oxo, amino, or carbamoyl, wherein R⁴ is optionally substituted with one or more, the same or different alkyl, halogen, cycloalkyl;

each R⁵ is independently selected from alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R⁵ is optionally substituted with one or more, the same or different, R⁶;

R⁶ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R⁶ is optionally substituted with one or more, the same or different, R⁷; and

R⁷ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, Ν,Ν-dimethylcarbamoyl, N,N-diethylcarbamoyl, N- methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N- dimethylsulfamoyl, Ν,Ν-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

Formula I

or pharmaceutically acceptable salts thereof wherein,

X is O or CH₂ or CD₂;

R¹ is a phosphate or polyphosphate, wherein the phosphate or a phosphate in the polyphosphate is a phosphorothioate or phosphoroamidate, and

wherein the phosphate, phosphorothiolate, or phosphoroamidate is optionally substituted with one or more, the same or different R⁵, and

wherein the phosphate, phosphorothiolate, or phosphoroamidate optionally forms a phosphorus containing heterocyclic ring, and

wherein the phosphate, phosphorothiolate, or phosphoroamidate optionally forms a phosphorus containing heterocyclic ring with the R² carbon;

R² is hydrogen, hydroxy, alkoxy, azide, or halogen;

Formula I

or pharmaceutically acceptable salts thereof wherein,

X is CH₂ or CD₂;

R¹ is a hydroxy;

R² is hydrogen, hydroxy, alkoxy, azide, or halogen;

R³ is hydroxy, halogen, cyano, -CH₂-R⁵, -CH(R⁵)₂, or C₂_₂₂ alkyl optionally substituted with one or more R⁵;

R⁷ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, Ν,Ν-diethylcarbamoyl, N- methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N- dimethylsulfamoyl, Ν,Ν-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R² is hydrogen or hydroxy.

In certain embodiments, R³ is methyl, fluoromethyl, hydroxymethyl, difluoromethyl, trifluoromethyl, acetylenyl, ethyl, vinyl, or cyano.

In certain embodiments, R² and R³ are hydrogen.

In certain embodiments, the R⁴ heterocyclyl is 7-deazapurine, 7-deaza-7-substituted purine, 5-azapyrimidine, tricyclic heterocycle, pyrazine, triazole, imidazole, or 5,6-dihydro-5- azapyrimidine, and heterocycles attached to the carbohydrate through a C-C bond.

In certain embodiments, X is methylene (CH₂) and R¹ is a phosphate, phosphonate, polyphosphate, or polyphosphonate substituent wherein the phosphate or a phosphate in the polyphosphate or polyphosphonate is optionally a phosphorothioate or phosphoroamidate, and the substituent is further substituted with hydrophobic group.

In certain embodiments, the hydrophobic group is a C₆_₂₂ alkyl, alkoxy, polyethylene glycol, or aryl substituted with an alkyl group.

In certain embodiments, the hydrophobic group is a lipid optionally substituted with one or more, the same or different R⁶. In certain embodiments, the lipid is a sphingolipid such as sphingosine, ceramide, or sphingomyelin, or 2-aminoalkyl optionally substituted with one or more substituents.

In certain embodiments, X is methylene (CH₂) and R¹ is a phosphate, phosphonate, polyphosphate, or polyphosphonate substituent wherein the phosphate or a phosphate in the polyphosphate or polyphosphonate is optionally a phosphoroborate, phosphorothioate, or phosphoroamidate, and the substituent is further substituted with a Lipid. In certain embodiments, the Lipid is a C₆-22 alkyl, alkoxy, polyethylene glycol, or aryl substituted with an alkyl group.

In certain embodiments, the Lipid is a fatty alcohol, fatty amine, or fatty thiol derivied from essential and non-essential fatty acids.

In certain embodiments, the Lipid is an unsaturated, polyunsaturated, omega unsaturated, or omega polyunsaturated fatty alcohol, fatty amine, or fatty thiol derivied from essential and non-essential fatty acids.

In certain embodiments, the Lipid is a fatty alcohol, fatty amine, or fatty thiol derivied from essential and non-essential fatty acids that has one or more of its carbon units substituted with an oxygen, nitrogen, or sulfur.

In certain embodiments, the Lipid is an unsaturated, polyunsaturated, omega unsaturated, or omega polyunsaturated fatty alcohol, fatty amine, or fatty thiol derivied from essential and non-essential fatty acids that has one or more of its carbon units substituted with an oxygen, nitrogen, or sulfur.

In certain embodiments, the Lipid is a fatty alcohol, fatty amine, or fatty thiol derivied from essential and non-essential fatty acids that is optionally substituted with one or more, the same or different R⁶.

In certain embodiments, the Lipid is an unsaturated, polyunsaturated, omega unsaturated, or omega polyunsaturated fatty alcohol, fatty amine, or fatty thiol derivied from essential and non-essential fatty acids that is optionally substituted with one or more, the same or different R⁶.

In certain embodiments, the Lipid is a fatty alcohol, fatty amine, or fatty thiol derivied from essential and non-essential fatty acids that has one or more of its carbon units substituted with an oxygen, nitrogen, or sulfur that is optionally substituted with one or more, the same or different R⁶.

In certain embodiments, the Lipid is an unsaturated, polyunsaturated, omega unsaturated, or omega polyunsaturated fatty alcohol, fatty amine, or fatty thiol derivied from essential and non-essential fatty acids that has one or more of its carbon units substituted with an oxygen, nitrogen, or sulfur that is also optionally substituted with one or more, the same or different R⁶.

In certain embodiments, the Lipid is hexadecyloxypropyl.

In certain embodiments, the Lipid is 2-aminohexadecyloxypropyl.

In certain embodiments, the Lipid is 2-aminoarachidyl. In certain embodiments, the Lipid is lauryl, myristyl, palmityl, stearyl, arachidyl, behenyl, or lignoceryl.

In certain embodiments, the Lipid is a sphingolipid.

In certain embodiments, the sphingolipid is optionally substituted with one or more, the same or different R⁶.

In certain embodiments, the sphingolipid is sphingosine, ceramide, or sphingomyelin, or 2-aminoalkyl optionally substituted with one or more substituents.

R⁶ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl^amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R⁶ is optionally substituted with one or more, the same or different, R⁷; and

In certain embodiments, the Lipid group of the formula:

wherein,

R⁸ is hydrogen, alkyl, C(=0)R¹², C(=0)OR¹², or C(=0)NHR¹²;

R⁹ is hydrogen, fluoro, OR¹², OC(=0)R¹², OC(=0)OR¹², or OC(=0)NHR¹²;

R¹⁰ is a saturated or unsaturated alkyl chain of greater than 6 and less than 22 carbons optionally substituted with one or more halogen or hydroxy or a structure of the following formula: CH₃(CH₂)_n CH₃(CH₂)_n'

n is 8 to 14 or less than or equal to 8 to less than or equal to 14, o is 9 to 15 or less than or equal to 9 to less than or equal to 15, the total or m and n is 8 to 14 or less than or equal to 8 to less than or equal to 14, the total of m and o is 9 to 15 or less than or equal to 9 to less than or equal to 15; or

n is 4 to 10 or less than or equal to 4 to less than or equal to 10, o is 5 to 11 or less than or equal to 5 to less than or equal to 11, the total of m and n is 4 to 10 or less than or equal to 4 to less than or equal to 10, and the total of m and o is 5 to 11 or less than or equal to 5 to less than or equal to 11 ; or

n is 6 to 12 or n is less than or equal to 6 to less than or equal to 12, the total of m and n is 6 to 12 or n is less than or equal to 6 to less than or equal to 12;

R¹¹ is OR¹², OC(=0)R¹², OC(=0)OR¹², or OC(=0)NHR¹²;

R¹² is hydrogen, a branched or strait chain Ci_i₂alkyl, Ci₃_₂₂alkyl, cycloalkyl, or aryl selected from benzyl or phenyl, wherein the aryl is optionally substituted with one or more, the same or different R¹³; and

R¹³is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, Ν,Ν-dimethylcarbamoyl, N,N-diethylcarbamoyl, N- methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N- dimethylsulfamoyl, Ν,Ν-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, the hydrophobic group is of the formula the formula:

wherein,

R⁸ is hydrogen, hydroxy, fiuoro, OR¹², OC(=0)R¹², OC(=0)OR¹², or OC(=0)NHR¹²; R⁹ is hydrogen, hydroxy, fiuoro, OR¹², OC(=0)R¹², OC(=0)OR¹², or OC(=0)NHR¹²;

R¹⁰ is a saturated or unsaturated alkyl chain of greater than 6 and less than 22 carbons optionally substituted with one or more halogens or a structure of the following formula:

CH₃(CH₂)_n ^/ CF₃(CF₂)_m(CH₂)_n ^/

n is 8 to 14 or less than or equal to 8 to less than or equal to 14, the total or m and n is 8 to 14 or less than or equal to 8 to less than or equal to 14; R is hydrogen, a branched or strait chain Ci_i₂alkyl, Ci₃_₂₂alkyl, cycloalkyl, or aryl selected from benzyl or phenyl, wherein the aryl is optionally substituted with one or more, the same or different R¹³; and

R¹³ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, Ν,Ν-dimethylcarbamoyl, N,N-diethylcarbamoyl, N- methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N- dimethylsulfamoyl, Ν,Ν-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, "salts" includes, for example, Li⁺, Na⁺, K⁺, Mg²⁺,

trialkylammonium, or other pharmaceutically acceptable salts.

In certain embodiments, the disclosure relates to a compound of the following formula:

Formula IA

or pharmaceutically acceptable salts thereof wherein,

X is CH₂ or CD₂;

R is one of the formula:

Y is O or S; R³ is hydrogen, hydroxy, halogen, cyano, C₂-₂₂ alkyl optionally substituted with one or more R⁵;

R⁴ is a heterocyclyl comprising two or more nitrogen heteroatoms substituted with an oxo, amino, or carbamoyl, wherein R⁴ is optionally substituted with one or more, the same or different alkyl, halogen, or cycloalkyl;

R⁵ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl^amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R⁵ is optionally substituted with one or more, the same or different, R⁶;

Formula IA

or pharmaceutically acceptable salts thereof wherein,

X is O;

R¹ is one of the formula:

Y is O or S;

R³ is hydrogen, hydroxy, halogen, cyano, or C₂-₂₂ alkyl optionally substituted with one or more R⁵;

Formula IA or pharmaceutically acceptable salts thereof wherein,

X is CH₂ or CD₂;

R¹ is hydroxyl;

R⁵ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfmyl, alkylsulfonyl,

arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R⁵ is optionally substituted with one or more, the same or different, R⁶;

R⁶ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfmyl, alkylsulfonyl,

arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R⁶ is optionally substituted with one or more, the same or different, R⁷; and

In certain embodiments, R³ is H, fluoro, methyl, fluoromethyl, hydroxymethyl, difluoromethyl, trifluoromethyl, acetylenyl, ethyl, vinyl, or cyano.

In certain embodiments, aryl is phenyl, monosubstituted phenyl, disubstituted phenyl, trisubstituted phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, or 1-naphthyl, 2-naphthyl.

In certain embodiments, R⁶ is alkyl, methyl, ethyl, propyl, n-butyl , branched alkyl, isopropyl, 2-butyl, 1-ethylpropyl, l-propylbutyl,cycloalkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, benzyl, or 2-butyl. In certain embodiments, the disclosure relates to a compound of the following formula:

1

Formula IA

Or pharmaceutically acceptable salts thereof wherein,

X is CH₂ or CD₂;

R¹ is one of the formula:

Y Y O Y O Y

I I H, I I I I .. H₂

Lipid- -p-o- Lipid-P-C Lipid-P-O-P-O- Lipid-

I I p I -o-p I -c

OH OH OH OH OH OH

Y is O or S;

Lipid is any of the formula described above or herein.

R³ is hydrogen, hydroxy, halogen, cyano, or C₂_₂₂ alkyl optionally substituted with one or more R⁵;

R⁵ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R⁵ is optionally substituted with one or more, the same or different, R⁶;

R⁷ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, Ν,Ν-dimethylcarbamoyl, N,N-diethylcarbamoyl, N- methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfmyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N- dimethylsulfamoyl, Ν,Ν-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

>1

Formula I A

or pharmaceutically acceptable salts thereof wherein,

X is O;

R¹ is one of the formula:

Y

I I H, O Y

I I H,

Lipid-P-C Lipid-P-O-P-C

I I

OH OH

Y is O or S;

Lipid is any of the formula described above or herein;

R⁵ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R⁵ is optionally substituted with one or more, the same or different, R⁶; R⁶ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R⁶ is optionally substituted with one or more, the same or different, R ; and

Formula IA

or pharmaceutically acceptable salts thereof wherein,

X is CH₂ or CD₂;

R¹ is hydroxyl;

R³ is hydroxy, halogen, cyano, -CH₂-R⁵, -CH(R⁵)₂, or C₂_₂₂ alkyl optionally substituted with one or more

R⁵ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R⁵ is optionally substituted with one or more, the same or different, R⁶; R⁶ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R⁶ is optionally substituted with one or more, the same or different, R⁷; and

In certain embodiments, the sphingolipid has the formula:

wherein,

R⁸ is hydrogen, alkyl, C(=0)R¹², C(=0)OR¹², or C(=0)NHR¹²;

R⁹ is hydrogen, fluoro, OR¹², OC(=0)R¹², OC(=0)OR¹², or OC(=0)NHR¹²;

R¹⁰ is a saturated or unsaturated alkyl chain of greater than 6 and less than 22 carbons optionally substituted with one or more halogen or hydroxy or a structure of the following formula:

CH₃(CH₂)^ CH₃(CH₂)_n-

CF₃(CF₂)_m(CH₂)_n ^/ CF₃(CF₂)_m(CH₂)n

R¹¹ is OR¹², OC(=0)R¹², OC(=0)OR¹², or OC(=0)NHR¹²;

R¹² is hydrogen, a branched or strait chain Ci_i₂alkyl, Ci3_₂₂alkyl, cycloalkyl, or aryl selected from benzyl or phenyl, wherein the aryl is optionally substituted with one or more, the same or different R¹³; and R is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, Ν,Ν-diethylcarbamoyl, N- methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N- dimethylsulfamoyl, Ν,Ν-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R¹² is H, alkyl, methyl, ethyl, propyl, n-butyl , branched alkyl, isopropyl, 2-butyl, l-ethylpropyl,l-propylbutyl, cycloalkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, benzyl, phenyl, monosubstituted phenyl, disubstituted phenyl, trisubstituted phenyl, or saturated or unsaturated C12-C19 long chain alkyl.

In certain embodiments, the sphingolipid has the formula:

wherein,

R⁸ is hydrogen, hydroxy, fiuoro, OR¹², OC(=0)R¹², OC(=0)OR¹², or OC(=0)NHR¹²; R⁹ is hydrogen, hydroxy, fiuoro, OR¹², OC(=0)R¹², OC(=0)OR¹², or OC(=0)NHR¹²; R¹⁰ is a saturated or unsaturated alkyl chain of greater than 6 and less than 22 carbons optionally substituted with one or more halogens or a structure of the following formula:

CH₃(CH₂)_n ^/ CFaiCFzWCH^ n is 8 to 14 or less than or equal to 8 to less than or equal to 14, the total or m and n is 8 to 14 or less than or equal to 8 to less than or equal to 14;

R¹² is hydrogen, a branched or strait chain Ci_i₂alkyl, Ci₃_₂₂alkyl, cycloalkyl, or aryl selected from benzyl or phenyl, wherein the aryl is optionally substituted with one or more, the same or different R¹³; and R is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, Ν,Ν-diethylcarbamoyl, N- methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N- dimethylsulfamoyl, Ν,Ν-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

In certain embodiment, the disclosure relates to a compound of the following formula:

Formula IB

or pharmaceutically acceptable salts thereof wherein,

X is CH₂ or CD₂;

R¹ is one of the formula:

Aryl

Y is O or S;

R³ is hydrogen, hydroxy, halogen, cyano, or C₂_₂₂ alkyl optionally substituted with one or more R⁵; R⁴ is a heterocyclyl comprising two or more nitrogen heteroatoms substituted with an oxo, amino, or carbamoyl, wherein R⁴ is optionally substituted with one or more, the same or different alkyl, halogen, cycloalkyl;

Formula IB

or pharmaceutically acceptable salts thereof wherein,

X is O;

R¹ is one of the formula:

or Y is O or S;

Formula IB

or pharmaceutically acceptable salts thereof wherein,

X is CH₂ or CD₂;

R¹ is hydroxyl; R³ is hydroxy, halogen, cyano, -CH₂-R⁵, -CH(R⁵)₂, or C₂_₂₂ alkyl optionally substituted with one or more R⁵;

In certain embodiments, aryl is phenyl, monosubstituted phenyl, disubstituted phenyl, trisubstituted phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 1-naphthyl, or 2-naphthyl.

In certain embodiments, R⁶ is alkyl, methyl, ethyl, propyl, n-butyl , branched alkyl, isopropyl, 2-butyl, 1-ethylpropyl, l-propylbutyl,cycloalkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, benzyl, or 2-butyl.

In certain embodiments, the sphingolipid is any of the formula described above or herein. In certain embodiments, R⁴ is any of the heterocycles described above or herein.

In certain embodiment, the disclosure relates to a compound of the following formula: HO F

Formula IB

or pharmaceutically acceptable salts thereof wherein,

X is CH₂ or CD₂;

R¹ is one of the formula:

Y Y u O Y Ο Υ μ

I I <, I I ⁿ2 I I II II I I ⁿ2 ¾

Lipid— P-0— Lipid— P-C -| Lipid-P-0-P-0- Lipid-P-O-P-C - OH OH OH OH OH OH

Y is O or S;

Lipid is any of the formula described above or herein;

R³ is hydrogen, hydroxy, halogen, cyano, or C₂_₂₂ alkyl optionally substituted with one or more the same or different R⁵;

HO F

Formula IB

or pharmaceutically acceptable salts thereof wherein,

X is O;

R¹ is one of the formula:

Y u μ

II ⁿ2 s O Y

I I II rl₂ ,

Lipid-P-C -{ Lipid-P-O-P-C -\

OH or OH OH ;

Y is O or S;

Lipid is is any of the formula described above or herein;

R³ is hydrogen, hydroxy, halogen, cyano, or C₂-₂₂ alkyl optionally substituted with one or more the same or different R⁵;

R⁶ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl^amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R⁶ is optionally substituted with one or more, the same or different, R⁷; and R is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, Ν,Ν-diethylcarbamoyl, N- methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N- dimethylsulfamoyl, Ν,Ν-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

Formula IB

or pharmaceutically acceptable salts thereof wherein,

X is CH₂ or CD₂;

R¹ is hydroxy;

R³ is hydrogen, hydroxy, halogen, cyano, -CH₂-R⁵, -CH(R⁵)₂, or C₂_₂₂ alkyl optionally substituted with one or more R⁵;

R⁶ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R⁶ is optionally substituted with one or more, the same or different, R⁷; and R is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, Ν,Ν-diethylcarbamoyl, N- methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N- dimethylsulfamoyl, Ν,Ν-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, the lipid is any of the formula described above or herein.

In certain embodiments, R⁴ is any of the heterocycles described above or herein.

Formula IC

or pharmaceutically acceptable salts thereof wherein,

X is CH₂ or CD₂;

Y is O or S;

R³ is hydrogen, hydroxy, halogen, cyano, or C₂_₂₂ alkyl optionally substituted with more the same or different R⁵;

R⁶ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R⁶ is optionally substituted with one or more, the same or different, R⁷;

R⁷ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, Ν,Ν-diethylcarbamoyl, N- methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N- dimethylsulfamoyl, Ν,Ν-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl;

R⁴⁰ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl^amino, alkylsulfmyl, alkylsulfonyl,

arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R⁴⁰ is optionally substituted with one or more, the same or different, R⁴¹; and

R⁴¹ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, Ν,Ν-dimethylcarbamoyl, N,N-diethylcarbamoyl, N- methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N- dimethylsulfamoyl, Ν,Ν-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R³ is hydrogen, methyl, fluoromethyl, hydroxymethyl, difluoromethyl, trifluoromethyl, acetylenyl, ethyl, vinyl, or cyano.

In certain embodiments, R⁴⁰ is alkyl, methyl, ethyl, propyl, n-butyl , branched alkyl, isopropyl, 2-butyl, 1-ethylpropyl, l-propylbutyl,cycloalkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, benzyl, or 2-butyl.

As referred to herein, the term "lipid" refers to any of the lipid compounds, derivatives or lipid formulas described above or herein.

Formula ID

or pharmaceutically acceptable salts thereof wherein,

X is CH2 or CD2;

Y is O or S;

R⁵⁰ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R is optionally substituted with one or more, the same or different, R⁵¹; and

R⁵¹ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, Ν,Ν-dimethylcarbamoyl, N,N-diethylcarbamoyl, N- methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N- dimethylsulfamoyl, Ν,Ν-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R⁵⁰ is alkyl, methyl, ethyl, propyl, n-butyl , branched alkyl, isopropyl, 2-butyl, 1-ethylpropyl, 1-propylbutyl, cycloalkyl, cyclopropyl, cyclobutyl,

cyclopentyl, cyclohexyl, benzyl, or 2-butyl.

In certian embodiments, R⁴ is any of the heterocycles described above or herein.

Formula IE

or pharmaceutically acceptable salts thereof wherein,

X is CH2 or CD2;

Y is O or S;

Lipid is any lipid described above or herein.

R³ is hydrogen, hydroxy, halogen, cyano, or C₂-₂₂ alkyl optionally substituted with one or more the same or different R⁵; R⁴ is a heterocyclyl comprising two or more nitrogen heteroatoms substituted with an oxo, amino, or carbamoyl, wherein R⁴ is optionally substituted with one or more, the same or different alkyl, halogen, cycloalkyl;

Formula II

or pharmaceutically acceptable salts thereof wherein,

X is O or CH₂ or CD₂;

wherein R¹ is a phosphate, phosphonate, polyphosphate, polyphosphonate substituent wherein the phosphate or a phosphate in the polyphosphate or poly phosphonate is optionally a phosphorothioate or phosphoroamidate, and the substituent is further substituted with an amino acid ester or sphingolipid or derivative optionally substituted with one or more, the same or different, R⁶;

R⁶ is the same or different at occurrence alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R⁶ is optionally substituted with one or more, the same or different, R⁷; and

In certain embodiments, the disclosure relates to a compound of the following formula

Formula III

or pharmaceutically acceptable salts thereof wherein,

X is CH₂ or CD₂;

Y¹ is O or S;

Y² is O or S;

Z is O or NH;

R² is hydrogen, hydroxy, alkoxy, azide, or halogen;

R³ is hydrogen, hydroxy, alkoxy, azide, or halogen; R⁴ is a heterocyclyl comprising two or more nitrogen heteroatoms substituted with an oxo, amino, or carbamoyl, wherein R⁴ is optionally substituted with one or more, the same or different alkyl, halogen, or cycloalkyl;

R²⁰ is an alkyl of 6 to 22 carbons optionally substituted with one or more, the same or different R²⁶;

R²¹ and R²² are each the same or different at each occurrence hydrogen, alkyl, or alkanoyl, wherein R²¹ and R²² are eachoptionally substituted with one or more, the same or different R²⁶;

R²⁴ and R²⁵are each the same or different at each occurrence hydrogen, alkyl, or aryl, wherein R 24 and R 25 are eachoptionally substituted with one or more, the same or different R 26 ;

R²⁶ is the same or different at occurrence alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl^amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R²⁶ is optionally substituted with one or more, the same or different, R²⁷;

R²⁷ is the same or different at occurrence alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl^amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R²⁷ is optionally substituted with one or more, the same or different, R²⁸; and

R²⁸ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, Ν,Ν-diethylcarbamoyl, N- methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N- dimethylsulfamoyl, Ν,Ν-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R²⁰ is an alkyl of between 8 and 16 carbons.

In certain embodiments, the disclosure relates to compounds of the following formula

Formula IIIA

or pharmaceutically acceptable salts thereof wherein,

the dotted line represents the presence of a single or double bond;

X is CH₂ or CD₂;

Z is O or NH;

Y¹ is O or S;

Y² is O or S;

R² is hydrogen, hydroxy, alkoxy, azide, or halogen;

R³ is hydrogen, hydroxy, alkoxy, azide, or halogen;

R 21 , R 22 , and R 23 are each the same or different at each occurrence hydrogen, alkyl, or

21 22 23

alkanoyl, wherein R , R , and R are eachoptionally substituted with one or more, the same or different R²⁶;

R²⁴ and R²⁵are each the same or different at each occurrence hydrogen, alkyl, or aryl, wherein R 24¾nd R 2"5 are eachoptionally substituted with one or more, the same or different R 26 ;

R²⁶ is the same or different at occurrence alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R²⁶ is optionally substituted with one or more, the same or different, R²⁷;

R²⁷ is the same or different at occurrence alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R²⁷ is optionally substituted with one or more, the same or different, R²⁸; and R is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, Ν,Ν-diethylcarbamoyl, N- methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N- dimethylsulfamoyl, Ν,Ν-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, the disclosure relates to the compound 2-amino-3- hydroxyoctadec-4-en-l-yldihydrogen (3-azido-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin- l(2H)-yl)tetrahydrofuran-2-yl)methyl diphosphate or pharmaceutically acceptable salts thereof.

In certain embodiments, the disclosure relates to a compound having the following formula:

Formula IV

or pharmaceutically acceptable salts thereof wherein,

X is O or NH;

Y is O or S;

Z is O or NH;

_R30

is an alkyl of 6 to 22 carbons optionally substituted with one or more, the same or different R³⁷;

R³³ and R³⁴ are each the same or different at each occurrence hydrogen, alkyl, or alkanoyl, wherein R³³ and R³⁴ are eachoptionally substituted with one or more, the same or different R³⁷;

R³⁵ is hydrogen, alkyl, or aryl wherein R³⁵ is optionally substituted with one or more, the same or different R³⁷; FT is alkyl;

R is the same or different at occurrence alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl^amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R³⁷ is optionally substituted with one or more, the same or different, R³⁸; and

R³⁸ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, Ν,Ν-dimethylcarbamoyl, N,N-diethylcarbamoyl, N- methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N- dimethylsulfamoyl, Ν,Ν-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

In certain embodiment, the disclosure relates to compounds having the following formula:

Formula IVA

or pharmaceutically acceptable salts thereof wherein,

X is O or NH;

Y is O or S;

Z is O or NH;

_R30

is an alkyl of 6 to 22 carbons optionally substituted with one or more, the same or different R³⁷; R 31 , R 32 , R 33 , and R 34 are each the same or different at each occurrence hydrogen, alkyl,

31 32 33 34

or alkanoyl, wherein R , R , R , and R are eachoptionally substituted with one or more, the same or different R³⁷;

R³⁵ is hydrogen, alkyl, or aryl wherein R³⁵ is optionally substituted with one or more, the same or different R³⁷;

R^3b is alkyl;

R is the same or different at occurrence alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R³⁷ is optionally substituted with one or more, the same or different, R³⁸; and

In certain embodiments, the disclosure relates to a the compound 2-amino-3,4- dihydroxyoctadecyl hydrogen ((3-(6-amino-purin-9-yl)-2-methylpropoxy)methyl)phosphonate or pharmaceutically acceptable salts thereof.

Formula V

Or pharmaceutically acceptable salts thereof wherein,

X is O or CH₂;

wherein R¹ is a phosphate, phosphonate, polyphosphate, polyphosphonate substituent wherein the phosphate or a phosphate in the polyphosphate or polyphosphonate is optionally a phosphorothioate or phosphoroamidate, and the substituent is further substituted with an amino acid ester or a sphingolipid or derivative optionally substituted with one or more, the same or different, R⁶;

R² is hydrogen, fluoro, hydroxy, alkoxy, or azide;

R³ is hydrogen, fluoro, hydroxy, alkoxy, or azide;

R⁶ is the same or different at occurrence alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl^amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R⁶ is optionally substituted with one or more, the same or different, R⁷; and

R⁷ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, Ν,Ν-diethylcarbamoyl, N- methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N- dimethylsulfamoyl, Ν,Ν-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl

Formula VA

or pharmaceutically acceptable salts thereof wherein,

Y¹ is O or S;

Y² is O or S;

R² is hydrogen, fluoro, hydroxy, or azide;

R³ is hydrogen, fluoro, hydroxy, or azide; R⁴ is a heterocyclyl comprising two or more nitrogen heteroatoms substituted with an oxo, amino, or carbamoyl, wherein R⁴ is optionally substituted with one or more, the same or different alkyl, halogen, or cycloalkyl;

R²⁷ is the same or different at occurrence alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, alkanoyl, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfmyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, or heterocyclyl, wherein each R²⁷ is optionally substituted with one or more, the same or different, R²⁸; and

R²⁸ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl, Ν,Ν-dimethylcarbamoyl, N,N-diethylcarbamoyl, N- methyl-N-ethylcarbamoyl, methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N- dimethylsulfamoyl, Ν,Ν-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

Formula VB

or pharmaceutically acceptable salts thereof wherein,

Lipid is a lipid of any formula described above or herein;

Y¹ is O or S;

Y² is O or S;

R² is hydrogen, fluoro, hydroxy, or azide;

R³ is hydrogen, fluoro, hydroxy, or azide;

In certain embodiments, the sphingolipid may be selected from: 2-aminooctadecane-3,5- diol; (2S,3S,5S)-2-aminooctadecane-3,5-diol; (2S,3R,5S)-2-aminooctadecane-3,5-diol; 2- (methylamino)octadecane-3,5-diol; (2S,3R,5S)-2-(methylamino)octadecane-3,5-diol; 2- (dimethylamino)octadecane-3,5-diol; (2R,3S,5S)-2-(dimethylamino)octadecane-3,5-diol; 1- (pyrrolidin-2-yl)hexadecane-l,3-diol; (lS,3S)-l-((S)-pyrrolidin-2-yl)hexadecane-l,3-diol; 2- amino-11,1 l-difluorooctadecane-3,5-diol; (2S,3S,5S)-2-amino-l 1,1 l-difluorooctadecane-3,5- diol; 11,1 l-difluoro-2-(methylamino)octadecane-3,5-diol; (2S,3S,5S)-11,1 l-difluoro-2- (methylamino)octadecane-3,5-diol; N-((2S,3S,5S)-3,5-dihydroxyoctadecan-2-yl)acetamide; N- ((2S,3S,5S)-3,5-dihydroxyoctadecan-2-yl)palmitamide;l-(l-aminocyclopropyl)hexadecane-l,3- diol; (lS,3R)-l-(l-aminocyclopropyl)hexadecane-l,3-diol; (1S,3S)-1-(1- aminocyclopropyl)hexadecane-l ,3-diol; 2-amino-2-methyloctadecane-3,5-diol; (3S,5S)-2- amino-2-methyloctadecane-3,5-diol; (3S,5R)-2-amino-2-methyloctadecane-3,5-diol; (3S,5S)-2- methyl-2-(methylamino)octadecane-3,5-diol; 2-amino-5-hydroxy-2-methyloctadecan-3-one; (Z)-2-amino-5-hydroxy-2-methyloctadecan-3-one oxime; (2S,3R,5R)-2-amino-6,6- difluorooctadecane-3,5-diol; (2S,3S,5R)-2-amino-6,6-difluorooctadecane-3,5-diol; (2S,3S,5S)-2- amino-6,6-difluorooctadecane-3,5-diol; (2S,3R,5S)-2-amino-6,6-difluorooctadecane-3,5-diol; and (2S,3S,5S)-2-amino-18,18,18-trifluorooctadecane-3,5-diol; which may be optionally substituted with one or more substituents.

In certain embodiments, the disclosure relates to compounds of Formula VIA:

R Base

Formula VIA

wherein R is:

Aryi

O Y

Cation iation

or the mono-, di- or tri-phosphate of

HQH C— 1

Ri is methyl, fluoromethyl, hydroxymethyl, difluoromethyl, trifluoromethyl, acetylenyl, ethyl, vinyl or cyano;

Base is P

WO 2014/124430

_s 7-deazapurines, 7-deaza-7-substituted purines, 5- azapyrimidines, tricyclic heterocycles, pyrazines, triazoles, imidazoles, or 5,6-dihydro-5- azapyrimidines;

Cation is a metal cation, e.g. Li⁺, Na⁺, ⁺, or Mg²⁺; or a quaternary amine, e.g.

trialkylammonium; or other pharmaceutically acceptable salt cation; Sphingoid is a sphingolipid;

Aryl is phenyl; monosubstituted phenyl, e.g. 4-fluorophenyl, 4-chlorophenyl, or 4-bromophi disubstituted phenyl; trisubstituted phenyl; 1-naphthyl; or 2-naphthyl;

and Y is O or S.

In certain embodiments, the disclosure relates to compounds of Formula VIB:

R _Λ Base

Formula VIB

wherein:

Ri is H, fluoro, methyl, fluoromethyl, hydroxymethyl, difluoromethyl, trifluoromethyl, acetylenyl, ethyl, vinyl, or cyano;

Base is P

WO 2014/124430

₅ 7-deazapurines, 7-deaza-7-substituted purines, 5- azapyrimidines, tricyclic heterocycles, pyrazines, triazoles, imidazoles, or 5,6-dihydro-5- azapyrimidines;

Cation is a metal cation, e.g. Li⁺, Na⁺, K⁺, or Mg²⁺; or a quaternary amine, e.g.

Aryl is phenyl; monosubstituted phenyl, e.g. 4-fluorophenyl, 4-chlorophenyl, or 4- bromophenyl; disubstituted phenyl; trisubstituted phenyl; 1-naphthyl; or 2-naphthyl;

Y is O or S;

and R₂ is linear or branched alkyl, e.g. methyl, ethyl, propyl, n-butyl, isopropyl, 2-butyl, 1-ethylpropyl, or 1-propylbutyl; cycloalkyl, e.g. cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; or benzyl.

In certain embodiments, the disclosure relates to compounds of Formula VIC:

Formula VIC

wherein:

Caiion Rati n

, or Caiion 6atfc>n

; Ri is H, fiuoro, methyl, fluoromethyl, hydroxymethyl, difluoromethyl, trifluoromethyl, acetylenyl, ethyl, vinyl, or cyano;

Base is P

WO 2014/124430

trialkylammonium; or other pharmaceutically acceptable salt cation; Y is O or S;

Sphingoid is

wherein R₂ is H; alkyl, e.g. methyl; C(0)R'; C(0)OR'; or C(0)NHR';

R₃ is H; hydroxyl; fluoro; OR'; OC(0)R'; OC(0)OR'; OC(0)NHR';

R' is H; straight or branched alkyl, e.g. methyl, ethyl, propyl, n-butyl, isopropyl, 2-butyl, 1-ethylpropyl, 1-propylbutyl, or a C12-19 long chain alkyl; cycloalkyl, e.g. cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; benzyl; phenyl; monosubstituted phenyl; disubstituted phenyl; trisubstituted phenyl;

or , wherein n is 8-14 and

o is 9-15; or

, wherein "m+n" is 8-14 and "m+o" is 9-15; or

wherein n is 4-10 and o is 5-11; or

, wherein "m+n" is 4-10 and "m+o" is 5-11; or

or wherein n is 6-12; or

, wherein

'm+n" is 6-12; and

R₅ is H, hydroxyl, fluoro, OR', OC(0)R', OC(0)OR', or OC(0)NHR'.

In certain embodiments, the disclosure relates to compounds of Formula VID: R _Λ Base

wherein:

Base is

P

WO 2014/124430

Sphingoid is

;

wherein Rg is H; hydroxyl; fluoro; OR'; OC(0)R'; OC(0)OR'; or OC(0)NHR';

Rv is H; hydroxyl; fluoro; OR'; OC(0)R'; OC(0)OR'; or OC(0)NHR';

R' is H; straight or branched alkyl, e.g. methyl, ethyl, propyl, n-butyl, isopropyl, 2-butyl, 1- ethylpropyl, 1-propylbutyl, or a C₁₂-19 long chain alkyl; cycloalkyl, e.g. cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; benzyl; phenyl; monosubstituted phenyl; disubstituted phenyl; trisubstituted phenyl;

Rg is a C9-15 alkyl chain, e.g.

wherein n is 8-14 or

wherein "m+n" is 8-14.

In certain embodiments, the disclosure relates to compounds of Formula VIIA:

R Base

Formula VIIA

wherein R is:

ΗΟΗ — <

H O H —

mono-, di- or tri-phosphate of ;

Ri is fluoromethyl, hydroxymethyl, difluoromethyl, trifluoromethyl, ethyl, vinyl or cyano;

Base is

70

, 7-deazapurines, 7-deaza-7-substituted purines, 5- azapyrimidines, tricyclic heterocycles, pyrazines, triazoles, imidazoles, or 5,6-dihydro-5- azapyrimidines;

trialkylammonium; or other pharmaceutically acceptable salt cation;

Sphingoid is a sphingolipid;

and Y is O or S.

In certain embodiments, the disclosure relates to compounds of Formula VIIB:

Formula VIIB

wherein R is:

HOH₂C— I

,

mono-, di- or tri-phosphate of ;

Ri is acetylenyl;

Base is

72

7-deazapurines, 7-deaza-7-substituted purines, 5- azapyrimidines, tricyclic heterocycles, pyrazines, triazoles, imidazoles, or 5,6-dihydro-5- azapyrimidines;

trialkylammonium; or other pharmaceutically acceptable salt cation;

Sphingoid is a sphingolipid;

and Y is O or S.

In certain embodiments, the disclosure relates to compounds of Formula VIIC:

Formula VIIC

wherein R is:

Aiyl

Ri is fluoromethyl, hydroxymethyl, difluoromethyl, trifiuoromethyl, acetylenyl, ethyl, vinyl or cyano;

Base is

75

trialkylammonium; or other pharmaceutically acceptable salt cation;

Sphingoid is a sphingolipid;

Aryl is phenyl; monosubstituted phenyl, e.g. 4-fiuorophenyl, 4-chlorophenyl, or 4- bromophenyl; disubstituted phenyl; trisubstituted phenyl; 1-naphthyl; or 2-naphthyl;

R₂ is straight or branched alkyl, e.g. methyl, ethyl, propyl, n-butyl, isopropyl, 2-butyl, 1- ethylpropyl, or 1-propylbutyl; cycloalkyl, e.g. cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; or benzyl;

and Y is O or S.

In certain embodiments, the disclosure relates to compounds of Formula VIID:

Formula VIID wherein R is:

Y

0ation

Ri is H, methyl, fluoromethyl, hydroxymethyl, difluoromethyl, trifluoromethyl, acetylenyl, ethyl, vinyl or cyano;

Base is



, 7-deazapurines, 7-deaza-7-substituted purines, 5 azapyrimidines, tricyclic heterocycles, pyrazines, triazoles, imidazoles, or 5,6-dihydro-5- azapyrimidines;

trialkylammonium; or other pharmaceutically acceptable salt cation;

Y is O or S;

Sphingoid is

;

wherein R₂ is H; alkyl, e.g. methyl; C(0)R'; C(0)OR'; or C(0)NHR';

R₃ is H; hydroxyl; fluoro; OR'; OC(0)R'; OC(0)OR'; OC(0)NHR';

_s or , wherein n is 8-14 and

o is 9-15; or

, wherein "m+n" is 8-14 and "m+o" is 9-15; or

o

wherein n is 4-10 and o is 5-11; or

, wherein "m+n" is 4-10 and "m+o" is 5-11; or

, or , wherein n is 6-12; or

'm+n" is 6-12; and

R₅ is H, hydroxyl, fluoro, OR', OC(0)R', OC(0)OR', or OC(0)NHR'.

In certain embodiments, the disclosure relates to compounds of Formula VIIE

Formula VIIE

wherein R is:

Y

SphingOid-P-OH₂C—

0

0atton

Base is

P

WO 2014/124430

Sphingoid is

;

wherein Rg is H; hydroxyl; fluoro; OR'; OC(0)R'; OC(0)OR'; or OC(0)NHR';

R₇ is H; hydroxyl; fluoro; OR'; OC(0)R'; OC(0)OR'; or OC(0)NHR';

R' is H; straight or branched alkyl, e.g. methyl, ethyl, propyl, n-butyl, isopropyl, 2-butyl, 1-ethylpropyl, 1-propylbutyl, or a C₁₂-19 long chain alkyl; cycloalkyl, e.g. cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; benzyl; phenyl; monosubstituted phenyl; disubstituted phenyl; trisubstituted phenyl;

Rg is a C9-15 alkyl chain, e.g.

wherein n is 8-14 or

wherein "m+n" is 8-14.

In certain embodiments, the disclosure relates to compounds of Formula VIII:

Formula VIII wherein Ri is H, methyl, fluoromethyl, hydroxymethyl, difluoromethyl, trifluoromethyl, acetylenyl, ethyl, vinyl or cyano;

Base is P

WO 2014/124430

Y is O or S; R is straight or branched alkyl, e.g. methyl, ethyl, propyl, n-butyl, isopropyl, 2-butyl, 1- ethylpropyl, 1-propylbutyl, or a C_{12- 1}9 long chain alkyl; cycloalkyl, e.g. cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; or benzyl.

In certain embodiments, the disclosure relates to compounds of Formula IX:

Formula IX wherein Ri is H, methyl, fluoromethyl, hydroxymethyl, difluoromethyl, trifluoromethyl, acetylenyl, ethyl, vinyl or cyano;

Base is

88

Y is O or S;

R is straight or branched alkyl, e.g. methyl, ethyl, propyl, n-butyl, isopropyl, 2-butyl, 1- ethylpropyl, 1-propylbutyl, or a C_{12- 1}9 long chain alkyl; cycloalkyl, e.g. cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; or benzyl.

In certain embodiments, the disclosure relates to compounds of Formula XA:

S hingoid

Formula XA

Base is P

WO 2014/124430

Y is O or S; R.

Sphingoid is HR₂

wherein R₂ is H; alkyl, e.g. methyl; C(0)R'; C(0)OR'; or C(0)NHR';

R₃ is H; hydroxyl; fluoro; OR'; OC(0)R'; OC(0)OR'; OC(0)NHR';

or wherein n is 8-14 and

o is 9-15 or

, wherein "m+n" is 8-14 and "m+o" is 9-15; or

, wherein n is 4-10 and o is 5-11; or

wherein "m+n" is 4-10 and "m+o" is 5-11; or

wherein n is 6-12; or

, wherein

'm+n" is 6-12; and

R₅ is H, hydroxyl, fluoro, OR', OC(0)R', OC(0)OR', or OC(0)NHR'.

In certain embodiments, the disclosure relates to compounds of Formula XB:

Sphi 1ngoid

Formula XB

wherein Ri is H, methyl, fluoromethyl, hydroxymethyl, difluoromethyl, trifluoromethyl, acetylenyl, ethyl, vinyl or cyano;

Base is

95

Y is O or S;

Sphingoid is

wherein g is H; hydroxyl; fluoro; OR'; OC(0)R'; OC(0)OR'; or OC(0)NHR';

Rv is H; hydroxyl; fluoro; OR'; OC(0)R'; OC(0)OR'; or OC(0)NHR';

R' is H; straight or branched alkyl, e.g. methyl, ethyl, propyl, n-butyl, isopropyl, 2-butyl, 1-ethylpropyl, 1-propylbutyl, or a C_12-1g long chain alkyl; cycloalkyl, e.g. cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; benzyl; phenyl; monosubstituted phenyl; disubstituted phenyl; trisubstituted phenyl;

Rg is a C9-15 alkyl chain, e.g.

wherein n is 8-14 or

Infectious Diseases

In some embodiments, the disclosure relates to treating or preventing an infection by viruses, bacteria, fungi, protozoa, and parasites. In some embodiments, the disclosure relates to methods of treating a viral infection comprising administering a compound herein to a subject that is diagnosed with, suspected of, or exhibiting symptoms of a viral infection.

Viruses are infectious agents that can typically replicate inside the living cells of organisms. Virus particles (virions) usually consist of nucleic acids, a protein coat, and in some cases an envelope of lipids that surrounds the protein coat. The shapes of viruses range from simple helical and icosahedral forms to more complex structures. Virally coded protein subunits will self-assemble to form a capsid, generally requiring the presence of the virus genome.

Complex viruses code for proteins that assist in the construction of their capsid. Proteins associated with nucleic acid are known as nucleoproteins, and the association of viral capsid proteins with viral nucleic acid is called a nucleocapsid.

Viruses are transmitted by a variety of methods including direct or bodily fluid contact, e.g., blood, tears, semen, preseminal fluid, saliva, milk, vaginal secretions, lesions; droplet contact, fecal-oral contact, or as a result of an animal bite or birth. A virus has either DNA or RNA genes and is called a DNA virus or a RNA virus respectively. A viral genome is either single-stranded or double-stranded. Some viruses contain a genome that is partially double- stranded and partially single-stranded. For viruses with RNA or single-stranded DNA, the strands are said to be either positive-sense (called the plus-strand) or negative-sense (called the minus-strand), depending on whether it is complementary to the viral messenger RNA (mRNA). Positive-sense viral RNA is identical to viral mRNA and thus can be immediately translated by the host cell. Negative-sense viral RNA is complementary to mRNA and thus must be converted to positive-sense RNA by an RNA polymerase before translation. DNA nomenclature is similar to RNA nomenclature, in that the coding strand for the viral mRNA is complementary to it (negative), and the non-coding strand is a copy of it (positive).

Antigenic shift, or reassortment, can result in novel strains. Viruses undergo genetic change by several mechanisms. These include a process called genetic drift where individual bases in the DNA or RNA mutate to other bases. Antigenic shift occurs when there is a major change in the genome of the virus. This can be a result of recombination or reassortment. RNA viruses often exist as quasispecies or swarms of viruses of the same species but with slightly different genome nucleoside sequences.

The genetic material within viruses, and the method by which the material is replicated, vary between different types of viruses. The genome replication of most DNA viruses takes place in the nucleus of the cell. If the cell has the appropriate receptor on its surface, these viruses enter the cell by fusion with the cell membrane or by endocytosis. Most DNA viruses are entirely dependent on the host DNA and RNA synthesizing machinery, and RNA processing machinery. Replication usually takes place in the cytoplasm. RNA viruses typically use their own RNA replicase enzymes to create copies of their genomes.

The Baltimore classification of viruses is based on the mechanism of mRNA production. Viruses must generate mRNAs from their genomes to produce proteins and replicate themselves, but different mechanisms are used to achieve this. Viral genomes may be single-stranded (ss) or double-stranded (ds), RNA or DNA, and may or may not use reverse transcriptase (RT).

Additionally, ssRNA viruses may be either sense (plus) or antisense (minus). This classification places viruses into seven groups: I, dsDNA viruses (e.g. adenoviruses, herpesviruses, poxviruses); II, ssDNA viruses (plus )sense DNA (e.g. parvoviruses); III, dsRNA viruses (e.g. reoviruses); IV, (plus)ssRNA viruses (plus)sense RNA (e.g. picornaviruses, togaviruses); V, (minus)ssRNA viruses (minus)sense RNA (e.g. orthomyxoviruses, Rhabdoviruses); VI, ssRNA- RT viruses (plus)sense RNA with DNA intermediate in life-cycle (e.g. retroviruses); and VII, dsDNA-RT viruses (e.g. hepadna viruses).

Human immunodeficiency virus (HIV) is a lentivirus (a member of the retrovirus family) that causes acquired immunodeficiency syndrome (AIDS). Lentiviruses are transmitted as single-stranded, positive-sense, enveloped RNA viruses. Upon entry of the target cell, the viral RNA genome is converted to double-stranded DNA by a virally encoded reverse transcriptase. This viral DNA is then integrated into the cellular DNA by a virally encoded integrase, along with host cellular co-factors. There are two species of HIV. HIV-1 is sometimes termed LAV or HTLV-III.

HIV infects primarily vital cells in the human immune system such as helper T cells (CD4+ T cells), macrophages, and dendritic cells. HIV infection leads to low levels of CD4+ T cells. When CD4+ T cell numbers decline below a critical level, cell-mediated immunity is lost, and the body becomes progressively more susceptible to other viral or bacterial infections.

Subjects with HIV typically develop malignancies associated with the progressive failure of the immune system.

The viral envelope is composed of two layers of phospholipids taken from the membrane of a human cell when a newly formed virus particle buds from the cell. Embedded in the viral envelope are proteins from the host cell and a HIV protein known as Env. Env contains glycoproteinsgpl20, and gp41. The RNA genome consists of at structural landmarks (LTR, TAR, RRE, PE, SLIP, CRS, and INS) and nine genes (gag, pol, and env, tat, rev, nef, vif, vpr, vpu, and sometimes a tenth tev, which is a fusion of tat env and rev) encoding 19 proteins. Three of these genes, gag, pol, and env, contain information needed to make the structural proteins for new virus particles. HIV-1 diagnosis is typically done with antibodies in an ELISA, Western blot, orimmunoaffinity assays or by nucleic acid testing (e.g., viral RNA or DNA amplification).

HIV is typically treated with a combination of antiviral agent, e.g., two nucleoside- analogue reverse transcription inhibitors and one non-nucleoside-analogue reverse transcription inhibitor or protease inhibitor. The three drug combination is commonly known as a triple cocktail. In certain embodiments, the disclosure relates to treating a subject diagnosed with HIV by administering a pharmaceutical composition disclosed herein in combination with two nucleoside-analogue reverse transcription inhibitors and one non-nucleoside-analogue reverse transcription inhibitor or protease inhibitor.

In certain embodiments, the disclosure relates to treating a subject by administering a compound disclosed herein, emtricitabine, tenofovir, and efavirenz. In certain embodiments, the disclosure relates to treating a subject by administering a compound disclosed herein, emtricitabine, tenofovir and raltegravir. In certain embodiments, the disclosure relates to treating a subject by administering a compound disclosed herein, emtricitabine, tenofovir, ritonavir and darunavir. In certain embodiments, the disclosure relates to treating a subject by administering a compound disclosed herein, emtricitabine, tenofovir, ritonavir and atazanavir.

In certain embodiments, the disclosure relates to methods of treating a subject by administering a lipid conjugated compound disclosed herein. In certain embodiments, the disclosure relates to methods of treating a subject by administering a sphingolipid conjugate.

Banana lectin (BanLec or BanLec-1) is one of the predominant proteins in the pulp of ripe bananasand has binding specificity for mannose and mannose-containing oligosaccharides. BanLec binds to the HIV-1 envelope protein gpl20. In certain embodiments, the disclosure relates to treating viral infections, such as HIV, by administering a compound disclosed herein in combination with a banana lectin.

The hepatitis C virus is a single-stranded, positive sense RNA virus. It is the only known member of the hepacivirus genus in the family Flaviviridae. There are six major genotypes of the hepatitis C virus, which are indicated numerically. The hepatitis C virus particle consists of a core of genetic material (RNA), surrounded by an icosahedral protective shell, and further encased in a lipid envelope. Two viral envelope glycoproteins, El and E2, are embedded in the lipid envelope. The genome consists of a single open reading frame translated to produce a single protein. This large pre-protein is later cut by cellular and viral proteases into smaller proteins that allow viral replication within the host cell, or assemble into the mature viral particles, e.g., El, E2, NS2, NS3, NS4, NS4A, NS4B, NS5, NS5A, and NS5B.

HCV leads to inflammation of the liver, and chronic infection leads to cirrhosis. Most people with hepatitis C infection have the chronic form. Diagnosis of HCV can occur via nucleic acid analysis of the 5'-noncoding region. ELISA assay may be performed to detect hepatitis C antibodies and RNA assays to determine viral load. Subjects infected with HCV may exhibit symptoms of abdominal pain, ascites, dark urine, fatigue, generalized itching, jaundice, fever, nausea, pale or clay-colored stools and vomiting.

Therapeutic agents in some cases may suppress the virus for a long period of time.

Typical medications are a combination of interferon alpha and ribavirin. Subjects may receive injections of pegylated interferon alpha. Genotypes 1 and 4 are less responsive to interferon- based treatment than are the other genotypes (2, 3, 5 and 6). In certain embodiments, the disclosure relates to treating a subject with HCV by administering a compound disclosed herein to a subject exhibiting symptoms or diagnosed with HCV. In certain embodiments, the compound is administered in combination with interferon alpha and another antiviral agent such as ribavirin, and/or a protease inhibitor such as telaprevir or boceprevir. In certain embodiments, the subject is diagnosed with genotype 2, 3, 5, or 6. In other embodiments, the subject is diagnosed with genotype 1 or 4.

In certain embodiments, the subject is diagnosed to have a virus by nucleic acid detection or viral antigen detection. Cytomegalovirus (CMV) belongs to the Betaherpesvirinae subfamily of Herpesviridae. In humans it is commonly known as HCMV or Human Herpesvirus 5 (HHV- 5). Herpesviruses typically share a characteristic ability to remain latent within the body over long periods. HCMV infection may be life threatening for patients who are

immunocompromised. In certain embodiments, the disclosure relates to methods of treating a subject diagnosed with cytomegalovirus or preventing a cytomegalovirus infection by

administration of a compound disclosed herein. In certain embodiments, the subject is immunocompromised. In typical embodiments, the subject is an organ transplant recipient, undergoing hemodialysis, diagnosed with cancer, receiving an immunosuppressive drug, and/or diagnosed with an HIV-infection. In certain embodiments, the subject may be diagnosed with cytomegalovirus hepatitis, the cause of fulminant liver failure, cytomegalovirus retinitis

(inflammation of the retina, may be detected by ophthalmoscopy), cytomegalovirus colitis

(inflammation of the large bowel), cytomegalovirus pneumonitis, cytomegalovirus esophagitis, cytomegalovirus mononucleosis, polyradiculopathy, transverse myelitis, and subacute encephalitis. In certain embodiments, a compound disclosed herein is administerd in

combination with an antiviral agent such as valganciclovir or ganciclovir. In certain

embodiments, the subject undergoes regular serological monitoring.

HCMV infections of a pregnant subject may lead to congenital abnormalities. Congenital HCMV infection occurs when the mother suffers a primary infection (or reactivation) during pregnancy. In certain embodiments, the disclosure relates to methods of treating a pregnant subject diagnosed with cytomegalovirus or preventing a cytomegalovirus infection in a subject at risk for, attempting to become, or currently pregnant by administering a compound disclosed herein.

Subjects who have been infected with CMV typically develop antibodies to the virus. A number of laboratory tests that detect these antibodies to CMV have been developed. The virus may be cultured from specimens obtained from urine, throat swabs, bronchial lavages and tissue samples to detect active infection. One may monitor the viral load of CMV-infected subjects using PCR. CMV pp65 antigenemia test is an immunoaffmity based assay for identifying the pp65 protein of cytomegalovirus in peripheral blood leukocytes. CMV should be suspected if a patient has symptoms of infectious mononucleosis but has negative test results for

mononucleosis and Epstein-Barr virus, or if they show signs of hepatitis, but have negative test results for hepatitis A, B, and C. A virus culture can be performed at any time the subject is symptomatic. Laboratory testing for antibody to CMV can be performed to determine if a subject has already had a CMV infection.

The enzyme-linked immunosorbent assay (or ELISA) is the most commonly available serologic test for measuring antibody to CMV. The result can be used to determine if acute infection, prior infection, or passively acquired maternal antibody in an infant is present. Other tests include various fluorescence assays, indirect hemagglutination, (PCR), and latex agglutination. An ELISA technique for CMV-specific IgM is available.

Hepatitis B virus is a hepadna virus. The virus particle, (virion) consists of an outer lipid envelope and an icosahedral nucleocapsid core composed of protein. The genome of HBV is made of circular DNA, but the DNA is not fully double-stranded. One end of the strand is linked to the viral DNA polymerase. The virus replicates through an RNA intermediate form by reverse transcription. Replication typically takes place in the liver where it causes inflammation

(hepatitis). The virus spreads to the blood where virus-specific proteins and their corresponding antibodies are found in infected people. Blood tests for these proteins and antibodies are used to diagnose the infection.

Hepatitis B virus gains entry into the cell by endocytosis. Because the virus multiplies via RNA made by a host enzyme, the viral genomic DNA has to be transferred to the cell nucleus by host chaperones. The partially double stranded viral DNA is then made fully double stranded and transformed into covalently closed circular DNA (cccDNA) that serves as a template for transcription of viral mRNAs.The virus is divided into four major serotypes (adr, adw, ayr, ayw) based on antigenic epitopes presented on its envelope proteins, and into eight genotypes (A-H) according to overall nucleotide sequence variation of the genome.

The hepatitis B surface antigen (HBsAg) is typically used to screen for the presence of this infection. It is the first detectable viral antigen to appear during infection. However, early in an infection, this antigen may not be present and it may be undetectable later in the infection if it is being cleared by the host. The infectious virion contains an inner "core particle" enclosing viral genome. The icosahedral core particle is made of core protein, alternatively known as hepatitis B core antigen, or HBcAg. IgM antibodies to the hepatitis B core antigen (anti-HBc IgM) may be used as a serological marker. Hepatitis B e antigen (HBeAg) may appear. The presence of HBeAg in the serum of the host is associated with high rates of viral replication. Certain variants of the hepatitis B virus do not produce the 'e' antigen,

If the host is able to clear the infection, typically the HBsAg will become undetectable and will be followed by IgG antibodies to the hepatitis B surface antigen and core antigen, (anti- HBs and anti HBc IgG). The time between the removal of the HBsAg and the appearance of anti-HBs is called the window period. A person negative for HBsAg but positive for anti-HBs has either cleared an infection or has been vaccinated previously. Individuals who remain HBsAg positive for at least six months are considered to be hepatitis B carriers. Carriers of the virus may have chronic hepatitis B, which would be reflected by elevated serum alanine aminotransferase levels and inflammation of the liver which may be identified by biopsy.

Nucleic acid (PCR) tests have been developed to detect and measure the amount of HBV DNA in clinical specimens.

Acute infection with hepatitis B virus is associated with acute viral hepatitis. Acute viral hepatitis typically begins with symptoms of general ill-health, loss of appetite, nausea, vomiting, body aches, mild fever, dark urine, and then progresses to development of jaundice. Chronic infection with hepatitis B virus may be either asymptomatic or may be associated with a chronic inflammation of the liver (chronic hepatitis), possibly leading to cirrhosis. Having chronic hepatitis B infection increases the incidence of hepatocellular carcinoma (liver cancer).

During HBV infection, the host immune response causes both hepatocellular damage and viral clearance. The adaptive immune response, particularly virus-specific cytotoxic T lymphocytes (CTLs), contributes to most of the liver injury associated with HBV infection. By killing infected cells and by producing antiviral cytokines capable of purging HBV from viable hepatocytes, CTLs eliminate the virus .Although liver damage is initiated and mediated by the CTLs, antigen-nonspecific inflammatory cells can worsen CTL-induced immunopathology, and platelets activated at the site of infection may facilitate the accumulation of CTLs in the liver.

Therapeutic agents can stop the virus from replicating, thus minimizing liver damage. In certain embodiments, the disclosure relates to methods of treating a subject diagnosed with HBV by administering a compound disclosed herein disclosed herein. In certain embodiments, the subject is immunocompromised. In certain embodiments, the compound is administered in combination with another antiviral agent such as lamivudine, adefovir, tenofovir, telbivudine, and entecavir, and/or immune system modulators interferon alpha-2a and pegylated interferon alpha-2a (Pegasys). In certain embodiments, the disclosure relates to preventing an HBV infection in an immunocompromised subject at risk of infection by administering a

pharmaceutical composition disclosed herein and optionally one or more antiviral agents. In certain embodiments, the subject is at risk of an infection because the sexual partner of the subject is diagnosed with HBV.

In one aspect of the disclosure, an "infection" or "bacterial infection" refers to an infection caused by acinetobacter spp, bacteroides spp, burkholderia spp, Campylobacter spp, chlamydia spp, chlamydophila spp, Clostridium spp, enterobacter spp, enterococcus spp, escherichia spp, fusobacterium spp, gardnerella spp, haemophilus spp, helicobacter spp, lebsiella spp, legionella spp, moraxella spp, morganella spp, mycoplasma spp, neisseria spp, peptococcus spp peptostreptococcus spp, proteus spp, pseudomonas spp, salmonella spp, serratia spp., staphylococcus spp, streptoccocus spp, stenotrophomonas spp, or ureaplasma spp.

In one aspect of the disclosure, an "infection" or "bacterial infection" refers to an infection caused by acinetobacter baumanii, acinetobacter haemolyticus, acinetobacter junii, acinetobacter johnsonii, acinetobacter Iwoffi, bacteroides bivius, bacteroides fragilis , burkholderia cepacia, Campylobacter jejuni, chlamydia pneumoniae, chlamydia urealyticus , chlamydophila pneumoniae, Clostridium difficile, enterobacter aerogenes, enterobacter cloacae, enterococcus faecalis, enterococcus faecium, escherichia coli, gardnerella vaginalis, haemophilus par influenzae, haemophilus influenzae, helicobacter pylori, Klebsiella

pneumoniae, legionella pneumophila, methicillin-resistant staphylococcus aureus, methicillin- susceptible staphylococcus aureus, moraxella catarrhalis, morganella morganii, mycoplasma pneumoniae, neisseria gonorrhoeae, penicillin-resistant streptococcus pneumoniae, penicillin- susceptible streptococcus pneumoniae, peptostreptococcus magnus, peptostreptococcus micros, peptostreptococcus anaerobius, peptostreptococcus asaccharolyticus , peptostreptococcus prevotii, peptostreptococcus tetradius, peptostreptococcus vaginalis, proteus mirabilis, pseudomonas aeruginosa, quino lone-resistant staphylococcus aureus, quinolone-resistant staphylococcus epidermis, salmonella typhi, salmonella paratyphi, salmonella enteritidis, salmonella typhimurium, serratia marcescens, staphylococcus aureus, staphylococcus epidermidis, staphylococcus saprophyticus, streptoccocus agalactiae, streptococcus

pneumoniae, streptococcus pyogenes, stenotrophomonas maltophilia, ureaplasma urealyticum, vancomycin-resistant enterococcus faecium, vancomycin-resistant enterococcus faecalis, vancomycin-resistant staphylococcus aureus, vancomycin-resistant staphylococcus epidermis, mycobacterium tuberculosis, Clostridium perfringens, Klebsiella oxytoca, neisseria miningitidis, proteus vulgaris, or coagulase-negative staphylococcus (including staphylococcus lugdunensis, staphylococcus capitis, staphylococcus hominis, or staphylococcus saprophytic ).

In one aspect of the disclosure "infection" or "bacterial infection" refers to aerobes, obligate anaerobes, facultative anaerobes, gram-positive bacteria, gram-negative bacteria, gram- variable bacteria, or atypical respiratory pathogens. In some embodiments, the disclosure relates to treating a bacterial infection such as a gynecological infection, a respiratory tract infection (RTI), a sexually transmitted disease, or a urinary tract infection.

In some embodiments, the disclosure relates to treating a bacterial infection such as an infection caused by drug resistant bacteria.

In some embodiments, the disclosure relates to treating a bacterial infection such as community-acquired pneumoniae, hospital-acquired pneumoniae, skin & skin structure infections, gonococcal cervicitis, gonococcal urethritis, febrile neutropenia, osteomyelitis, endocarditis, urinary tract infections and infections caused by drug resistant bacteria such as penicillin-resistant streptococcus pneumoniae, methicillin- resistant staphylococcus aureus, methicillin-resistant staphylococcus epidermidis and vancomycin-resistant enterococci, syphilis, ventilator-associated pneumonia, intra-abdominal infections, gonorrhoeae, meningitis, tetanus, or tuberculosis.

In some embodiments, the disclosure relates to treating a fungal infections such as infections caused by tinea versicolor,microsporum, trichophyton, epidermophyton, candidiasis, cryptococcosis,or aspergillosis.

In some embodiments, the disclosure relates to treating an infection caused by protozoa including, but not limited to, malaria, amoebiasis, giardiasis, toxoplasmosis, cryptosporidiosis, trichomoniasis, leishmaniasis, sleeping sickness, or dysentery.

Certain compounds disclosed herein are useful to prevent or treat an infection of a malarial parasite in a subject and/or for preventing, treating and/or alleviating complications and/or symptoms associated therewith and can then be used in the preparation of a medicament for the treatment and/or prevention of such disease. The malaria may be caused by Plasmodium falciparum, P. vivax, P. ovale, or P. malariae.

In one embodiment, the compound is administered after the subject has been exposed to the malaria parasite. In another embodiment, a compound disclosed hereinis administered before the subject travels to a country where malaria is endemic.

In certain embodiments, the disclosure relates to the use of the compounds as topical microbicides. In certain embodiments, the disclosure relates to methods of treating a subject by using the lipid conjugates described herein as topical microbicides. In particular embodiments, the disclosure relates to methods of treating a subject by using the sphingo lipid conjugates described herein as topical microbicides.

The compounds or the above-mentioned pharmaceutical compositions may also be used in combination with one or more other therapeutically useful substances selected from the group comprising antimalarials like quinolines (e.g., quinine, chloroquine, amodiaquine, mefloquine, primaquine, tafenoquine); peroxide antimalarials (e.g.,artemisinin, artemether, artesunate); pyrimethamine-sulfadoxine antimalarials (e.g., Fansidar); hydroxynaphtoquinones (e.g., atovaquone); acroline-type antimalarials (e.g., pyronaridine); and antiprotozoal agents such as ethylstibamine, hydroxystilbamidine, pentamidine, stilbamidine, quinapyramine, puromycine, propamidine, nifurtimox, melarsoprol, nimorazole, nifuroxime, aminitrozole and the like.

In an embodiment, compounds disclosed herein can be used in combination one additional drug selected from the group consisting of chloroquine, artemesin, qinghaosu, 8- aminoquinoline, amodiaquine, arteether, artemether, artemisinin, artesunate, artesunic acid, artelinic acid, atovoquone, azithromycine, biguanide, chloroquine phosphate, chlorproguanil, cycloguanil, dapsone, desbutyl halofantrine, desipramine, doxycycline, dihydro folate reductase inhibitors, dipyridamole, halofantrine, haloperidol, hydroxychloroquine sulfate, imipramine, mefloquine, penfluridol, phospholipid inhibitors, primaquine, proguanil, pyrimethamine, pyronaridine, quinine, quinidine, quinacrineartemisinin, sulfonamides, sulfones, sulfadoxine, sulfalene, tafenoquine, tetracycline, tetrandine, triazine, salts or mixture thereof.

Cancer

In a typical embodiment, the disclosure relates to a method treating cancer comprising administering to a patient a compound disclosed herein. In some embodiments, the disclosure relates to a compound disclosed herein, or a pharmaceutically acceptable salt thereof for uses in treating cancer.

In some embodiments, the disclosure relates to a compound disclosed herein, or a pharmaceutically acceptable salt thereof, as defined herein for use in the treatment of cancer of the breast, colorectum, lung (including small cell lung cancer, non- small cell lung cancer and bronchioalveolar cancer) and prostate.

In some embodiments, the disclosure relates to a compound disclosed herein, or a pharmaceutically acceptable salt thereof, as defined herein for use in the treatment of cancer of the bile duct, bone, bladder, head and neck, kidney, liver, gastrointestinal tissue, oesophagus, ovary, endometrium, pancreas, skin, testes, thyroid, uterus, cervix and vulva, and of leukaemias (including ALL and CML), multiple myeloma and lymphomas.

In some embodiments, the disclosure relates to a compound disclosed herein, or a pharmaceutically acceptable salt thereof, as defined herein for use in the treatment of lung cancer, prostate cancer, melanoma, ovarian cancer, breast cancer, endometrial cancer, kidney cancer, gastric cancer, sarcomas, head and neck cancers, tumors of the central nervous system and their metastases, and also for the treatment of glioblastomas.

In some embodiments, compounds disclosed herein could be used in the clinic either as a single agent by itself or in combination with other clinically relevant agents. This compound could also prevent the potential cancer resistance mechanisms that may arise due to mutations in a set of genes.

The anti-cancer treatment defined herein may be applied as a sole therapy or may involve, in addition to the compound of the disclosure, conventional surgery or radiotherapy or chemotherapy. Such chemotherapy may include one or more of the following categories of anti- tumour agents:

(i) antiproliferative/antineoplastic drugs and combinations thereof, as used in medical oncology, such as alkylating agents (for example cis-platin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulfan and nitrosoureas); antimetabolites (for example antifolates such as fluoropyrimidines like 5-fluorouracil and gemcitabine, tegafur, raltitrexed, methotrexate, cytosine arabinoside and hydroxyurea); antitumour antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic agents (for example vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine and taxoids like taxol and taxotere); and topoisomerase inhibitors (for example epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan and camptothecin); and proteosome inhibitors (for example bortezomib [Velcade®]); and the agent anegrilide [Agrylin®]; and the agent alpha- interferon;

(ii) cytostatic agents such as antioestrogens (for example tamoxifen, toremifene, raloxifene, droloxifene and iodoxyfene), oestrogen receptor down regulators (for example fulvestrant), antiandrogens (for example bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH antagonists or LHRH agonists (for example goserelin, leuprorelin and buserelin), progestogens (for example megestrol acetate), aromatase inhibitors (for example as anastrozole, letrozole, vorazole and exemestane) and inhibitors of 5 -reductase such as finasteride;

(iii) agents which inhibit cancer cell invasion (for example metalloproteinase inhibitors like marimastat and inhibitors of urokinase plasminogen activator receptor function);

(iv) inhibitors of growth factor function, for example such inhibitors include growth factor antibodies, growth factor receptor antibodies (for example the anti-erbb2 antibody trastuzumab [Herceptin™] and the anti-erbbl antibody cetuximab) , farnesyl transferase inhibitors, tyrosine kinase inhibitors and serine/threonine kinase inhibitors, for example inhibitors of the epidermal growth factor family (for example EGFR family tyrosine kinase inhibitors such as: N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin- 4-a mine (gefitinib), N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine

(erlotinib), and 6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazolin- 4-amine (CI 1033), for example inhibitors of the platelet-derived growth factor family and for example inhibitors of the hepatocyte growth factor family, for example inhibitors or

phosphotidylinositol 3-kinase (PI3K) and for example inhibitors of mitogen activated protein kinase kinase (MEKl/2) and for example inhibitors of protein kinase B (PKB/Akt), for example inhibitors of Src tyrosine kinase family and/or Abelson (Abl) tyrosine kinase family such as dasatinib (BMS-354825) and imatinib mesylate (Gleevec™); and any agents that modify STAT signalling;

(v) antiangiogenic agents such as those which inhibit the effects of vascular endothelial growth factor, (for example the anti-vascular endothelial cell growth factor antibody

bevacizumab [Avastin™]) and compounds that work by other mechanisms (for example linomide, inhibitors of integrin οσνβ3 function and angiostatin);

(vi) vascular damaging agents such as Combretastatin A4;

(vii) antisense therapies, for example those which are directed to the targets listed above, such as an anti-ras antisense;

(viii) gene therapy approaches, including for example approaches to replace aberrant genes such as aberrant p53 or aberrant BRCAl or BRCA2, GDEPT (gene-directed enzyme pro- drug therapy) approaches such as those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme and approaches to increase patient tolerance to chemotherapy or radiotherapy such as multi-drug resistance gene therapy; and

(ix) immunotherapy approaches, including for example ex-vivo and in-vivo approaches to increase the immunogenicity of patient tumour cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor, approaches to decrease T-cell anergy, approaches using transfected immune cells such as cytokine- transfected dendritic cells, approaches using cytokine-transfected tumour cell lines and approaches using anti-idiotypic antibodies, and approaches using the immunomodulatory drugs thalidomide and lenalidomide [Revlimid®].

Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment. Such combination products employ the compounds of this disclosure, or pharmaceutically acceptable salts thereof, within the dosage range described hereinbefore and the other pharmaceutically-active agent within its approved dosage range.

Formulations

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

When the compounds of the disclosure contain an acidic group as well as a basic group, the compounds of the disclosure may also form internal salts, and such compounds are within the scope of the disclosure. When a compound of the disclosure contains a hydrogen-donating heteroatom (e.g., NH), the disclosure also covers salts and/or isomers formed by the transfer of the hydrogen atom to a basic group or atom within the molecule.

Pharmaceutically acceptable salts of the compounds include the acid addition and base salts thereof. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts. Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts. Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts. For a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley- VCH, 2002), incorporated herein by reference.

The compounds described herein may be administered in the form of prodrugs. A prodrug can include a covalently bonded carrier which releases the active parent drug when administered to a mammalian subject. Prodrugs can be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compounds. Prodrugs include, for example, compounds wherein a hydroxyl group is bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxyl group. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol functional groups in the compounds. Methods of structuring a compound as prodrugs can be found in the book of Testa and Mayer, Hydrolysis in Drug and Prodrug Metabolism, Wiley (2006). Typical prodrugs form the active metabolite by transformation of the prodrug by hydrolytic enzymes, the hydrolysis of amide, lactams, peptides, carboxylic acid esters, epoxides or the cleavage of esters of inorganic acids. It is well within the ordinary skill of the art to make an ester prodrug, e.g., acetyl ester of a free hydroxyl group. It is well known that ester prodrugs are readily degraded in the body to release the corresponding alcohol. See e.g., Imai, Drug Metab Pharmacokinet. (2006)

21(3): 173-85, entitled "Human carboxylesterase isozymes: catalytic properties and rational drug design."

Pharmaceutical compositions for use in the present disclosure typically comprise an effective amount of a compound and a suitable pharmaceutical acceptable carrier. The preparations may be prepared in a manner known per se, which usually involves mixing the at least one compound according to the disclosure with the one or more pharmaceutically acceptable carriers, and, if desired, in combination with other pharmaceutical active compounds, when necessary under aseptic conditions. Reference is made to U.S. Pat. No. 6,372,778, U.S. Pat. No. 6,369,086, U.S. Pat. No. 6,369,087 and U.S. Pat. No. 6,372,733 and the further references mentioned above, as well as to the standard handbooks, such as the latest edition of Remington's Pharmaceutical Sciences.

Generally, for pharmaceutical use, the compounds may be formulated as a

pharmaceutical preparation comprising at least one compound and at least one pharmaceutically acceptable carrier, diluent or excipient, and optionally one or more further pharmaceutically active compounds.

The pharmaceutical preparations of the disclosure are preferably in a unit dosage form, and may be suitably packaged, for example in a box, blister, vial, bottle, sachet, ampoule or in any other suitable single-dose or multi-dose holder or container (which may be properly labeled); optionally with one or more leaflets containing product information and/or instructions for use. Generally, such unit dosages will contain between 1 and 1000 mg, and usually between 5 and 500 mg, of the at least one compound of the disclosure, e.g., about 10, 25, 50, 100, 200, 300 or 400 mg per unit dosage.

The compounds can be administered by a variety of routes including the oral, ocular, rectal, transdermal, subcutaneous, intravenous, intramuscular or intranasal routes, depending mainly on the specific preparation used. The compound will generally be administered in an "effective amount", by which is meant any amount of a compound that, upon suitable

administration, is sufficient to achieve the desired therapeutic or prophylactic effect in the subject to which it is administered. Usually, depending on the condition to be prevented or treated and the route of administration, such an effective amount will usually be between 0.01 to 1000 mg per kilogram body weight of the patient per day, more often between 0.1 and 500 mg, such as between 1 and 250 mg, for example about 5, 10, 20, 50, 100, 150, 200 or 250 mg, per kilogram body weight of the patient per day, which may be administered as a single daily dose, divided over one or more daily doses. The amount(s) to be administered, the route of

administration and the further treatment regimen may be determined by the treating clinician, depending on factors such as the age, gender and general condition of the patient and the nature and severity of the disease/symptoms to be treated. Reference is made to U.S. Pat. No.

I l l 6,372,778, U.S. Pat. No. 6,369,086, U.S. Pat. No. 6,369,087 and U.S. Pat. No. 6,372,733 and the further references mentioned above, as well as to the standard handbooks, such as the latest edition of Remington's Pharmaceutical Sciences.

For an oral administration form, the compound can be mixed with suitable additives, such as excipients, stabilizers or inert diluents, and brought by means of the customary methods into the suitable administration forms, such as tablets, coated tablets, hard capsules, aqueous, alcoholic, or oily solutions. Examples of suitable inert carriers are gum arabic, magnesia, magnesium carbonate, potassium phosphate, lactose, glucose, or starch, in particular, corn starch. In this case, the preparation can be carried out both as dry and as moist granules. Suitable oily excipients or solvents are vegetable or animal oils, such as sunflower oil or cod liver oil.

Suitable solvents for aqueous or alcoholic solutions are water, ethanol, sugar solutions, or mixtures thereof. Polyethylene glycols and polypropylene glycols are also useful as further auxiliaries for other administration forms. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.

When administered by nasal aerosol or inhalation, the compositions may be prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. Suitable pharmaceutical formulations for administration in the form of aerosols or sprays are, for example, solutions, suspensions or emulsions of the compounds of the disclosure or their physiologically tolerable salts in a pharmaceutically acceptable solvent, such as ethanol or water, or a mixture of such solvents. If required, the formulation may additionally contain other pharmaceutical auxiliaries such as surfactants, emulsifiers and stabilizers as well as a propellant.

For subcutaneous or intravenous administration, the compounds, if desired with the substances customary therefore such as solubilizers, emulsifiers or further auxiliaries are brought into solution, suspension, or emulsion. The compounds may also be lyophilized and the lyophilizates obtained used, for example, for the production of injection or infusion preparations. Suitable solvents are, for example, water, physiological saline solution or alcohols, e.g. ethanol, propanol, glycerol, sugar solutions such as glucose or mannitol solutions, or mixtures of the various solvents mentioned. The injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.

When rectally administered in the form of suppositories, the formulations may be prepared by mixing the compounds of formula I with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug.

For topical administration, the compounds can be in various forms known to the art, including liquid form or in lotion form, either oil-in- water or water-in-oil emulsions, in aqueous gel compositions, in the form of foams, films, sprays, ointments, pessary, suppository, capsules, tablets, jellies, creams, liposomes or in other forms embedded in a matrix for the slow or controlled release of the biologically active material to the skin or surface onto which it has been applied or in contact. In one embodiment, the topical compositions of the present invention are aqueous compositions. In another embodiment, the topical compositions are aqueous gel compositions.

In certain embodiments, it is contemplated that these compositions can be extended release formulations. Typical extended release formations utilize an enteric coating. Typically, a barrier is applied to oral medication that controls the location in the digestive system where it is absorbed. Enteric coatings prevent release of medication before it reaches the small intestine. Enteric coatings may contain polymers of polysaccharides, such as maltodextrin, xanthan, scleroglucan dextran, starch, alginates, pullulan, hyaloronic acid, chitin, chitosan and the like; other natural polymers, such as proteins (albumin, gelatin etc.), poly-L-lysine; sodium

poly(acrylic acid); poly(hydroxyalkylmethacrylates) (for example

poly(hydroxyethylmethacrylate)); carboxypolymethylene (for example Carbopol™); carbomer; polyvinylpyrrolidone; gums, such as guar gum, gum arabic, gum karaya, gum ghatti, locust bean gum, tamarind gum, gellan gum, gum tragacanth, agar, pectin, gluten and the like; poly( vinyl alcohol); ethylene vinyl alcohol; polyethylene glycol (PEG); and cellulose ethers, such as hydroxymethylcellulose (HMC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), methylcellulose (MC), ethylcellulose (EC), carboxyethylcellulose (CEC),

ethylhydroxyethylcellulose (EHEC), carboxymethylhydroxyethylcellulose (CMHEC), hydroxypropylmethyl-cellulose (HPMC), hydroxypropylethylcellulose (HPEC) and sodium carboxymethylcellulose (Na CMC); as well as copolymers and/or (simple) mixtures of any of the above polymers. Certain of the above-mentioned polymers may further be crosslinked by way of standard techniques.

The choice of polymer will be determined by the nature of the active ingredient/drug that is employed in the composition of the disclosure as well as the desired rate of release. In particular, it will be appreciated by the skilled person, for example in the case of HPMC, that a higher molecular weight will, in general, provide a slower rate of release of drug from the composition. Furthermore, in the case of HPMC, different degrees of substitution of methoxyl groups and hydroxypropoxyl groups will give rise to changes in the rate of release of drug from the composition. In this respect, and as stated above, it may be desirable to provide

compositions of the disclosure in the form of coatings in which the polymer carrier is provided by way of a blend of two or more polymers of, for example, different molecular weights in order to produce a particular required or desired release profile.

Microspheres of polylactide, polyglycolide, and their copolymers poly(lactide-co- glycolide) may be used to form sustained-release protein delivery systems. Proteins can be entrapped in the poly(lactide-co-glycolide) microsphere depot by a number of methods, including formation of a water-in-oil emulsion with water-borne protein and organic solvent- borne polymer (emulsion method), formation of a solid-in-oil suspension with solid protein dispersed in a solvent-based polymer solution (suspension method), or by dissolving the protein in a solvent-based polymer solution (dissolution method). One can attach poly(ethylene glycol) to proteins (PEGylation) to increase the in vivo half-life of circulating therapeutic proteins and decrease the chance of an immune response. EXAMPLES

Example 1.

Conjugate Preparation

Mono and diphosphate prodrugs have been prepared by several groups. See Jessen et al, Bioreversible Protection of Nucleoside Diphosphates, Angewandte Chemie-International Edition English 2008, 47 (45), 8719-8722, hereby incorporated by reference. In order to prevent rupture of the P-O-P anhydride bond, one utilizes a pendant group that fragments rapidly (e.g. bis-(4- acyloxybenzyl)-nucleoside diphosphates (BAB-NDP) that is deacylated by an endogenous esterase) to generate a negative charge on the second phosphate. See also Routledge et al, Synthesis, Bioactivation and Anti-HIV Activity of 4-Acyloxybenzyl-bis(nucleosid-5'-yl)

Phosphates, Nucleosides & Nucleotides 1995, 14 (7), 1545-1558 and Meier et al, Comparative study of bis(benzyl)phosphate triesters of 2',3'-dideoxy-2',3'-didehydrothymidine (d4T) and cycloSal-d4TMP -hydrolysis, mechanistic insights and anti-HIV activity, Antiviral Chemistry and Chemotherapy 2002, 13,101-114, both hereby incorporated by reference. Once this occurs, the P-O-P anhydride bond is less susceptible to cleavage and the remaining protecting group can then do its final unraveling to produce the nucleoside diphosphate.

Other methods to prepare diphosphate and monothiodiphosphate prodrugs are shown in Figure 7. Standard coupling conditions are used to prepare sphingolipid-cyclobutyl nucleoside monophosphate prodrugs. The corresponding diphosphate prodrugs may be prepared according to the protocols shown in Figure 7 and as provided in Smith et al, Substituted Nucleotide Analogs. U.S. Patent Application 2012/0071434; Skowronska et al., Reaction of

Oxophosphorane-Sulfenyl and Oxophosphorane-Selenenyl Chlorides with Dialkyl Trimethylsilyl Phosphites - Novel Synthesis of Compounds Containing a Sulfur or Selenium Bridge Between 2 Phosphoryl Centers, Journal of the Chemical Society-Perkin Transactions 1 1988, 8, 2197-2201; Dembinski et al, An Expedient Synthesis of Symmetrical Terra- Alkyl Mono- thiopyrophosphates, Tetrahedron Letters 1994, 35 (34), 6331-6334; Skowronska et al, Novel Synthesis of Symmetrical Tetra-Alkyl Monothiophosphates, Tetrahedron Letters 1987, 28 (36), 4209-4210; and Chojnowski et al, Methods of Synthesis of 0,0-Bis TrimethylSilyl

Phosphorothiolates. Synthesis- Stuttgart 1977, 10, 683-686, all hereby incorporated by reference in there entirety .Using the above approaches, the phytosphingosine-tenofovir monophosphate and sphingosine-AZT diphosphate conjugates shown in Figure 8 have been prepared.

Example 2.

Activity of 2-Fluoronucleosides

The sphingo lipid containing nucleosides tested for RdRp inhibition are 2'-

Fluoronucleosides. Ribonucleoside analogs when activated to their corresponding triphosphate inhibit RNAdependent RNA viral replication by acting as competitive substrate inhibitors of the virally encoded RdRp. Compounds in this therapeutic class are useful in the treatment of viruses found in but not limited to the arenaviridae, bunyaviridae, flaviviridae, orthomyxoviridae, paramyxoviridae, and togaviridae viral families. Certain compounds disclosed herein are contemplated to have advantages such as a high genetic barrier for antiviral resistance; broad spectrum activity within viral families; and high oral bioavailability with targeted delivery to sites of infection.

The nucleoside analogs were designed with a 2 '-alpha-fluorine substituent to mimic natural ribonucleosides. The C-F bond length (1.35 A) is similar to the C-0 bond length (1.43 A) and fluorine is a hydrogen-bond acceptor making the fluorine substituent an isopolar and isosteric replacement of a hydroxyl group. Unlike ribonucleoside analogs currently in clinical trials for treating HCV infections, in certain embodiments, the 2',3'-dideoxy-2'-fluoronucleoside analogs covered by this disclosure lack a 3 '-hydroxyl group and are thus obligate chain terminators of viral replication. Once the nucleosides are converted to their triphosphates, they act as competitive substrate inhibitors of the virally encoded RdRp. After incorporation of the chain terminator into nascent RNA, viral replication ceases. One advantage to obligate chain terminators is that they are not mutagenic to the host when treating chronic diseases.

Several 2',3'-dideoxy-2'-fluoro nucleoside analogs were screened in a HCV replicon assay in Huh-7 cells at 5 μΜ. No antiviral activity was observed for any of the analogs.

However, the triphosphate of ECBN-01485 was prepared and screened against the isolated HCV NS5B RdRp in three separate experiments. ECBN-01485-5'-triphosphate showed inhibition of the HCV NS5B RdRp with IC50 = 0.66 μΜ, 0.39 μΜ, and 0.04 μΜ.

The table below shows activity of select analog triphosphates against the HCV NS5B polymerase (confirmatory assays).

NS5B RNA-dependent RNA polymerase reaction conditions

Compounds were assayed for inhibition of NS5B-521 from HCV GT-lb Con-1. Reactions included purified recombinant enzyme, 1 u/μί negative-strand HCV IRES RNA template, and ΙμΜ NTP substrates including either [³²P]-CTP or [³²P]-UTP. Assay plates ere incubated at 27 °C for 1 hour before quench. [³²P] incorporation into macromolecular product was assessed by filter binding. The table below shows activity of select analog triphosphates against the HCV NS5B polymerase.

HCV Replicon GT-lb Luciferase Assay Results

uM)

CEM

Infectious Dengue Type 2 Assay Results

The tables below shows data for activity of analogs and triphosphates against the parainfluenza and for influenza A/PR/8/34 polymerase.

enza_5F<f

The table below shows broad spectrum antiviral activity

The tables below show broad spectrum antiviral activity

The tables below show antiviral activity for cytidine analogs

Influenza A

H1N1 H3N2 H5N1 (low path) Influenza B RSV SARS

Structure

EC50 EC50 EC50 EC50 EC50 EC50

70 uM

ENUC-01841 > 100 uM > 100 uM > 100 uM > 100 uM > 100 uM

CC50 > 89 uM

ENUC-01853 > 100 uM > 100 uM > 100 uM > 100 uM > 100 uM > 100 uM

ENUC-01854 > 100 uM > 100 uM > 100 uM > 100 uM > 100 uM > 100 uM

ENUC-01855 > 100 uM > 100 uM > 100 uM > 100 uM > 100 uM > 100 uM

ENUC-01856 · > 100 uM > 100 uM > 100 uM > 100 uM > 100 uM > 100 uM

The tables below shows HIV inhibition data:

HIV-1 (92HT599)

HIV-l_Lai PBMCs

PBMCs

ID Structure EC₅₀ (μΜ) <Χ₅₀(μΜ) Ε0₅₀ (μΜ) <Χ₅₀(μΜ)

ECBN-01052 20% inh. @ lOuM

ECBN-01798 10% inh. @ lOuM

ECBN-01799 - o 0%inh. @10uM

ECBN-01800 12% inh. @ lOuM

ENSC-01792 ¾ ° ² 0.362 >10

ENSC-01801 0.0231 >10

HIV-l (92HT599)

HIV-l_Lai PBMCs

PBMCs

ID Structure EC₅₀ (μΜ) ^₅₀(μΜ) EC₅₀ (μΜ) <Χ₅₀(μΜ)

^N— ft \

X-

ENSC-01979 0.00119 >10

ENSC-01978 0.00719 >10

ENSC-01977 0.158 >10

ENSC-01976 0.132 >10

HIV-1 (92HT599)

HIV-l_Lai PBMCs

PBMCs

ID Structure EC₅₀ |μΜ) CC₅₀ (μM) EC₅₀ |μΜ) CC₅₀ |μΜ)

ENSC-01975 0.0668 > 10

ENSC-01974 0.00467 7.26

ENSC-01973 0.00241 6.43

ENSC-01972 0.00134 5.65

The tables below shows HBV inhibition data:

Example 3.

Sphingolipid Conjugates

The HIV activity of the compounds shown in Figure 8 were assessed. Tenofovir was inactive because of its inability to permeate into cells (EC₅₀ = >1 μΜ; CC50 = n.d.). Conversely, Tenofovir Disoproxil Fumarate (TDF) (EC₅₀ = 17 nM; CC₅₀ = >1, SI = 58.8), CMX-157 (EC₅₀ = 53 nM; CC₅₀ = >10, SI >189) and the Phytosphingosine-Tenofovir conjugate (EC₅₀ = 1 nM; CC₅₀ = >10, SI = >10,000) all exhibited low nanomolar activity. It is notable here that the sphingolipid conjugate is substantially more potent and has a much better Selectivity Index than the other compounds.

Phytosphingosine-Tenofovir conjugate (Figure 9) was found to exhibit good plasma stability (79.1% present after 120 minutes at 37°C at pH 7.4). The Phytosphingosine-Tenofovir conjugate does not undergo oxidative catabolism (i.e., oxidation of the terminal methyl group, followed by sequential loss of two carbon fragments leading to inactive, water soluble derivatives) as does CMX-157. Unlike phospholipids, no oxidative catabolism of the terminal methyl group of sphingoid bases has been observed with any sphingolipid derivatives.

Several AZTDP prodrugswere prepared, which are shown in Figure 9. Two of them, 16a and 16b, are alkylated glycerol and deoxyglycerol derivatives, the third, 17, is a "McGuigan" version of a diphosphate prodrug. A Sphingosine- AZTDP conjugate, 18 was also prepared. In the HIV-1 assay 17 provided EC₅₀ = 1 nM; CC₅₀ = >10, SI = > 10,000 when compared to AZT itself EC50 = 5 nM; CC50 = >1, SI = 167. Preliminary assessment of 18 indicates that it has good stability in human plasma and that it rapidly permeates the cell membrane of hepatocytes.

Example 4. Synthesis of Sphingolipids and Derivatives

The preparation of sphingolipids is provided for in PCT/US 12/57448 hereby

incorporated by reference in its entirety.

Example 5.

General Synthesis of 2',3'-Dideoxy-2'-P-Substituted-2'-a-Fluoronucleosides

L-Glutamic Acid

Reagents and conditions: a) NaN0₂, HCl_(aq); b) BH₃ SMe₂; c) TBDPSC1, DMAP, pyridine; d) i. LiHMDS, ii. RX (X = CI, Br, I, etc.); e) i. TBSOTf, Et₃N, ii. NFSi; f) DIBAL; g) Ac₂0, DMAP; h) HMPT, CC1₄

Base Coupling and Deprotection

8 9 10

Reagents and conditions: a) silylated base, TMSOTf, DCE; b) TBAF, THF; c) nucleobase, TDA-

Ι, ΚΟΗ, MeCN

References:

1) Tetrahedron: Asymmetry (1993), 4 (1), 121-131.

2) JOC (1998), 63, 2161-2167.

3) Tetrahedron (1987), 43 (10), 2355-2368. Synthesis of 2',3'-Dideoxy-2'-a-Fluoronucleosides

L-Glutamic Acid

14

Reagents and conditions: a) NaN0₂, HCl_(aq); b) BH₃ SMe₂; c) TBDPSC1, DMAP, pyridine; d) i. LiHMDS, ii. NFSi; e) DIBAL; f) Ac₂0, DMAP; g) HMPT, CC1₄

Base Coupling and Deprotection

13 15 16

14 15 16

Reagents and conditions: a) silylated base, TMSOTf, DCE; b) TBAF, THF; c) nucleobase, TDA- Ι, ΚΟΗ, MeCN References:

1) Tetrahedron: Asymmetry (1993), 4 (1), 121-131.

2) JOC (1998), 63, 2161-2167.

3) Tetrahedron (1987), 43 (10), 2355-2368.

Exemplary syntheses:

l-Chloro-2,3-dideoxy-2-fluoro-5-fert-butyldimethylsilylribose 17

A mixture of 2,3-dideoxy-2-fluoro-5-fert-butyldimethylsilylribose (840 mg, 2.24 mmol) and carbon tetrachloride (1.55g, 10.09 mmol) in anhydrous toluene (15 mL) at -50°C was treated dropwise with a solution of hexamethylphosphorous triamide (440 mg, 2.69 mmol) in toluene (15 mL) over a 35 min period. The mixture was stirred with gradual warming to 0°C and maintained at this temperature for 3h. After cooling to -20°C, the mixture was diluted with cold toluene (50 mL) and quenched by dropwise addition of cold brine (5mL at -10°C). After 10 min the organic layer was separated and washed again with cold brine (10 ML). After drying over sodium sulfate, the organic phase was filtered and concentrated by rotary evaporator (bath set at 20°C) to give crude 17 (900 mg) in a 9: 1 α:β ratio. Crude material was used in next step without further purification.

1H NMR (400 MHz, Chloroform-;/) δ 7.65 - 7.58 (m, 5H), 7.46 - 7.32 (m, 7H), 6.28 (d, J = 4.1 Hz, 1H), 5.36 (td, J = 8.4, 4.2 Hz, 1H), 5.22 (td, J = 8.3, 4.1 Hz, 1H), 4.55 (ddt, J = 7.6, 5.2, 2.8 Hz, 1H), 3.78 (ddd, J = 11.5, 2.7, 1.8 Hz, 1H), 3.60 (dd, J = 11.5, 2.8 Hz, 1H), 2.44 - 2.35 (m, 2H).

2 ' ,3 ' -Dideoxy-2 ' -fluoro-5 ' -O-tert-butyldimethylsilyl-4-chloro-7-deazaguanosine 18

A mixture of l-Chloro-2,3-dideoxy-2-fluoro-5-tert-butyldimethylsilylribose 17(880 mg, 2.24 mmol), 7-deazaguanidine (755 mg, 4.48 mmol), and powdered potassium hydroxide (377 mg, 6.72 mmol) in anhydrous acetonitrile (15 mL) was treated with catalytic amount of tris(2-(2- methoxyethoxy)ethyl) amine (72 mg, 0.224 mmol). After 12 h at rt, the mixture was concentrated and the resulting residue partitioned between ethyl acetate (100 mL) and water (50 mL). The aqueous phase was back extracted with ethyl acetate (2 x 25 mL) and the combined organic phases dried over sodium sulfate, filtered and concentrated to dryness. The crude material was purified by flash column chromatography (25 mm x 170 mm) over silica gel using 9: 1 hexanes: ethyl acetate to give 18 (540 mg, 46%) as a white foam.

1H NMR (400 MHz, Chloroform-;/) δ 7.70 - 7.60 (m, 3H), 7.48 - 7.30 (m, 8H), 6.33 (d, J = 18.1 Hz, 1H), 6.25 (d, J = 3.8 Hz, 1H), 5.29 (dd, J = 51.9, 4.3 Hz, 1H), 4.96 (s, 2H), 4.48 (ddt, J = 11.1, 5.5, 3.1 Hz, 1H), 4.07 (dd, J = 11.8 Hz, 3.0 Hz, 1H), 3.76 (dd, J = 11.8, 3.3 Hz, 1H), 2.57 - 2.37 (m, 1H), 2.20 (ddd, J= 19.8, 14.3, 5.2 Hz, 1H), 1.07 (s, 9H).

2 ' ,3 ' -Dideoxy-2 ' -fluoro-4-chloro-7-deazaguanosine 19

A solution of 2',3'-Dideoxy-2'-fluoro-5'-O-tert-butyldimethylsilyl-6-chloro-7- deazaguanosine 18(970 mg, 1.85 mmol) in THF (20 mL) at 4°C was treated dropwise with a solution of tetrabutylammonium fluoride (1.0 M in THF, 2.4 mL, 2.40 mmol) over a 20 min period. The mixture continued stirring at 0°C for lh after which time tic (1 : 1 hexanes: ethyl acetate) indicated complete reaction. The mixture was diluted with ethyl acetate (125 mL) and washed with saturated ammonium chloride solution (2 x 50 mL). The organic layer was dried over sodium sulfate, filtered and concentrated. The crude material was purified by flash column chromatography (30 mm x 110 mm) over silica gel using a solvent gradient from 50 to 95% ethyl acetate in hexanes to give 19 (420 mg, 79%>) as a white solid.

1H NMR (400 MHz, Chloroform-^) δ 6.93 (d, J = 3.8 Hz, 1H), 6.41 (d, J = 3.7 Hz, 1H), 5.95 (dd, J = 18.5, 2.8 Hz, 1H), 5.50 (ddt, J = 53.8, 6.4, 3.3 Hz, 1H), 5.10 (s, 3H), 4.54 (tt, J = 7.3, 2.1 Hz, 1H), 4.05 (dt, J = 12.5, 1.7 Hz, 1H), 3.64 (dd, J = 12.5, 2.2 Hz, 1H), 2.76 (dddd, J = 28.0, 14.1, 7.8, 6.3 Hz, 1H), 2.32 (dddd, J= 23.2, 14.0, 7.0, 3.6 Hz, 1H).

2 ' ,3 ' -Dideoxy-2 ' -fluoro-7-deazaguanosine 20

A suspension of 2 ',3' -Dideoxy-2 '-fluoro-4-chloro-7-deazaguanosine 19 (310 mg, 1.08 mmol) in 2N NaOH (15 mL) was heated to 115°C for 4h after which time tic (ethyl acetate) indicated no remaining starting material. The reaction mixture was neutralized to pH 7 with acetic acid and then concentrated. The crude material was purified by reverse-phase chromatography (19 mm x 130 mm) over Amberchrom CG-161M resin using a solvent gradient from 0 to 50% methanol in water to yield 20 (250 mg, 87%) as a white solid.

1H NMR (400 MHz, DMSO-d₆) δ 10.40 (s, 1H), 6.94 (d, J = 3.6 Hz, 1H), 6.31 (s, 2H), 6.27 (d, J = 3.6 Hz, 1H), 6.14 (d, J = 19.7 Hz, 1H), 5.31 (dd, J = 52.5, 4.5 Hz, 1H), 5.03 (t, J = 5.4 Hz, 1H), 4.27 (dq, J = 9.4, 4.6 Hz, 1H), 3.69 (ddd, J = 12.0, 5.7, 3.4 Hz, 1H), 3.55 (dt, J = 12.0, 4.7 Hz, 1H), 2.36 - 2.11 (m, 2H).

¹³C NMR (101 MHz, Chloroform-^) δ 159.05 , 153.19 , 150.58 , 117.40 , 102.83 , 100.61 , 97.86 (d, J= 177.1 Hz), 88.23 (d, J= 35.4 Hz), 80.74 , 62.35 , 33.02 (d, J= 20.4 Hz).

¹⁹F NMR (376 MHz, Chloroform-^ ) δ 44.17 (ddt, J= 52.3, 41.7, 20.7 Hz).

HRMS C₁₁H₁₄FN₄O₃ [M+H⁺]; calculated: 269.1.0445, found: 269.10434.

2\3'-Dideoxy-2'-fluoro-5'-O-tert-butyldimethylsilyl-4-chloro-7-fluoro-7-deaza-purine 21

A mixture of l-Chloro-2,3-dideoxy-2-fluoro-5-tert-butyldimethylsilylribose 17(1.06 g, 2.7 mmol), 7-fluoro-7-deaza-adenine (463 mg, 2.7 mmol), and powdered potassium hydroxide (454 mg, 8.1 mmol) in anhydrous acetonitrile (18 mL) was treated with catalytic amount of tris(2-(2-methoxyethoxy)ethyl) amine (87 mg, 0.2704 mmol). After 12 h at rt, the mixture was concentrated and the resulting residue partitioned between ethyl acetate (150 mL) and water (50 mL). The aqueous phase was back extracted with ethyl acetate (2 x 30 mL) and the combined organic phases dried over sodium sulfate, filtered and concentrated to dryness. The crude material was purified by flash column chromatography (25 mm x 170 mm) over silica gel using 9: 1 hexanes: ethyl acetate to give 21 (767 mg, 54%) as a white foam.

1H NMR (400 MHz, Chloroform-;/) δ 8.61 (s, 1H), 7.76 - 7.61 (m, 6H), 7.50 (d, J = 2.5 Hz, 1H), 7.46 - 7.32 (m, 8H), 6.54 (dd, J = 16.7, 1.3 Hz, 1H), 5.33 (dd, J = 51.7, 4.1 Hz, 1H), 4.51 (ddt, J = 10.7, 5.3, 2.7 Hz, 1H), 4.13 (dd, J = 11.9, 2.6 Hz, 1H), 3.74 (dd, J = 11.9, 3.0 Hz, 1H), 2.48 (dddd, J= 41.6, 14.8, 10.8, 4.3 Hz, 1H), 2.20 (ddd, J= 19.6, 14.4, 5.2 Hz, 1H), 1.09 (s, 9H).

2 ' ,3 ' -Dideoxy-2 ' -fluoro-5 ' -O-tert-butyldimethylsilyl-7-fluoro-7-deaza-adenosine 22

A solution of 2 ' ,3 ' -Dideoxy-2 ' -fluoro-5 ' -O-fert-butyldimethylsilyl-4-chloro-7-fluoro-7- deaza-purine 21(310 mg, 0.587 mmol) dissolve in 2M ammonia in dioxane was heated to 75°C in a thick- wall glass pressure vessel. After 12 h, the mixture was cooled to 0°C and then concentrated to dryness. The crude material was purified by flash chromatography (19 mm x 170 mm) over silica gel using 1 : 1 hexanes: ethyl acetate to give 22(255 mg, 85%) as a white solid.

1H NMR (400 MHz, Chloroform-^) δ 8.25 (s, 1H), 7.71 - 7.58 (m,4H), 7.45 - 7.32 (m, 6H), 7.14 (d, J = 1.9 Hz, 1H), 6.48 (dd, J = 17.8, 1.5 Hz, 1H), 5.52 (s, 2H), 5.30 (dd, J = 51.9, 4.4 Hz, 1H), 4.47 (ddt, J = 11.0, 5.6, 3.1 Hz, 1H), 4.09 (dd, J = 11.8, 2.9 Hz, 1H), 3.74 (dd, J = 11.8, 3.2 Hz, 1H), 2.46 (dddd, J = 41.4, 14.8, 10.8, 4.4 Hz, 1H), 2.18 (ddd, J = 19.7, 14.3, 5.1 Hz, 1H), 1.08 (s, 8H).

2 ' ,3 ' -Dideoxy-2 ' -fluoro-7-fluoro-7-deaza-adenosine 23

A solution of 2',3'-Dideoxy-2'-fluoro-5'-0-tert-butyldimethylsilyl-7-fluoro-7-deaza- adenosine 22(97 mg, 0.19 mmol) in THF (3 mL) at 4°C was treated dropwise with a solution of tetrabutylammonium fluoride (1.0 M in THF, 0.29 mL, 0.29 mmol) over a 5 min period. The mixture continued stirring at 0°C for 4h after which time tic (5% methanol in methylene chloride) indicated complete reaction. The mixture was diluted with ethyl acetate (50 mL) and washed with saturated ammonium chloride solution (2 x 10 mL). The organic layer was dried over sodium sulfate, filtered and concentrated. The crude material was purified by flash column chromatography (19 mm x 170 mm) over silica gel using 5% methanol in methylene chloride to give 23 (23 mg, 45%) as a white solid.

1H NMR (400 MHz, Methanol-^) δ 8.07 (s, 1H), 7.25 (d, J = 2.1 Hz, 1H), 6.39 (dt, J = 18.5, 1.3 Hz, 1H), 5.29 (ddt, J = 52.3, 4.9, 1.3 Hz, 1H), 4.57 (s, 1H), 4.52 - 4.29 (m, 1H), 3.88 (dd, J = 12.4, 2.9 Hz, 1H), 3.66 (dd, J = 12.3, 3.8 Hz, 1H), 2.41 (dddd, J = 39.0, 14.8, 10.2, 4.9 Hz, 1H), 2.24 (dddd, J= 20.0, 14.4, 5.7, 1.5 Hz, 1H).

HRMS C11H13F2N4O3 [M+H⁺]; calculated: 271.10011, found: 271.10004.

Example 6.

Synthesis of 2',3'-Dideoxy-2'-P-Methyl-2'-a-Fluoronucleosides

L-Glutamic Acid 2

28

Reagents and conditions: a) NaN0₂, HCl_(aq); b) BH₃ SMe₂; c) TBDPSC1, DMAP, pyridine; d) i. LiHMDS, ii. Mel; e) i. TBSOTf, Et₃N, ii. NFSi; f) DIBAL; g) Ac₂0, DMAP; h) HMPT, CC1₄

Base coupling and Deprotection

27 29 30

28 29 30

Reagents and conditions: a) silylated base, TMSOTf, DCE; b) TBAF, THF; c) nucleobase, TDA-1, KOH, MeCN References:

1) Tetrahedron: Asymmetry (1993), 4 (1), 121-131.

2) JOC (1998), 63, 2161-2167.

3) Tetrahedron (1987), 43 (10), 2355-2368.

Exemplary syntheses:

(3R,5S)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-3-methyldihydrofuran-2(3H)-one (31)

Tetrahedron: Asymmetry (1993), 4 (1), 121-131.

To a solution of diisopropylamine (2.01 ml, 14.1 mmol) in dry THF (20 ml) at 0°C, under an inert atmosphere, was added n-butyllithium (8.83 ml of a 1.6 M solution in hexane, 14.13 mmol). After 30 minutes of stirring, the solution was cooled to -78°C, and a solution of (4S)-4-tert-butyldiphenylsiloxymethyl-4-butanolide (5.01 g, 14.13 mmol) in dry THF (5 ml) was added drop-wise over a period of 5 minutes. After stirring for a further 30 minutes at -78°C, iodomethane (1.31 ml, 21.0 mmol) was added, and the reaction vessel removed from the ice bath. After 30 minutes at ambient temperature, deionised water (40 ml) was added to the solution, and the organics extracted with ether (3 x 15 ml). The combined organic layer was washed with 1M HCl (3 x 20 ml) and once more with brine, before being dried over Mg₂S0₄. The crude product was purified by silica chromatography (product Rf = 0.26 in 4: 1 hexane:ethyl acetate), eluting with 85: 15 hexane:ethyl acetate to afford the final product as a white, crystalline solid. Stereochemistry was established based on comparison of NMR data with reported data.

1H NMR (400 MHz, CDC1₃): δ 7.67-7.65 (m, 4H), 7.48-7.39 (m, 6H), 4.58-4.53 (m, 1H),

3.86 (dd, J= 3.2, 10.8 Hz, 1H), 3.68 (dd, J= 3.2, 11.2 Hz, 1H), 2.90-2.81 (m, 1H), 2.45 (ddd, J = 3.2, 9.2, 12.8 Hz, 1H), 1.98 (dt, J= 8.8, 12.4 Hz, 1H), 1.30 (d, J= 7.2 Hz, 3H), 1.06 (s, 9H).

(3R,5S)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-3-fluoro-3-methyldihydrofuran-2(3H)-one (32) Compound 31 (0.1000 g, 0.27 mmol) was placed in a dry flask under argon atmosphere and was dissvolved in dry DCM (5 mL). Next, TBSOTf (0.075 mL, 0.33 mmol) was added dropwise to the stirring DCM solution of lactone at room temperature followed by the dropwise addition of neat triethylamine (0.057 mL, 0.41 mmol) also at room temp. The reaction mixture was allowed to stir at room temp under nitrogen for 2 hours with monitoring by TLC. Next, NFSi (0.1280 g, 0.41 mmol) was dissolved in 2 mL of dry DCM and was added dropwise to the silyl enol ether at room temp under nitrogen. The reaction mixture turned dark red. The reaction mixture was allowed to stir over night. The reaction mixture was quenched with sat. NH4C1 and was diluted with ether. The organic layer was washed with brine, dried over MgS04, filtered, and concentrated. The product was purified on silica eluting with 8: 1 hexanes/ethyl acetate.

1H NMR (400 MHz, CDC1₃): δ 7.67-7.64(m, 4H), 7.48-7.39 (m, 6H), 4.75-4.70 (m, 1H), 3.96 (dd, J = 3.6, 12 Hz, 1H), 3.71 (dd, J = 3.6, 11.6 Hz, 1H), 2.53 (ddd, J = 6.4, 14.6, 22.8 Hz, 1H), 2.37 (ddd, J= 8.8, 14.6, 35.2 Hz, 1H), 1.66 (d, J= 22.8 Hz, 3H), 1.05 (s, 9H).

The X-ray Structure of compound 32 is shown in Figure 10.

(2R,3R,5S)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-3-fluoro-3-methyltetrahydrofuran-2-ol (33) Compound 33 was prepared following the procedure outlined by JOC (1998), 63, 2161-2167.

H NMR (400 MHz, CDC1₃): δ 7.70-7.67 (m, 4H), 7.47-7.39 (m, 6H), 5.10 (t, J lH), 4.50 (m, 1H), 3.87 (dd, J= 2.4, 11.2 Hz, 1H), 3.46 (dd, J

2H), 1.57 (d, J = 21.6 Hz, 3H), 1.09 (s, 9H).

(2S,3R,5S)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-3-fluoro-3-methyltetrahydrofuran-2-yl acetate (34) Compound 34 was prepared following the procedure outlined by JOC (1998), 63, 2161-2167.

1H NMR (400 MHz, CDC1₃): δ 7.69-7.66 (m, 4H), 7.46-7.37 (m, 6H), 6.13 (d, J = 10.4 Hz, 1H), 4.53-4.47 (m, 1H), 3.79 (dd, J = 4.4, 10.8 Hz, 1H), 3.72 (dd, J = 4.4, 11.6 Hz, 1H), 2.27-2.02 (m, 2H), 1.92 (s, 3H), 1.50 (d, J = 21.6 Hz, 3H), 1.07 (s, 9H). General Nucleobase Coupling Conditions

The desired nucleobase (5 equivalents) was transferred to a dry flask under an argon atmosphere and suspended in HMDS (2 mL/mmol nucleobase). Catalytic ammonium sulfate (1- 3 mgs) was added to the reaction vessel, and the suspension was allowed to reflux for 1-3 hours. During the course of reaction, the white suspension turned clear. The reaction vessel was allowed to cool to room temperature, and the excess HMDS was removed under reduced pressure. The resulting residue was dissolved in dry DCE (5 mL/mmol compound 34) followed by the addition of compound 34 at room temperature. Finally, neat TMSOTf (5.5 equivalents) was added to the stirring solution. The reaction was quenched with saturated sodium bicarbonate. The organic layer was collected, dried over MgS0₄, filtered, and concentrated under reduced pressure. The desired protected nucleoside was purified on silica gel eluting with 9: 1 DCM/MeOH.

General Deprotection Conditions

A solution of protected nucleosidedissolved in dry THF (10 ml/mmol of protected nucleoside) was treated with tetrabutylammonium fluoride (TBAF, 1 M solution in THF, 1.1 equivalents), and let to stir at room temperature for 3 hours. The crude mixture was concentrated in vacuo, and the resulting residue was purified on silica gel (0-10% methanol in dichloromethane) to give the desired nucleoside.

9-((2R,3R,5S)-5-(((tert-butyldipheny

9H-purin-6-amine (35)

Prepare following the general nucleobase coupling conditions.

1H NMR (400 MHz, CDC1₃): 58.46 (s, 1H), 8.02 (s, 1H), 7.67-7.64 (m, 4H), 7.48-7.39

(m, 6H), 6.85 (br, 2H), 5.83 (d, J = 18.8 Hz, 1H), 4.73-4.69 (m, 1H), 4.13 (dd, J = 2.8, 12 Hz, 1H), 3.75 (dd, J= 2.4, 11.6 Hz, 1H), 2.62-2.37 (m, 2H), 1.47 (d, J= 20.8 Hz, 3H), 1.12 (s, 9H).

((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-fluoro-4-methyltetrahydrofuran-2-yl)methanol (36)

Prepared following the general deprotection conditions.

1H NMR (400 MHz, CD₃OD): 58.49 (s, 1H), 8.27 (s, 1H), 6.20 (d, J = 19.6 Hz, 1H), 4.76-4.70 (m, 1H), 3.88 (dd, J = 2.8, 12 Hz, 1H), 3.66 (dd, J = 4.4, 12.4 Hz, 1H), 2.60-2.28 (m, 2H), 1.44 (d, J= 21.2 Hz, 3H).

l-((2R,3R,5S)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-3-fluoro-3-methyltetrahydrofuran-2- yl)pyrimidine-2,4(lH,3H)-dione (37) Prepare following the general nucleobase coupling conditions.

1H NMR (400 MHz, CDC1₃): 58.20 (br, 1H), 7.68-7.64 (m, 4H), 7.48-7.39 (m, 6H), 6.12 (d, J= 19.6 Hz, 1H), 5.73 (d, J= 8.4 Hz, 1H), 4.57-4.51 (m, 1H), 3.89 (dd, J= 3.2, 11.2 Hz, 1H), 3.67 (dd, J= 4.0, 11.6 Hz, 1H), 2.39-2.23 (m, 2H), 1.56 (d, J= 21.6 Hz, 3H), 1.08 (s, 9H).

l-((2R,3R,5S)-3-fluoro-5-(hydroxymethyl)-3-methyltetrahydroi iran-2-yl)pyrimidine- 2,4(lH,3H)-dione (38)

Prepared following the general deprotection conditions.

1H NMR (400 MHz, CD₃OD): 57.62 (dd,J = 3.2, 8.4 Hz, 1H), 6.13 (d, J = 19.6 Hz, 1H), 5.69 (d, J= 8.0 Hz, 1H), 4.60-4.54 (m, 1H), 3.78 (dd, J= 2.8, 12 Hz, 1H), 3.55 (dd, J= 4.4, 12.0 Hz, 1H), 2.39-2.16 (m, 2H), 1.53 (d, J=

4-amino-l-((2R,3R,5S)-3-fluoro-5-(hydroxymethyl)-3-methyltetrahydrofuran-2-yl)pyrimidin- 2(lH)-one (39)

Prepared following the general nucleobase coupling and deprotection conditions.

1H NMR (400 MHz, CD₃OD): 57.61 (dd, J = 3.2, 7.6 Hz, 1H), 6.22 (d, J = 19.6 Hz, 1H), 5.88 (d, J = 7.6 Hz, 1H), 4.57-4.51 (m, 1H), 3.76 (dd, J = 3.2, 12.0 Hz, 1H), 3.56 (dd, J = 4.4, 12.0 Hz, 1H), 2.38-2.14 (m, 2H), 1.52 (d, J= 21.6 Hz, 3H).

Example 7. Alternative Route for the Synthesis of 2',3'-Dideoxy-2'-P-Substituted-2'-a- Fluoronucleosides

Reagents and conditions: a) AcCl, pyridine, DCM; b) TCDI, pyridine, DCM; c) Bu₃SnH, AIBN; d) Ac₂0, AcOH, H₂S0₄; e) i. silylated base, TMSOTf, ii. K₂C0₃, MeOH; f) BzCl; g) DMP; h) RLi or RMgBr; i) DAST; j) NH₃, MeOH

References:

1) Tetrahedron: Asymmetry (1993), 4 (1), 121-131.

2) JOC (1998), 63, 2161-2167.

3) Tetrahedron (1987), 43 (10), 2355-2368.

4) WO2010057103

5) JOC (1985), 50, 3457-3462.

6) Nucleosides, Nucleotides and Nucleic Acids (2012), 31, 286-292. Example 8.

Alternative Route to 2',3'-Dideoxy-2'-a-Fluoronucleosides

Reagents and conditions: a) AcCl, pyridine, DCM; b) TCDI, pyridine, DCM; c) Bu₃SnH, AIBN; d) Ac₂0, AcOH, H₂S0₄; e) i. silylated base, TMSOTf, ii. K₂C0₃, MeOH; f) BzCl; g) DMP; h) NaBH₄; i) DAST; j) NH₃, MeOH

References:

1) Tetrahedron: Asymmetry (1993), 4 (1), 121-131.

2) JOC (1998), 63, 2161-2167.

3) Tetrahedron (1987), 43 (10), 2355-2368.

4) WO2010057103

5) JOC (1985), 50, 3457-3462.

6) Nucleosides, Nucleotides and Nucleic Acids (2012), 31, 286-292. Example 9.

2 ' ,3 '-Dideoxy-2 '-P-Ethynyl-2 '-α-Fluoronucleosides

Reagents and conditions: a) AcCl, pyridine, DCM; b) TCDI, pyridine, DCM; c) Bu₃SnH, AIBN; d) Ac₂0, AcOH, H₂S0₄; e) i. silylated base, TMSOTf, ii. K₂C0₃, MeOH; f) BzCl; g) DMP; h) i.trimethylsilylacetylene, BuLi, ii. NH₄F, MeOH; i) DAST; j) NH₃, MeOH

References:

1) Tetrahedron: Asymmetry (1993), 4 (1), 121-131.

2) JOC (1998), 63, 2161-2167.

3) Tetrahedron (1987), 43 (10), 2355-2368.

4) WO2010057103

5) JOC (1985), 50, 3457-3462.

6) Nucleosides, Nucleotides and Nucleic Acids (2012), 31, 286-292.

Example 10.

2',3'-Dideoxy-2'-P-Fluoromethyl-2'-a-Fluoronucleosides

Reagents and conditions: a) AcCl, pyridine, DCM; b) TCDI, pyridine, DCM; c) Bu₃SnH, AIBN; d) Ac₂0, AcOH, H₂S0₄; e) i. silylated base, TMSOTf, ii. K₂C0₃, MeOH; f) BzCl; g) DMP; h) Me₃S(0)I, NaH; i) KF, 18-crown-6; j) DAST; k) NH₃, MeOH

References:

1) Tetrahedron: Asymmetry (1993), 4 (1), 121-131.

2) JOC (1998), 63, 2161-2167.

3) Tetrahedron (1987), 43 (10), 2355-2368.

4) WO2010057103

5) JOC (1985), 50, 3457-3462.

6) Nucleosides, Nucleotides and Nucleic Acids (2012), 31, 286-292.

7) Org. Lett. (2003), 5(6), 807-810.

8) Org. Lett. (2001), 3(7), 1025-1028.

9) Org. Lett. (2007), 9(16), 3009-3012.

10) JOC (2005), 70, 7902-7910. Example 11.

2',3'-Dideoxy-2'-P-Difluoromethyl OR Trifluoromethyl-2'-a-Fluoronucleosides

Reagents and conditions: a) AcCl, pyridine, DCM; b) TCDI, pyridine, DCM; c) Bu₃SnH, AIBN; d) Ac₂0, AcOH, H₂S0₄; e) i. silylated base, TMSOTf, ii. K₂C0₃, MeOH; f) BzCl; g) DMP; h) CHF₂: i. PhS0₂CF₂H, LiHMDS, ii. Sml₂ or CF₃: TMSCF₃, TBAF; i) DAST; j) NH₃, MeOH

References:

1) Tetrahedron: Asymmetry (1993), 4 (1), 121-131.

2) JOC (1998), 63, 2161-2167.

3) Tetrahedron (1987), 43 (10), 2355-2368.

4) WO2010057103

5) JOC (1985), 50, 3457-3462.

6) Nucleosides, Nucleotides and Nucleic Acids (2012), 31, 286-292.

7) Org. Lett. (2003), 5(6), 807-810.

8) Org. Lett. (2001), 3(7), 1025-1028.

9) Org. Lett. (2007), 9(16), 3009-3012.

10) JOC (2005), 70, 7902-7910. Example 12. Alternative Route to 2',3'-Dideoxy-2'-P-Substituted-2'-a-Fluoronucleosides

Reagents and conditions: a) AcCl, pyridine, DCM; b) TCDI, pyridine, DCM; c) Bu₃SnH, AIBN; d) i. H₂S0₄, MeOH ii. K₂C0₃, MeOH; e) BzCl, pyridine; f) DMP; g) RLi or RMgBr; h) DAST; i) Ac₂0, AcOH, H₂S0₄; j) silylated base, TMSOTf; k) NH₃, MeOH

References:

1) Tetrahedron: Asymmetry (1993), 4 (1), 121-131.

2) JOC (1998), 63, 2161-2167.

3) Tetrahedron (1987), 43 (10), 2355-2368.

4) WO2010057103

5) JOC (1985), 50, 3457-3462.

6) Nucleosides, Nucleotides and Nucleic Acids (2012), 31, 286-292.

7) Org. Lett. (2003), 5(6), 807-810.

8) Org. Lett. (2001), 3(7), 1025-1028.

9) Org. Lett. (2007), 9(16), 3009-3012.

10) JOC (2005), 70, 7902-7910. Example 13.

Synthesis of 2'-Deoxy-2'-a-Fluororibonucleosides

7⁵

Reagents and conditions: a) TBDPSCl, imidazole; b) acetone, CuS0₄, H₂S0₄; c) KOH, BnCl; d)

CSA, MeOH; e) i. Tf₂0, ii. TBAF; f) 90% TFA_(aq); g) Ac₂0, DMAP; h) HMPT, CC1₄ Base Coupling and Deprotection

74 76 77

75 76 77 Reagents and conditions: a) silylated base, TMSOTf, DCE; b) BCI₃, DCM; c) nucleobase, TDA- Ι, ΚΟΗ, MeCN

References:

1) Tetrahedron: Asymmetry (1993), 4 (1), 121-131.

2) JOC (1998), 63, 2161-2167.

3) Tetrahedron (1987), 43 (10), 2355-2368.

4) Acta Chemica Scandinavica (1986), B 40, 15-20.

5) JACS (2005), 127, 10879-10884.

6) Antimicrobial Agents and Chemotherapy (2004), 48(10), 3944-3953.

7) JACS (2008), 130, 13639-13648.

Exemplary Syntheses:

A stirred suspension of fluorocytidine (1.207g, 9.35mmol) and ammonium formate ( lOmg, 0.077mmol) in HMDS ( 8mL, 37.4mmol) was heated to 140°C for 2 hours, until the reaction became a clear solution. Reaction was then cooled to room temperature and concentrated in vacuo. The resulting solid was stored under vacuum for 2 hours. The silylated base was then suspended in 6mL dichloroethane and was charged with acetate79 (700mg, 1.87mmol) as a solution in 6mL dichloroethane. Resulting suspension was cooled to 0°C and charged with trimethylsilane trifluoromethanesulfonate ( 1.86mL, 10.28mmol). The reaction was allowed to warm to room temperature and was stirred 18 hours. The reaction was then poured into sodium bicarbonate (50mL sat aq.) and stirred with dichloromethane (50mL) for 20 min. The resulting colloidal suspension was filtered through a fritted funnel and then extracted with dichloromethane 3xl00mL. The combined organics were dried with sodium sulfate and concentrated and purified by silica gel chromatography 2-10% to afford 990mg of nucleoside.

1H NMR (400 MHz, Chloroform-;/) δ 8.26 (d, J = 6.4 Hz, 1H), 7.67 (dd, J = 6.0, 1.4 Hz, 1H), 7.42 - 7.16 (m, 20H), 6.22 (ddd, J = 17.3, 3.2, 1.6 Hz, 1H), 6.01 (d, J = 15.1 Hz, 1H), 5.38 - 5.15 (m, 1H), 5.06 (dd, J= 51.8, 3.7 Hz, 1H), 4.69 (dd, J= 11.6, 10.0 Hz, 2H), 4.63 - 4.29 (m, 5H), 4.30 - 4.06 (m, 2H), 3.99 (dd, J= 11.1, 2.1 Hz, 1H), 3.69 (ddd, J= 22.4, 11.1, 2.1 Hz, 2H), 3.55 (dd, J= 11.1, 3.4 Hz, 1H). MS C23H23F2N3O4 [M-H⁺]; calculated: 444.2, found: 444.3.

80 81

A stirred solution of benzyl ether 80 (990mg, 2.233mmol) in dichloromethane (56 mL) was cooled to -78°C and charged with boron trichloride (22.3mL, 1M solution in dichloromethane) via addition funnel over 20 min. After 1.5 hoursthe reaction was complete by tic and was quenched with the careful addition of methanol 20mL. Reaction was then concentrated and purified by silica gel chromatography 10 - 25% methanol in dichloromethane to afford 500mg 85%> yield of product.

1H NMR (400 MHz, DMSO-d₆) δ 8.96 (s, 2H), 8.68 (s, 2H), 8.56 (d, J = 7.2 Hz, 4H), 8.07 (d, J= 6.8 Hz, 1H), 6.07 (ddd, J= 16.9, 3.6, 1.6 Hz, 3H), 5.81 (d, J = 15.9 Hz, 4H), 5.12 (t, J = 3.7 Hz, 1H), 4.99 (dd, J = 9.4, 4.0 Hz, 1H), 4.87 (d, J = 4.0 Hz, 1H), 4.19 (dddd, J = 37.8, 24.9, 8.1, 3.9 Hz, 1H), 4.05 - 3.75 (m, 2H), 3.63 (ddd, J = 12.5, 9.9, 2.3 Hz, 1H), 3.45 (dd, J = 12.4, 3.8 Hz, 1H). MS C9H11F2N3O4 [M-H⁺]; calculated:264.2, found: 264.1

A solution of lactol 83 (lg, 3.01mmol) in THF (12mL) and acetonitrile (48mL) was cooled to -50°C and was charged with carbon tetrachloride. A solution of hexamethyl phosphoryl triamine (607mL, 3.32mmol) in THF (2mL) was added drop wise over 30 min resulting in a red solution. In a separate flask compound 82 (924mg, 6.02mmol) was slurried in acetonitrile (15mL) at 0°C and was charged with sodium hydride (247mg, 6.17mmol, 60%in oil). After 1 hourthe lactol reaction was warmed to -5°C and deprotonated base was added via cannula (2x4mL acetonitrile wash). The resulting brown red solution was allowed to warm to room temperature. After 16 hoursthe reaction mixture was quenched with water (3mL) and concentrated. The resulting oil was diluted in lOOmL water and extracted with ethyl acetate (3x lOOmL). The combined organics were dried with sodium sulfate, filtered and concentrated. The brown oil was purified by silica gel chromatography 10-40% ethyl acetate in hexanes to provide a complex mixture (900mg) containing desired product.

The resulting oil in dichloromethane (48mL) was cooled to -78°C and charged with boron trichloride (9.6mL, 1M solution in dichloromethane) via addition funnel over 20 minutes. After 1 hour the reaction was complete by tic and the reaction was quenched with the careful addition of methanol (5mL). Reaction was then concentrated and purified by silica gel chromatography 2 - 15% methanol in dichloromethane to afford 490 mg 57% yield of product over two steps.

1H NMR (400 MHz, DMSO-d₆) δ 8.67 (s, 1H), 7.84 (dd, J = 3.8, 2.8 Hz, 1H), 6.77 (d, J = 3.9 Hz, 0.5H), 6.73 (dd, J = 4.0, 1.5 Hz, 1.5H), 5.20 (dt, J = 54.6, 4.0 Hz, 1H), 4.38 (ddd, J = 20.5, 7.2, 4.2 Hz, 1H), 4.21 (tt, J= 5.1, 2.2 Hz, 1H), 3.66 (dd, J= 12.2, 2.6 Hz, 1H), 3.50 (dd, J = 12.3, 4.1 Hz, 1H). MS C9H11F2N3O4 [M-H⁺]; calculated:288.1, found: 288.1

A stirred suspension of chloride 85 (254mg, .883mmol) in dioxane (l .lmL) and ammonium hydroxide (3.3mL) was prepared. The reaction vessel was sealed and heated to 60°C. After 18 hours the reaction was cooled and concentrated. The resulting paste was purified by silica gel chromatography 10-25% methanol in dichloromethane to afford 230mg 97% of product as a white solid.

1H NMR (400 MHz, DMSO-d₆) δ 8.09 (s, 1H), 7.30 (d, J = 4.0 Hz, 1H), 7.18 (s, 2H),

6.64 (d, J = 3.7 Hz, 1H), 6.58 (dd, J = 19.7, 3.6 Hz, 1H), 5.75 (d, J = 6.3 Hz, 1H), 5.07 (dt, J = 54.9, 3.9 Hz, 1H), 4.91 (s, 1H), 4.33 (m 1H), 4.18 - 4.04 (m, 1H), 3.70 - 3.42 (m, 2H).

MS C9H11F2N3O4 [M-H⁺]; calculated:269.1, found: 269.1

In a 50 mL round-bottomed flask charged with (2R,3R,4R,5R)-4-(benzyloxy)-5- ((benzyloxy)methyl)-3-fluorotetrahydrofuran-2-ol(0.31 g, 0.94 mmol), N-(4-chloro-7H- pyrrolo[2,3-d]pyrimidin-2-yl)pivalamide (0.26 g, 1.03 mmol) and Ph₃P (0.54 g, 2.07 mmol) was added dry THF (5 mL) to give a colorless solution under N₂. This was cooled to 0°C and then DIAD (0.4 mL, 2.07 mmol) was added dropwise to give a yellow solution. After 20 min, ice- water bath was removed and after stirring for 22 hours, solvent was removed in vacuo. The crude material was purified by Si0₂ column chromatography eluting from hexanes/EtOAc = 6/1 to 5/1 to 4/1 to afford N-(7-((2R,3R,4R,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-3- fluorotetrahydrofuran-2-yl)-4-chloro-7H-pyrrolo[2,3-d]pyrimidin-2-yl)pivalamide (0.22 g, 41%) as a white solid.

1H NMR (400 MHz, CDC1₃) 58.08 (s, 1H), 7.40 - 7.24 (m, 11H), 6.47 (dd, J = 3.8, 0.4

Hz, 1H), 6.26 (dd, J = 19.6, 1.6 Hz, 1H), 5.59 (ddd, J = 53.2, 4.4, 2.0 Hz, 1H), 4.93 (ddd, J = 19.6, 8.0, 4.4 Hz, 1H), 4.83 (d, J= 11.6 Hz, 1H), 4.75 (d, J = 11.6 Hz, 1H), 4.54 (d, J = 11.6 Hz, 1H), 4.49 (d, J= 12.0 Hz, 1H), 4.37 - 4.34 (m, 1H), 3.85 (dd, J= 10.8, 2.4 Hz, 1H), 3.70 (dd, J = 10.8, 4.8 Hz, 1H), 1.33 (d, J= 0.8 Hz, 9H).

HRMS C₃oH₃₂ClFN₄0₄ [M+H⁺]; calculated: 567.2096, found 567.2180.

88

In a 250 mL round-bottomed flask charged with N-(7-((2R,3R,4R,5R)-4-(benzyloxy)-5- ((benzyloxy)methyl)-3-fluorotetrahydrofuran-2-yl)-4-chloro-7H-pyrrolo[2,3-d]pyrimidin-2- yl)pivalamide(0.58 g, 1 mmol) was added dry DCM (24 mL) under argon to give a colorless solution. This was cooled to -78 °C and then BC1₃ (10 mL, 10 mmol, 1.0 M in DCM) was added dropwise over 25 min. After stirring at -78 °C for 50 min, dry ice-acetone bath was replaced by an ice-water bath. After stirring at 0°C for 75 min, TLC showed no SM. Then the crude mixture was quenched with MeOH (18 mL) and ice-water bath was removed. After warming up to rt, solvent was removed in vacuo and the crude material was purified by Si0₂ column chromatography eluting from 3% MeOH in DCM to 4% MeOH in DCM to affordN-(4-chloro-7- ((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-7H-pyrrolo[2,3- d]pyrimidin-2-yl)pivalamide (0.34 g, 85%>) as a white solid. 1H NMR (400 MHz, CDC1₃) 58.21 (s, 1H), 7.23 (d, J = 4.0 Hz, 1H), 6.54 (d, J = 4.0 Hz, 1H), 6.06 (dd, J= 17.2, 4.0 Hz, 1H), 5.64 (dt, J= 53.6, 4.0 Hz, 1H), 5.22 - 5.16 (m, lH), 4.18 (d, J= 5.2 Hz, 1H), 3.99 - 3.85 (m, 3H), 3.30 (bs, 1H), 1.33 (s, 9H). HRMS C₁₆H₂₀CIFN₄O₄ [M+H⁺]; calculated: 387.1157, found 387.1232.

89

In a 100 mL round-bottomed flask charged with N-(4-chloro-7-((2R,3R,4R,5R)-3-fluoro- 4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-2- yl)pivalamide(0.33 g, 0.85 mmol) was added 0.5 N NaOMe in MeOH (20 mL) to give a colorless solution. This was heated to refluxing, after 2.5 h, TLC showed no SM. Then the reaction was cooled to rt and solvent was removed in vacuo. The crude material was purified by Si0₂ column chromatography eluting with 3% MeOH in DCM to 4% MeOH in DCM to 5% MeOH in DCM to afford (2R,3R,4R,5R)-5-(2-amino-4-methoxy-7H-pyrrolo[2,3-d]pyrimidin-7- yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol (0.2 g, 80%) as a white solid.

1H NMR (600 MHz, CDC1₃) 57.00 (d, J= 7.8 Hz, 1H), 6.72 (d, J= 3.6 Hz, 1H), 6.35 (d, J= 3.6 Hz, 1H), 5.91 (dd, J= 14.4, 7.2 Hz, 1H), 5.73 (dt, J= 52.8, 4.8 Hz, 1H), 4.97 (s, 2H), 4.65 (s, 1H), 4.27 (s, 1H), 3.99 (s, 3H), 3.96 (d, J= 12.6 Hz, 1H), 3.74 (t, J= 9.0 Hz, 1H), 3.58 (s, 1H).

HRMS Ci₂Hi₅FN₄04 [M+H⁺]; calculated: 299.1077, found 299.1149.

In a 100 mL round-bottomed flask charged with (2R,3R,4R,5R)-5-(2-amino-4-methoxy- 7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol (0.12 g, 0.42 mmol) was added dry CH₃CN (23 mL) to give a colorless solution under argon. This was then treated with TMSI (0.07 mL, 0.50 mmol) dropwise. The colorless solution became a yellow solution. After 1 h, LC-MS showed no SM. Then this was cooled to 0°C and the reaction was quenched by adding dry MeOH (1 mL). After stirring for 5 min, solvent was removed in vacuo. The crude material was purified by Si0₂ column chromatography eluting with DCM and then 15% MeOH in DCM to 20% MeOH in DCM to afford 2-amino-7-((2R,3R,4R,5R)-3-fiuoro-4- hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (0.1 g, 85%>) as a light yellow solid.

1H NMR (400 MHz, 6-DMSO) 510.42 (bs, 1H), 6.97 (d, J= 3.6 Hz, 1H), 6.33 (bs, 2H), 6.29 (d, J= 3.6 Hz, 1H), 6.15 (dd, J= 16.4, 4.0 Hz, 1H), 5.64 (d, J= 5.2 Hz, 1H), 5.10 (dt, J = 53.2, 4.0 Hz, 1H), 5.09 (t, J= 4.8 Hz, 1H), 4.32 - 4.24 (m, 1H), 3.87 - 3.86 (m, 1H), 3.69 - 3.64 (m, 1H), 3.57 - 3.64 (m, 1H).

HRMS CiiHi₃FN₄0₄ [M+H⁺]; calculated: 285.0921, found 285.0992.

91 In a 50 mL round-bottomed flask charged with 6-fiuoro-3-((trimethylsilyl)oxy)pyrazine- 2-carboxamide (0.18 g, 0.76 mmol) and a crystal of (NH₄)₂S0₄ was added HMDS (5 mL) under argon. This was heated to 140 °C for 2 hours and then solvent was removed in vacuo and was dried under oil pump for 3 h. The residue was dissolved in dry 1,2-DCE (4 mL) and treated with a 1,2-DCE solution (4 mL) of (2S,3R,4R,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-3- fluorotetrahydrofuran-2-yl acetate (0.26 g, 0.69 mmol). This was then cooled to 0 °C, TMSOTf (0.14 mL, 0.76 mmol) was added dropwise. After stirring at 0 °C for 3 hours, saturated NaHC0₃ was added. Then aqueous phase was separated and extracted with DCM (X 3). The combined organic phase was dried (Na₂S0₄) and concentrated in vacuo. The crude material was purified by Si0₂ column chromatography eluting from hexanes/EtOAc =1/1 to 1/2 to 1/3 to afford 4- ((2R,3R,4R,5R)-4-(benzyloxy)-5-((benzyloxy)methyl)-3-fluorotetrahydrofuran-2-yl)-6-fluoro-3- oxo-3,4-dihydropyrazine-2-carboxamide (0.17 g, 52%) as a yellow solid.

1H NMR (600 MHz, CDC1₃) 59.12 (bs, 1H), 8.37 (d, J= 5.4 Hz, 1H), 7.38 - 7.25 (m, 10H), 6.23 (d, J= 15.0 Hz, 1H), 6.07 (bs, 1H), 5.04 (dd, J= 51.6, 4.2 Hz, 1H), 4.70 (d, J= 12.0 Hz, lH), 4.62 (d, J= 11.4 Hz, 1H), 4.50 (dd, J= 12.0, 7.2 Hz, 2H), 4.45 (d, J= 9.0 Hz, 1H), 4.17 (ddd, J= 23.4, 9.0, 3.6 Hz, 1H), 4.07 (dd, J= 11.4, 1.8 Hz, 1H), 3.70 (d, J= 10.8 Hz, 1H).

HRMS C2₄H₂₃F₂N₃0₅ [M+H⁺]; calculated: 472.1606, found 472.1679.

92

In a 50 mL oven-dried round-bottomed flask charged with 4-((2R,3R,4R,5R)-4- (benzyloxy)-5 -((benzyloxy)methyl)-3 -fluorotetrahydrofuran-2-yl)-6-fluoro-3 -oxo-3 ,4- dihydropyrazine-2-carboxamide(0.12 g, 0.25 mmol) was added dry DCM (10 mL) to give a bright yellow solution under argon. This was cooled to -78 °C and then BC1₃ (1.2 mL, 1.2 mmol, 1.0 M in DCM) dissolved in 2.4 mL dry DCM was added dropwise over 30 min. After stirring for another 20 min, the reaction was quenched with 7 N ammonia in MeOH (0.53 mL), followed by dry MeOH (5 mL). After stirring for 45 min, dry-ice acetone bath was removed and 30 min later, solvent was removed in vacuo. The yellow residue was washed with acetone 3 times and the filtrate was concentrated in vacuo and was purified by Si0₂ column chromatography eluting from 5% MeOH in DCM to 10% MeOH in DCM to 12% MeOH in DCM to afford 6-fluoro-4- ((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3-oxo-3,4- dihydropyrazine-2-carboxamide (30 mg, 42%) as a light yellow solid.

1H NMR (400 MHz, CD₃OD) δ 8.70 (d, J = 5.2 Hz, 1H), 6.18 (d, J = 15.6 Hz, 1H), 5.02 (dd, J = 52.0, 4.0 Hz, 1H), 4.32 (ddd, J = 26.0, 9.6, 4.0 Hz, 1H), 4.16 (d, J = 9.2 Hz, 1H), 4.1 1 (dd, J= 12.4, 2.0 Hz, 1H), 3.85 (dd, J= 12.4, 2.0 Hz, 1H).

HRMS CioHnF₂N₃0₅Na [M+Na]; calculated: 314.0667, found 314.0559.

Example 14.

Synthesis of 2'-Deoxy-2'-P-Substituted-2'-a-Fluororibonucleosides

102

Reagents and conditions: a) i. I₂, acetone, ii. K₂C0₃, MeOH; b) KOH, BnCl; c) H₂S0₄, MeOH; d) DMP; e) RLi or RMgBr; f) DAST; g) H₂S0₄, H₂0; h) Ac₂0, DMAP; i) HMPT, CC1₄ Base Coupling and Deprotection

101 103 104

102 103 104

Reagents and conditions: a) silylated base, TMSOTf, DCE; b) BC1₃, DCM; c) nucleobase, TDA- l, KOH, MeCN

References:

1) Tetrahedron: Asymmetry (1993), 4 (1), 121-131.

2) JOC (1998), 63, 2161-2167.

3) Tetrahedron (1987), 43 (10), 2355-2368.

4) Acta Chemica Scandinavica (1986), B 40, 15-20.

5) JACS (2005), 127, 10879-10884.

6) Antimicrobial Agents and Chemotherapy (2004), 48(10), 3944-3953.

7) JACS (2008), 130, 13639-13648.

8) Carbohydrate Research (1975), 39, 227-236.

9) Tetrahedron Letters (1974), 16, 1519-1521.

10) Carbohydrate Research (2009), 344, 2240-2244.

11) J. Med. Chem. (2005), 48, 5504-5508.

12) Tetrahedron (2002), 58, 1279-1288.

13) JOC (1971), 36(26), 4113-4116. Example 15.

Alternative Synthesis for 2'-Deoxy-2'-a-Fluororibonucleosides

97 71 72 7 74

75

Reagents and conditions: a) i. I₂, acetone, ii. K₂C0₃, MeOH; b) KOH, BnCl; c) H₂S0₄, MeOH; d) DMP; e) NaBH₄; f) DAST; g) H₂S0₄, H₂0; h) Ac₂0, DMAP; i) HMPT, CC1₄

Base Coupling and Deprotection

74 76 77

75 76 77

References:

1) Tetrahedron: Asymmetry (1993), 4 (1), 121-131.

2) JOC (1998), 63, 2161-2167.

3) Tetrahedron (1987), 43 (10), 2355-2368.

4) Acta Chemica Scandinavica (1986), B 40, 15-20.

5) JACS (2005), 127, 10879-10884.

6) Antimicrobial Agents and Chemotherapy (2004), 48(10), 3944-3953.

7) JACS (2008), 130, 13639-13648.

8) Carbohydrate Research (1975), 39, 227-236.

9) Tetrahedron Letters (1974), 16, 1519-1521.

10) Carbohydrate Research (2009), 344, 2240-2244.

11) J. Med. Chem. (2005), 48, 5504-5508.

12) Tetrahedron (2002), 58, 1279-1288.

13) JOC (1971), 36(26), 4113-4116.

Example 16.

Synthesis of 2'-Deoxy-2'-P-Fluoromethyl-2'-a-Fluororibonucleosides

107

Reagents and conditions: a) i. I₂, acetone, ii. K₂C0₃, MeOH; b) KOH, BnCl; c) H₂S0₄, MeOH; d) DMP; e) i. Me₃S(0)I, NaH, ii. KF, 18-crown-6; f) DAST; g) H₂S0₄, H₂0; h) Ac₂0, DMAP; i) HMPT, CC1₄

Base Coupling and Deprotection

106 108 109

107 108 109

Reagents and conditions: a) silylated base, TMSOTf, DCE; b) BC1₃, DCM; c) nucleobase, TDA- Ι, ΚΟΗ, MeCN References:

1) Tetrahedron: Asymmetry (1993), 4 (1), 121-131.

2) JOC (1998), 63, 2161-2167.

3) Tetrahedron (1987), 43 (10), 2355-2368.

4) Acta Chemica Scandinavica (1986), B 40, 15-20.

5) JACS (2005), 127, 10879-10884.

6) Antimicrobial Agents and Chemotherapy (2004), 48(10), 3944-3953.

7) JACS (2008), 130, 13639-13648.

8) Carbohydrate Research (1975), 39, 227-236.

9) Tetrahedron Letters (1974), 16, 1519-1521.

10) Carbohydrate Research (2009), 344, 2240-2244.

11) J. Med. Chem. (2005), 48, 5504-5508.

12) Tetrahedron (2002), 58, 1279-1288.

13) JOC (1971), 36(26), 41 13-41 16.

14) Org. Lett. (2003), 5(6), 807-810.

15) Bioorganic and Medicinal Chemistry Letters (1994), 4(5), 721-724.

Example 17.

Synthesis of 2'-Deoxy-2'-P-Difluoromethyl OR Trifluoromethyl-2'-a- Fluororibonucleosides

113

Reagents and conditions: a) i. I₂, acetone, ii. K₂C0₃, MeOH; b) KOH, BnCl; c) H₂S0₄, MeOH; d) DMP; e) CHF₂: i. PhS0₂CF₂H, LiHMDS, ii. Sml₂ or CF₃: TMSCF₃, TBAF; f) DAST; g) H₂S0₄, H₂0; h) Ac₂0, DMAP; i) HMPT, CC1₄

Base Coupling and Deprotection

113 114 115 Reagents and conditions: a) silylated base, TMSOTf, DCE; b) BC1₃, DCM; c) nucleobase, TDA- Ι, ΚΟΗ, MeCN

References:

1) Tetrahedron: Asymmetry (1993), 4 (1), 121-131.

2) JOC (1998), 63, 2161-2167.

3) Tetrahedron (1987), 43 (10), 2355-2368.

4) Acta Chemica Scandinavica (1986), B 40, 15-20.

5) JACS (2005), 127, 10879-10884.

6) Antimicrobial Agents and Chemotherapy (2004), 48(10), 3944-3953.

7) JACS (2008), 130, 13639-13648.

8) Carbohydrate Research (1975), 39, 227-236.

9) Tetrahedron Letters (1974), 16, 1519-1521.

10) Carbohydrate Research (2009), 344, 2240-2244.

11) J. Med. Chem. (2005), 48, 5504-5508.

12) Tetrahedron (2002), 58, 1279-1288.

13) JOC (1971), 36(26), 4113-4116.

14) Org. Lett. (2003), 5(6), 807-810.

15) Bioorganic and Medicinal Chemistry Letters (1994), 4(5), 721-724.

16) JOC (2005), 70, 7902-7910.

17) Org. Lett. (2011), 13(19), 5342-5345.

18) Org. Lett. (2001), 3(7), 1025-1028.

19) Org. Lett. (2007), 9(16), 3009-3012.

Example 18.

Alternative Synthesis of 2',3'-Dideoxy-2'-P-Substituted-2'-a-Fluoronucleosides

104 116 117 Reagents and conditions: a) TBSC1, Et₃N, DMAP; b) i. phenylchlorothiono formate, ii. AIBN; c) TBAF

References:

1) WO2008082601

2) Tetrahedron Letters (2001), 42, 2321-2324.

3) JOC (2002), 67, 8794-8797.

Example 19. Monophosphate and Diphosphate Prodrug Synthesis

123 X = H, OH

Reagents and conditions: a) chlorophosphoramidate, imidazole; b) DIC, sphingoid base-1- phosphate; c) POCl₃, 0=P(OMe)₃; d) i. DIC, morpholine, ii. Sphingoid base- 1 -phosphate, tetrazole

References:

1) J. Med. Chem. (2010), 53, 7202-7218.

2) Nucleosides, Nucleotides and Nucleic Acids (2008), 27, 460-468.

3) Journal of Lipid Research (1994), 35, 2305.

4) US 20120071434

5) Synthesis (2009), 5, 0759-0766.

6) Antiviral Chemistry and Chemotherapy (2012), 22, 181-203. 7) Bull. Korean Chem. Soc. (2003), 24(3), 267-268.

8) Tetrahedron Letters (2000), 41, 7821-7824.

9) Angew. Chem. Int. Ed. (2008), 47, 8719-8722.

10) Nucleic Acids Symposium Series (2008), 52, 83-84.

11) Nucleosides, Nucleotides and nucleic Acids (2008), 27, 43-56.

12) Carbohydrate Research (2004), 339, 2641-2649.

13) Tetrahedron Letters (1989), 30(1), 35-38.

Exemplary Syntheses:

124

N-tert-Butyloxycarbonyl-sphingosine (124)

Prepared according to Boumendjel, Ahcene and Miller, Stephen Journal of Lipid Researchl994, 35, 2305.

A mixture of sphingosine (450 mg, 1.50 mmol) and di-tert-butyl dicarbonate (0.656 g, 3.01 mmol) in methylene chloride (100 mL) at 4°C was treated dropwise with diisopropylethylamine (0.53 mL, 3.01 mmol). After gradual warming to rt, the mixture was stirred for an additional 12 h and then diluted with methylene chloride (100 mL) followed by a wash with water (30 mL) and brine (30 mL). The organic phase was dried over sodium sulfate, filtered and concentrated to dryness. The crude residue was purified by flash column chromatography over silica gel (19 mm x 175 mm) using 50% ethyl acetate in hexanes to give N- tert-butyloxycarbonyl-sphingosine (540 mg, 90%>) as a white solid.

1H NMR (300 MHz, Chloroform-;/) δ 5.77 (dt, J = 15.4, 8.4 Hz, 1H), 5.52 (dd, j= 15.4, 8.4 Hz, 1H), 3.93 (dd, J = 11.4, 3.7 Hz, 1H), 3.70 (dd, J = 11.4, 3.7 Hz, 1H), 3.59 (s, 3H), 2.05 (q, J= 7.0 Hz, 2H), 1.52 (s, 9H), 1.25 (s, 22 H), 0.87 (t, J= 6.5 Hz, 3H).

125

N-tert-Butyloxycarbonyl-sphingosine- 1 -O-dimethylphosphate (125)

N-tert-Butyloxycarbonyl-sphingosine 124(540 mg, 1.35 mmol) was rendered anhydrous by co-evaporation with anhydrous pyridine (2 x 12 mL). The residue was then dissolved in anhydrous pyridine and treated with carbon tetrabromide (622 mg, 1.88 mmol). The mixture was cooled to 0°C and treated dropwise with a solution of trimethylphosphite (0.25 mL, 2.10 mmol) in anhydrous pyridine (3 mL) over a 30 min period. After an additional 12 h at rt, both LCMS and tic (5% methanol in methylene chloride) analysis indicated complete conversion. The mixture was quenched with water (2 mL) and then concentrated to dryness. The resulting dark oil was dissolved in ethyl acetate (150 mL) and washed with 3% HCL solution ( 2 x 20 mL) followed by saturated sodium bicarbonate solution (30 mL). The organic layer was dried over sodium sulfate, filtered and concentrated. The crude residue was purified by flash column chromatography over silica gel (19 mm x 175 mm) using 2% methanol in methylene chloride to give TV-fert-butyloxycarbonyl-sphingosine-l-O-dimethylphosphate 125 (350 mg, 51%) as a gum.

H NMR (400 MHz, Chloroform-^) δ 5.82 (dt, J = 15.4, 7.1 Hz, 1H), 5.48 (dd, J = 15.4,

7.1 Hz, 1H), 4.99 (d, J = 8.9 Hz, 1H), 4.32 (ddd, J = 10.7, 8.0, 4.6 Hz, 1H), 4.11 (ddt, J = 10.7, 7.4, 3.1 Hz, 2H), 3.77 (dd, J = 11.1, 2.1 Hz, 6H), 2.01 (q, J = 7.1 Hz, 2H), 1.41 (s, 9H), 1.34 (m, 2H), 1.23 (m, 20H), 0.86 (t, J= 6.4 Hz, 3H).

3¹P NMR (162 MHz, Chloroform-^/) δ 2.00.

MS C17H25N04 [M+Na+]; calculated: 330.2, found: 330.2.

126 Sphingosine-1 -phosphate 126 A solution of N-tert-butyloxycarbonyl-sphingosine-l-O-dimethylphos hate 125 (350 mg, 0.689 mmol) in anhydrous methylene chloride (8 mL) was treated dropwise with trimethylsilyl bromide (0.45 mL, 3.45 mmol) at 0°C. After warming to room temperature, the mixture was allowed to stir at rt for 6h and then concentrated to dryness. The resulting residue was co- evaporated with methylene chloride to remove excess trimethylsilyl bromide and then treated with 66% aqueous THF (6 mL). The resulting precipitate was collected by filtration to give sphingosine-1 -phosphate 126 (218 mg, 83%) as a white solid.

1H NMR (400 MHz, Methanol-d₄+ CD₃C0₂D) δ 5.84 (dt, J= 15.5, 6.7 Hz, 1H), 5.46 (dd, J= 15.5, 6.7 Hz, 1H), 4.33 (t, J= 6.0 Hz, 1H), 4.13 (ddd, J= 11.8, 7.7, 3.6 Hz, 1H), 4.03 (dt, J = 11.8, 8.4 Hz, 1H), 3.47 (ddd, J = 8.3, 4.8, 3.2 Hz, 1H), 2.10 - 1.99 (m, 2H), 1.37 (m, 2H), 1.24 (m, 20H), 0.83 (t, J= 6.4 Hz, 3H).

³¹P NMR (162 MHz, Chloroform-^/) δ 0.69.

MS Ci₈H₃₈N0₅P [M-H⁺]; calculated: 378.2, found: 378.2.

AZT-phosphomorpholidate (amidate salt, 127)

A solution of 5'-phospho-AZT [bis(triethylammonium)salt] (440 mg, 0.8 mmol) in deionized water (4 mL) was eluted through a column of Dowex 50WX8 (10 mL) using deionized water (100 mL). The elutent was concentrated to dryness and then dissolved in t-BuOH/water (8 ml/8 ml). After addition of morpholine (0.35 mL, 4 mmol), the mixture was heated to reflux and then treated dropwise with a solution of DCC (0.66 g, 3.2 mmol) in t-butanol (3 mL) over a period of lh. After refluxing for an additional 4h, the mixture was allowed to stir at rt for 4h and then filtered to remove precipitated DCU. The filtrate was concentrated to remove t-butanol, and the resulting aqueous solution diluted with water (30 mL) and extracted with ether (2 x 75 mL). The aqueous layer was concentrated to dryness to afford AZT-phosphomorpholidate 127 (amidate salt, 560 mg, 99%) as an off-white solid that was used in the next step without further purification.

1H NMR (300 MHz, Deuterium Oxide) δ 7.66 (d, J = 1.0 Hz, 1H), 6.23 (t, J = 6.7 Hz, 1H), 4.46 (q, J = 5.4 Hz, 1H), 4.15 (d, J = 4.0 Hz, 1H), 4.09 - 3.87 (m, 2H), 3.64 (t, J = 4.8 Hz, 5H), 3.42 (t, J = 4.7 Hz, 6H), 3.31 (dd, J = 10.4, 4.6 Hz, 2H), 3.02 (q, J = 4.8 Hz, 5H), 2.98 - 2.85 (m, 5H), 2.48 (t, J = 6.3 Hz, 2H), 1.74 (dd, J = 9.4, 4.2 Hz, 5H), 1.59 (d, J = 12.6 Hz, 3H), 1.46 - 1.03 (m, 16H).

³¹P NMR (121 MHz, Deuterium Oxide) δ 7.43.

MS C14H20N6O7P [M-H⁺]; calculated: 415.1, found: 415.0.

3 ' -Azido-3 ' -deoxy-5 ' -(Sphingosine- 1 -diphospho-)-thymidine 128 (triethylammonium form)

Sphingosine- 1 -phosphate 126 (190 mg, 0.5 mmol) in deionized water (5 mL) was treated with excess triethylamine (2 mL) and then gently heated to fully dissolve. After 10 min at rt the mixture was concentrated to dryness and the residue was then co-evaporation with pyridine (3 x 10 mL). Added AZT-phosphomorpholidate (355 mg, 0.5 mmol) and tetrazole (8 mL of a 3% solution in acetonitrile, 2.5 mmol) and then concentrated to dryness. The resulting residue was co-evaporated with pyridine (3 x 5 mL), dissolved in pyridine (1 mL) and stirred for 60h at rt. The mixture was concentrated to dryness and then purified by flash column chromatography over silica gel (11 mm x 180 mm) using a solvent gradient from 3 to 25% methanol in methylene chloride containing 3% triethylamine. Fractions containing pure product were concentrated and the resulting solid co-evaporated with acetone (2 x 20 ml) and then dried under high vacuum for 16 h to give 3'-Azido-3'-deoxy-5'-(sphingosine-l-diphospho-)-thymidine 128 (triethylammonium form, 110 mg, 27%) as a white solid.

1H NMR (400 MHz, Methanol-^) δ 7.80 (s, 1H), 6.23 (dd, J= 8.1, 5.8 Hz, 1H), 5.85 (dt, J = 14.2, 6.7 Hz, 1H), 5.46 (dd, J= 15.4, 7.0 Hz, 1H), 4.59 (s, 1H), 4.28 (t, J= 6.3 Hz, 1H), 4.26 - 4.11 (m, 3H), 4.08 (t, J = 2.7 Hz, 1H), 3.41 - 3.33 (m, 1H), 3.22 - 3.14 (q, 5H), 2.44 (dt, J = 14.4, 7.1 Hz, 1H), 2.30 (ddd, J= 13.8, 6.0, 2.7 Hz, 1H), 2.14 - 2.00 (m, 2H), 1.93 (s, 3H), 1.50 - 1.36 (m, 2H), 1.32 - 1.24 (m, 29H), 0.87 (t, J= 7.0 Hz, 3H). ³ *P NMR ( 162 MHz, Methanol- d₄) δ - 11.05 (d, J = 21.3 Hz), - 11.42 (d, J = 21.1 Hz). HRMS C₂9H4₅Nio0₇P2 [M-H⁺]; calculated: 707.29534, found: 707.29526.

5'-(5-hexadecyl-2-oxido-l,3,2-dioxaphosphinan-2-yl) phospho-AZT (129)

A mixture of 2-chloro-5-hexadecyl-l,3,2-dioxaphosphinane 2-oxide (200 mg, 0.525 mmol) and 5 '-phospho-AZT [bis(triethylammonium)salt] (310 mg, 0.564 mmol) was suspended in anhydrous 1/1 dioxane/acetonitrile (12 mL) and treated with triethylamine (0.15 mL, 1.05 mmol). After 36h at rt, the mixture was concentrated and purified by column chromatography over silica gel. First the column was deactivated with 5% methanol in DCM with 3% (v/v) triethyamine and then conditioned with 5% methanol in DCM without triethylamine. After loading crude product, the column was eluted with a solvent gradient from 5 to 25% methanol in DCM. Fractions containing pure product were combined and concentrated to give 5 '-(5- hexadecyl-2-oxido-l,3,2-dioxaphosphinan-2-yl) phospho-AZT 129 (205 mg, 57%>) as a diastereomeric mixture.

1H NMR (400 MHz, Chloroform-^/) δ 11.45 (s, 1H), 8.84 (s, 1H), 7.69 (s, 1H), 6.26 (m, 1H), 4.60 (dd, J= 10.6, 5.7 Hz, 1H), 4.49 (q, J= 4.6 Hz, 1H), 4.21 (m, 5H), 4.00 (m, 1H), 3.22 - 2.97 (q, J = 7.4 Hz, 6H), 2.50 - 2.10 (m, 5H), 1.94 (s, 3H), 1.31 (t, J = 7.4 Hz, 9H), 1.21 (m, 29H), 0.84 (t, J = 6.9 Hz, 3H).

³¹P NMR (162 MHz, Chloroform-^ ) δ -12.72 (d, J = 19.0 Hz, isomerl), -17.33 (d, J = 18.9 Hz, isomer 1 and 2), -17.81 (d, J= 18.9 Hz, isomer 2).

HRMS C₂9H₅oN₅OioP₂ [M-H⁺]; calculated: 690.30437, found: 690.30384.

5 '-(Phosphoamidate-phospho)-AZT (130)

A mixture of (2S)-isopropyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate (224 mg,

0.513 mmol) and 5 '-phospho-AZT [bis(triethylammonium)salt] (282 mg, 0.513 mmol) was suspended in anhydrous 1/1 dioxane/acetonitrile (6 mL) and treated with triethylamine (0.22 mL, 1.53 mmol). After 18h the mixture was concentrated and then purified by column chromatography (19 mm x 175 mm) over silica gel. The column was first deactivated with 5% methanol in DCM with 3% (v/v) triethylamine and then conditioned with 5% methanol in DCM without triethylamine. After loading crude product, the column was eluted with a gradient from 5 to 10% methanol in DCM. Fractions containing pure product were combined and concentrated to give a 5 '-(Phosphoamidate-phospho)-AZT 130 (220 mg, 60%) as a diastereomeric mixture.

1H NMR (400 MHz, Methanol-^, diastereomeric mixture) δ 7.73 (s, 1H, isomer 1) 7.72 (s, 1H, isomer 2), 7.36 - 7.08 (m, 5H), 6.21 (dd, J= 7.7, 6.0 Hz, 1H), 4.46 - 4.41 (m, 1H), 4.20 - 4.16 (m, 1H), 4.1 1 (ddd, J = 1 1.4, 5.6, 2.9 Hz, 1H), 4.04 (m, 1H), 4.01 (dtd, J = 13.6, 5.4, 4.1 , 2.6 Hz, 1H), 3.16 (q, J = 7.3 Hz, 6H), 2.50 - 2.18 (m, 2H), 1.88 (s, 3H, isomer 1), 1.87 (s, 3H, isomer 2), 1.36 (d, J= 3.9 Hz, 3H), 1.28 (t, J = 7.3 Hz, 9H), 1.23 - 1.12 (m, 7H).

³¹P NMR (162 MHz, Methanol-^) δ -7.59 (dd, J= 54.6, 20.6 Hz), -12.40 (t, J= 19.4 Hz). HRMS C₂3H2₅Nio0₇P2 [M-H⁺]; calculated: 615.13884, found: 615.13530.

N-Trifluoroacetyl-phytosphingosine (131)

To a slurry of phytosphingosine (4 g, 12.6 mmol) and anhydrous powdered potassium carbonate (5.22 g, 37.8 mmol) in methylene chloride (85 mL) was added trifluoroacetic anhydride (1.96 mL, 13.9 mmol). The mixture was stirred at rt for 18 h and then diluted with methylene chloride (500 mL). The mixture was washed with water (100 mL). Methanol (60 mL) was added to break the emulsion. The organic phase was then dried over sodium sulfate, filtered and concentrated to give 131 (4.9 g, 94 %) as a white solid

1H NMR (400 MHz, DMSO-d₆) δ 8.90 (s, 1H), 4.90 - 4.68 (m, 1H), 4.56 (d, J = 6.1 Hz, 1H), 4.43 (s, 1H), 3.97 (d, J = 7.6 Hz, 1H), 3.65 (d, J = 10.8 Hz, 1H), 3.46 (t, J = 10.2 Hz, 1H), 3.32 - 3.16 (m, 1H), 1.42 (tt, J= 15.7, 7.5 Hz, 2H), 1.20 (s, 24H), 0.83 t, J= 6.8 Hz, 3H).

l-O-tert-Butyldiphenylsilyl-2-N-trifluoroacetyl-phytosphingosine (132)

N-Trifluoroacetyl-phytosphingosine (131, 1.88 g, 4.5 mmol) in anhydrous pyridine (23 mL) was treated with DMAP (56 mg, 0.45 mmol) and then dropwise with tert-butyldiphenylsilyl chloride (1.38 g, 5.0 mmol). After 18 h concentrated to dryness. The resulting residue was dissolved in ethyl acetate (200 mL) and washed with saturated ammonium chloride (2x 50 mL) and then brine (50 mL). The aqueous phases was back-extracted with ethyl acetate (50 mL). Combined organic phases were dried over sodium sulfate and concentrated to give crude l-O- tert-Butyldiphenylsilyl-2-N-trifluoroacetyl-phytosphingosine 132 (3g, 100%) as a gum. The material was used in the next step without further purification.

1H NMR (400 MHz, Chloroform-;/) δ 7.62 (m, 2H), 7.60 - 7.56 (m, 2H), 7.47 - 7.31 (m, 6H), 7.07 (d, J = 8.4 Hz, 1H), 4.23 (dd, J= 8.5, 4.1 Hz, 1H, 4.04 (dt, J = 11.0, 2.5 Hz, 1H), 3.82 (ddd, J = 11.0, 4.3, 1.8 Hz, 1H), 3.64 (dq, J = 10.6, 6.0, 4.3 Hz, 2H), 1.45 (m, 2H), 1.39 - 1.15 (m, 24H), 1.05 (m, 9H), 0.94 - 0.80 (t, J= 6.9 Hz 3H).

1 -O-fert-Butyldiphenylsilyl-3 ,4-0-isopropylidene-2-N-trifluoroacetyl-phytosphingosine 133 A solution of l-O-tert-Butyldiphenylsilyl-2-N-trifluoroacetyl-phytosphingosine 132

(3g,4.5 mmol) in 1/1 (v/v) 2,2-dimethoxypropane/THF was treated with catalytic amount of p- toluenesulfonic acid (87 mg, 0.45 mmol) and allowed to stir for 16h at rt. The mixture was quenched with saturated sodium bicarbonate (30 mL) and then excess THF/2,2- dimethoxypropane was removed under vacuum. The mixture was extracted with ethyl acetate (200 mL). After washing with brine, the organic layer was dried over sodium sulfate, filtered and concentrated. The crude oil was purified by column chromatography (25 mm x 175mm) over silica gel with a hexanes/ethyl acetate mobile phase to give 133(2.45 g, 78%).

1H NMR (400 MHz, Chloroform-^) δ 7.68 - 7.63 (m, 2H), 7.63 - 7.57 (m, 2H), 7.39 (m, 6H), 6.54 (d, J = 9.4 Hz, 1H), 4.23 (dd, J = 8.2, 5.6 Hz, 1H), 4.12 (ddd, J = 13.3, 6.9, 3.8 Hz, 2H), 3.96 (dd, J = 10.5, 3.9 Hz, 1H), 3.69 (dd, J = 10.5, 2.9 Hz, 1H), 1.52 - 1.36 (m, 2H), 1.33 (s, 3H), 1.31 (s, 3H), 1.24 (m, 24H), 1.03 (s, 9H), 0.86 (t, J= 53.7, 6.9 Hz, 3H).

3 ,4-0-Isopropylidene-2-N-Trifluoroacetyl-phytosphingosine 134

A solution of l-O-tert-Butyldiphenylsilyl-3,4-0-isopropylidene-2-N-trifluoroacetyl- phytosphingosine 133(2.45 g, 3.54 mmol)in THF (18 mL) was treated with tetrabutylammonium fluoride (4.25 mL of a 1.0 M solution in THF, 4.25 mmol) and stirred at rt for 12h. The mixture was diluted with ethyl acetate (100 mL) and saturated ammonium chloride (2 x 50 mL) and then brine (50 mL). The organic phase was dried over sodium sulfate, filtered, and concentrated to give a white solid that was further purified by column chromatography (25 mm x 175 mm) over silica gel with a 9: 1 hexanes: ethyl acetate mobile phase to afford 134(1.5g, 93%) as a white solid.

1H NMR (300 MHz, Chloroform-;/) δ 6.92 (d, J = 8.7 Hz, 1H), 4.31 - 4.16 (m, 2H), 4.11 (dq, J = 11.7, 3.7 Hz, 1H), 4.00 (dd, J = 11.5, 2.6 Hz, 1H), 3.70 (dd, J = 11.5, 3.6 Hz, 1H), 1.48 (s, 3H), 1.35 (s, 3H), 1.25 (m, 26H = 6.9 Hz 3H).

3 ,4-O-Isopropylidene-2-N-trifluoroacetyl-phytosphingosine- 1 -O-dimethylphosphate 135

A solution of 3,4-O-Isopropylidene-2-N-Trifluoroacetyl-phytosphingosine 134(630 mg, 1.39 mmol) was rendered anhydrous by co-evaporation with anhydrous pyridine (2 x 12 mL). The residue was then dissolved in anhydrous pyridine (12 mL) and treated with carbon tetrabromide (533 mg, 1.67 mmol). The mixture was cooled to 0°C and treated dropwise with a solution of trimethylphosphite (0.23 mL, 1.95 mmol) in anhydrous pyridine (3 mL) over a 30 min period. After an additional 12 h at rt, both LCMS and tic (5% methanol in methylene chloride) analysis indicated complete conversion. The mixture was quenched with water (2 mL) and then concentrated to dryness. The resulting dark oil was dissolved in ethyl acetate (100 mL) and washed with 3% HCL solution ( 2 x 20 mL) followed by saturated sodium bicarbonate solution (30 mL). The organic layer was dried over sodium sulfate, filtered and concentrated. The crude residue was purified by flash column chromatography over silica gel (19 mm x 175 mm) using 2% methanol in methylene chloride to give 135 (650 mg, 83%).

1H NMR (300 MHz, Chloroform-^ ) δ 7.42 (d, J= 8.8 Hz, 1H), 4.36 (td, J= 10.9, 5.0 Hz, 1H), 4.25 (m, 1H), 4.19 (m, J = 6.5, 2.0 Hz, 3H), 3.77 (dd, J = 11.2, 7.5 Hz, 6H), 1.44 (s, 3H), 1.33 (s, 3H), 1.25 (m, 26H), 0.87 (t, J= 6.6 Hz, 3H).

³¹P NMR (121 MHz, Chloroform-^/) δ 1.69.

MS C₂₅H₄₇F₃NO₇P [M-H⁺]; calcul

3 ,4-O-Isopropylidene-2-N-trifluoroacetyl-phytosphingosine- 1 -phosphate 136

A solution of 3,4-0-Isopropylidene-2-N-trifluoroacetyl-phytosphingosine-l-0- dimethylphosphate 135(650 mg, 1.16 mmol) in anhydrous methylene chloride (12 mL) was treated dropwise with trimethylsilyl bromide (0.81 mL, 6.23 mmol) at 0°C. After 12h at rt, the mixture was concentrated to dryness and the resulting residue co-evaporated with methylene chloride (3 x 50 mL) to remove excess trimethylsilyl bromide. The residue then was dissolved in cold (4°C) solution of 1% NH₄OH while maintaining pH 7-8. After 10 min at rt, the mixture was concentrated to dryness, and the resulting solid triturated with methanol/acetonitrile. The solid was collected by filtration, washed with acetonitrile, and dried under high vacuum to give 136(500 mg, 75%) as a white solid.

1H NMR (300 MHz, Methanol-i/4) δ 4.31 (dd, J= 8.7, 5.4 Hz, 1H), 4.09 (m, 4H), 1.42 (s, 3H), 1.36 (s, 3H), 1.31 (m, 26H), 0.89 (t, J= 6.4 Hz, 3H).

³¹P NMR (121 MHz, Methanol-i/4) δ 1.28. ¹⁹F NMR (282 MHz, Methanol-i/4) δ -77.13. HRMS C₂₃H₄₂F₃NO₇P [M-H⁺]; calculated: 532.26565, found: 532.26630.

2 ' ,3 ' -dideoxy-2 ' -fiuoro-5 ' -(N-trifluoroacetyl-3 ,4-O-isopropylidene-phytosphingosine- 1 - phospho)-7-deazaguanosine 137

A mixture of N-trifluoroacetyl-phytosphingosine-1 -phosphate 136(200mg, 0.373 mmol) and 2 ',3' -dideoxy-2 '-fluoro-7-deazaguanine (100 mg, 0.373 mmol) was rendered anhydrous by co-evaporation with anhydrous pyridine (3 x 10 mL). The resulting residue then was dissolved in anhydrous pyridine (4 mL) and treated with diisopropylcarbodiimide (127 mg, 1.01 mmol) and HOBt (60 mg, 0.447 mmol). After 24 h at 75°C, the reaction mixture was cooled to rt and concentrated to dryness. The crude material was purified by flash column chromatography (19 mm x 170 mm) over silica gel using a solvent gradient from 5 to 7.5% methanol in chloroform with 1% (v/v) NH4OH to give 137(80 mg, 27%) as a white solid.

H NMR (300 MHz, Methanol-^) δ 6.88 (d, J = 3.8 Hz, 1H), 6.46 (d, J = 3.8 Hz, 1H), 6.24 (d, J= 19.9 Hz, 1H), 5.34 (dd, J = 52.4, 4.6 Hz, 1H), 4.53 (s, 1H), 4.34 - 3.97 (m, 6H), 2.63 - 2.17 (m, 2H), 1.40 (s, 3H), 1.30 (s, 3H), 1.27 (m, 26H), 0.89 (t, J= 6.6 Hz, 3H).

³¹P NMR (121 MHz, Methanol-i 4) δ 12.50. ¹⁹F NMR (282 MHz, Methanol- d₄) δ -77.10 , -179.69 - -180.25 (m).

MS C₃₄H₅₂2F₄N₅0₉P [M-H⁺]; calculated: 781.3, found: 782.2

2 ' ,3 ' -dideoxy-2 ' -fluoro-5 ' -(phytosphingosine- 1 -phospho)-7-deazaguanosine 138

A solution of 2 ',3' -dideoxy-2' -fluoro-5 '-( N-trifluoroacetyl-phytosphingosine-1- phospho)-7-deazaguanosine 137(70 mg, 0.089 mmol) in cold (4°C) 70% aqueous TFA (1 mL) was stirred for 3h with gradual warming to rt. The mixture was concentrated under high vacuum and then co-evaporated with methanol (3 x 5 mL) to remove residual TFA. The resulting residue was dissolved in methanol (0.5 mL) and then treated with cone. NH₄OH (5 mL). The mixture was transferred to sealed tube and heated to 45°C for 2.5 h. After cooling to rt, the mixture was concentrated to dryness and then purified by flash column chromatography (11 mm x 160 mm) over silica gel using a solvent gradient from 25% to 35% methanol in chloroform with 2% aqueous NH₄OH (v/v). The nucleoside conjugate was further purified by semi-preparative HPLC equipped with a Dynamax C-18 (21 mm x 250 mm) using gradient from 75 to 95% methanol in water over 40 min to give 138(9 mg, 16%>) as a white solid.

1H NMR (400 MHz, Methanol-^) δ 6.91 (d, J = 3.7 Hz, 1H), 6.43 (d, J = 3.7 Hz, 1H), 6.22 (d, J= 19.6 Hz, 1H), 5.28 (d, J= 52.8, 4.7 Hz, 1H), 4.52 (m, 1H), 4.28 - 4.13 (m, 1H), 4.14 - 4.00 (m, 2H), 3.89 (dd, J = 11.7, 4.1 Hz, 1H), 3.73 (dd, J = 11.7, 8.6 Hz, 1H), 3.65 - 3.56 (m, 1H), 3.49 (m, 1H), 2.58 - 2.20 (m, 2H), 1.26 (m, 26H), 0.94 - 0.88 (t, J= 6.6 Hz, 3H).

³¹P NMR (162 MHz, Methanol-^) δ 0.44. HRMS C₂9H₅₂FN₅0₈P [M+H⁺]; calculated: 648.35320, found: 648.35336.

Experimental procedure for synthesis of McGuigan prodrugs

A solution of isopropyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate (0.397 g, 1.300 mmol) in anhydrous THF (5 ml) was added to a -78 °C stirred solution of 2'-deoxy-2'- fluoronucleoside (0.812 mmol) and 1 -methyl- lH-imidazole (0.367 ml, 4.63 mmol) in pyridine (10.00 ml). After 15 min the reaction was allowed to warm to room temperature and was stirred for an additional 3 hours. Next, the solvent was removed under reduced pressure. The crude product was dissolved in 120 ml of DCM and was washed with 20 ml 1 N HCl solution followed by 10 ml water. The organic phase was dried over sodium sulfate, filtered and concentrated in vacuo. The residues were separated over silica column (neutralized by TEA) using 5%> MeOH in DCM as a mobile phase to yield the respective products as diastereomers.

1H NMR (400 MHz, CDC1₃) δ 1.48 - 1.06 (m, 9H), 4.04 - 3.84 (m, 1H), 4.60 - 4.14 (m, 3H),4.86 - 4.64 (m, 1H), 5.11 - 4.90 (m, 1H), 5.61 - 5.19 (m, 1H), 6.32 - 5.94 (m, 3H), 7.44 - 7.02 (m, 5H), 8.11 - 7.89 (m, 1H), 8.46 - 8.20 (m, 1H). LC-MS m/z 589.4 (M+H⁺).

1H NMR (400 MHz, CDC1₃) δ 1.14 - 1.29 (m, 6H), 1.31 - 1.43 (m, 3H), 3.83 - 4.07 (m, 2H), 4.15 - 4.54 (m, 3H), 4.91 - 5.11 (m, 1H), 5.61 - 5.74 (m, 1H), 5.81 - 5.97 (m, 1H), 7.14 - 7.24 (m, 3H), 7.27 - 7.44 (m, 2H), 7.48 - 7.51 (m, 1H), 7.80 (t, J = 7.96, 7.96 Hz, OH), 9.30 (s, 1H). LC-MS m/z 516.3 (M+l⁺)

Exam le 20. Phosphonate Synthesis

10 141 142 143

Reagents and conditions: a) BAIB, TEMPO; b) Pb(OAc)₄; c) (EtO)₂POCH₂OH, pTSA

146

Reagents and conditions: a) i. Pt/C, 0₂, ii. DMF-dineopentyl acetal; b) IBr, (EtO)₂POCH₂OH; c) AIBN, Bu₃SnH; d) i. AgOAc, ii. NaOMe, MeOH, iii. PPh₃, DIAD, 4-N0₂C₆H₄COOH, iv.

154 146

Reagents and conditions: a) i. I₂, acetone, ii. K₂C0₃, MeOH; b) Pt/C, 0₂; c) i. Pb(OAc)₄, ii.

Ac₂0, DMAP; d) i. (EtO)₂POCH₂OH, pTSA, ii. K₂C0₃, MeOH; e) BnCl, KOH; f) Ac₂0, AcOH, H₂S0₄; g) i. silylated base, TMSOTf, ii.K₂C0₃, MeOH; h) DMP; i) i. RLi or RMgBr, ii. DAST; j) H₂, Pd/C

1 3

Reagents and conditions: a) i. I₂, acetone, ii. K2CO3, MeOH; b) Pt/C, 0₂; c) i. Pb(OAc)₄, ii. Ac₂0, DMAP; d) i. (EtO)₂POCH₂OH, pTSA, ii. K₂C0₃, MeOH; e) i. TCDI, pyridine, ii.Bu₃SnH, AIBN; f) Ac₂0, AcOH, H₂S0₄; g) i. silylated base, TMSOTf, ii.K₂C0₃, MeOH; h) DMP; i) i. RLi or RMgBr, ii. DAST

Reagents and conditions: a) i. SOCl₂, ii. NaI0₄, RuCl₃; b) TBAF; c) BF₃ OEt₂, DIBAL; d) Ac₂0, AcOH, H₂S0₄; e) (EtO)₂POCH₂OH, pTSA; f) i. DIBAL, ii. Ac₂0, Et₃N, DMAP; g) i. silylated base, TMSOTf, ii. K₂C0₃, MeOH References:

1) Bioorganic and Medicinal Chemistry (2010), 18, 3606-3617.

2) Bioorganic and Medicinal Chemistry (2010), 18, 7186-7192.

3) Carbohydrate Research (1991), 210, 51-70.

4) JACS (1988), 110, 7538-7539.

5) Tetrahedron (2007), 63, 4516-4534.

6) JACS (1972), 94(9), 3213-3218.

7) US20110288053

Example 21.

Phos honate Prodrug Synthesis

171

X = H, OH

Reagents and conditions: a) TMSBr; b) amino ester, ArOH, Et₃N, 2,2'-dithiodipyridine, PPh₃ i. DIC, sphingoid base, ii. TFA; d) chlorophosphoramidate, Et₃N; e) DIC, sphingoid base-1- phosphate

References:

1) Bioorganic and Medicinal Chemistry (2010), 18, 3606-3617.

2) Bioorganic and Medicinal Chemistry (2010), 18, 7186-7192.

3) Carbohydrate Research (1991), 210, 51-70.

4) JACS (1988), 110, 7538-7539.

5) Tetrahedron (2007), 63, 4516-4534.

6) JACS (1972), 94(9), 3213-3218.

7) US20110288053 8) ChemMedChem (2009), 4, 1779-1791.

9) Bioorganic and Medicinal Chemistry Letters (2012), 22, 5924-5929.

10) European Journal of Medicinal Chemistry (2009), 44, 3765-3770.

Exemplary Syntheses:

Protected-phytosphingosine-cyclic-cidofovir conjugate 172

A mixture of 4-N-benzoyl-cyclic-cidofovir (600mg, 1.64 mmol) and 2-N-Trifluoroacetyl- phytosphingosine-dimethylacetal 134(745 mg, 1.64 mmol) was rendered anhydrous by co- evaporation with anhydrous pyridine (3 x 35 mL). The resulting residue then was dissolved in anhydrous pyridine (16 mL) and treated with diisopropylcarbodiimide (352 mg, 2.79 mmol) and HOBt (266 mg, 1.97 mmol). After 16 h at 70°C, the reaction mixture was cooled to rt and concentrated to dryness. The crude material was purified by flash column chromatography (19 mm x 175 mm) over silica gel using a solvent gradient from 2 to 5% methanol in methylene chloride to give a diastereomeric mixture of 172(1.2 g, 92%) as a colorless foam. The diasteromeric mixture was used in the next reaction without further purification.

1H NMR (400 MHz, Chloroform-;/) 59.50-9.0 (br s, 1H, diastereomer 1), 8.65 - 8.52 (m, 1H, diastereomer 2), 8.47 (d, J = 8.9 Hz, 1H, diastereomer 1), 8.28 (d, J = 8.7 Hz, 1H, diastereomer 2), 7.98 - 7.86 (m, 2H), 7.75 - 7.35 (m, 5H), 4.56 - 4.42 (m, 2H), 4.34 (m, 2H), 4.16 (m, 5H), 3.89 - 3.80 (m, 1H), 3.81 - 3.72 (m, 1H), 3.62 (dd, J = 13.8, 8.1 Hz, 1H, diastereomer 1), 3.49 (dd, J = 13.8, 8.2 Hz, 1H, diastereomer 2), 1.56 - 1.43 (m, 2H), 1.40 (s, 3H, diastereomer 1) 1.37 (s, 3H, diastereomer 2), 1.27 (s, 3H, diastereomer 1), 1.25 (s, 3H, diastereomer 2), 1.20 (d, J= 3.8 Hz, 24H), 0.83 (t, J= 7.0Hz, 3H). ³¹P NMR (162 MHz, Methanol- δ 13.22 , 11.31.

Phytosphingosine-cidofovir conjugate 173

A solution of protected-phytosphingosine-cyclic-cidofovir conjugate 172(650 mg, 0.812 mmol) in 70% aqueous TFA (10 mL) was stirred for 2.5 h at rt. The mixture was concentrated under high vacuum and then co-evaporated with methanol (3 x 25 mL) to remove residual TFA. The white solid was dissolved in cone. NH₄OH (12 mL) and transferred to sealed tube and heated to 70°C for 3.5 h. After cooling to rt, the mixture was concentrated to dryness and then purified by flash column chromatography (11 mm x 190 mm) over silica gel using a solvent gradient from 10% to 30% methanol in methylene chloride with 2% (v/v) aqueous NH₄OH. The nucleoside conjugate was further purified by reverse-phase chromatography (11 mm x 170 mm) over Amberchrom CG-161M resin (Rohm & Haas) using gradient from 15 to 100% methanol in water. Pure nucleoside-conjugated eluted from column with 70%> methanol to give 173(55 mg, 12%)) as a white solid.

1H NMR (400 MHz, Methanol-^) δ 7.69 (d, J = 7.3 Hz, 1H), 5.90 (d, J = 7.3 Hz, 1H), 4.24 (dt, J = 6.8, 3.2 Hz, 1H), 4.08 - 3.86 (m, 3H), 3.80 - 3.66 (m, 5H), 3.60 (dd, J = 12.8, 9.7 Hz, 1H), 3.55 - 3.47 (m, 2H), 1.71 (tt, J = 9.9, 4.8 Hz, 1H), 1.61 (tq, J = 11.2, 5.4 Hz, 1H), 1.43 (ddd, J= 25.3, 11.7, 5.8 Hz, 1H), 1.26 (s, 23H), 0.88 (t, J= 6.8 Hz, 3H).

³¹P NMR (162 MHz, Methanol-^) δ 15.27.

HRMS C₂6H₅oN₄0₈P [M-H⁺]; calculated: 577.33718, found: 577.33706.

N-tert-Butyloxycarbonyl-phytosphingosine (174)

A suspension of phytosphingosine (10.6 g, 33.5 mmol) and triethylamine (5.6 ml, 40.2 mmol) in THF (250 mL) was treated dropwise with di-tert-butyl dicarbonate (8.6 mL, 36.9 mmol). After 12h at rt, the mixture was concentrated to dryness and the resulting white solid was recrystallized from ethyl acetate (80 mL) and then dried under high vacuum at 35°C for 12h to give 174(10.5 g, 75%).

1H NMR (400 MHz, Chloroform-;/) δ 5.31 (d, J= 8.5 Hz, 1H), 3.89 (d, J = 11.1 Hz, 1H), 3.83 (s, 2H), 3.74 (dd, J = 11.1, 5.2 Hz, 1H), 3.65 (d, J = 8.3 Hz, 1H), 3.61 (d, J = 3.9 Hz, 1H), 1.43 (s, 9H), 1.23 (s, 27H), 0.86 (t, J= 6.4 Hz, 3H).

2-O-tert-Butyldiphenylsilyl- 1 -N-tert-butyloxycarbonyl-phytosphingosine (175)

A solution of N-tert-Butyloxycarbonyl-phytosphingosine 174 (9.5 g, 22.65 mmol) and triethylamine (3.8 mL, 27.2 mmol) in anhydrous methylene chloride/DMF (120 mL/10 mL) was treated dropwise with tert-butylchlorodiphenylsilane (7 mL, 27.25 mmol). After 18h at rt, the mixture was diluted with methylene chloride (200 mL) and washed with 0.2N HCl (100 mL) and then brine (100 mL). The organic phase was dried over sodium sulfate, filtered and then concentrated to give 175 (14.9 g) as an oil which was used in the next reaction without further purification.

1H NMR (400 MHz, Chloroform- d) δ 5.31 (d, J= 8.5 Hz, 1H), 3.89 (d, J = 11.1 Hz, 1H), 3.83 (m, 1H), 3.74 (dd, J= 11.1, 5.2 Hz, 1H), 3.65 (d, J = 8.3 Hz, 1H), 3.61 (d, J = 3.9 Hz, 1H), 1.43 (s, 9H), 1.23 (s, 27H), 0.86 (t, J= 6.4 Hz, 3H).

2-O-tert-Butyldiphenylsilyl- 1 -N-tert-butyloxycarbonyl-3 ,4-O-isopropylidene-phytosphingosine (176)

A solution of 2-O-tert-Butyldiphenylsilyl-l-N-tert-butyloxycarbonyl-phytosphingosine (175, 14.9 g, 22.65 mmol) in 1/1 (v/v) THF/2,2-dimethoxypropane was treated with catalytic /?ara-toluenesulfonic acid (860 mg, 4.53 mmol). After 24h, the mixture was quenched with saturated sodium bicarbonate solution (50 mL). The mixture was concentrated and then dissolved in ethyl acetate (200 mL) and washed with brine (2 x 50 mL). The organic phase was dried over sodium sulfate, filtered and concentrated to give 176 (15.7 g) as a gum which was used in the next step without further purification.

1H NMR (400 MHz, Chloroform-;/) δ 7.66 (m, 4H), 7.51 - 7.27 (m, 6H), 4.78 (d, J = 10.0 Hz, 1H), 4.18 (dd, J = 9.3, 5.5 Hz, 1H), 3.89 (dd, J = 9.9, 3.3 Hz, 1H), 3.80 (d, J = 9.9 Hz, 1H), 3.72 (d, J= 9.9 Hz, 1H), 1.45 (s, 9H), 1.42 (s, 3H), 1.35 (s, 3H), 1.25 (s, 27H), 1.05 (s, 9H), 0.87 (t, J= 6.5 Hz, 3H).

l-N-tert-butyloxycarbonyl-3,4-0-isopropylidene-phytosphingosine (177)

A solution of 2-0-tert-Butyldiphenylsilyl-l-N-tert-butyloxycarbonyl-3,4-O- isopropylidene-phytosphingosine 176 (15.7 g,22.6 mmol) in THF at 0°C was treated dropwise with a solution of tetrabutylammonium fluoride (1.0 M in THF, 24.9 mL, 24.9 mmol) over a 20 min period. After 16h at rt, tic (3: 1 hexanes: ethyl acetate) indicated complete conversion. The mixture was concentrated to dryness and the resulting residue was dissolved in ethyl acetate (300 mL) and washed with water (3 x 100 mL). The organic phase was dried over sodium sulfate, filtered and concentrated. The resulting oil purified by flash column chromatography (35 mm x 180 mm) using a solvent gradient from 25 to 50% ethyl acetate in hexanes to give 177 (7.3 g, 71 ) over 3 steps) as a white solid.

1H NMR (400 MHz, Chloroform-;/) δ 4.93 (d, J = 9.1, 1H), 4.16 (q, J= 7.1, 6.4 Hz, 1H),

4.07 (t, J= 6.5 Hz, 1H), 3.83 (dd, J= 11.1, 2.4 Hz, 1H), 3.76 (m, 1H), 3.67 (dd, J= 11.2, 3.6 Hz, 1H), 1.43 (s, 3H), 1.42 (s, 9H), 1.32 (s, 3H), 1.23 (s, 27H), 0.86 (t, J= 6.9 Hz, 3H).

l-N-tert-butyloxycarbonyl-3,4-0-isopropylidene-phytosphingosine-tenofovir conjugate 178

A mixture of tenofovir (1.2 g, 4.18 mmol) and l-N-tert-butyloxycarbonyl-3,4-0- isopropylidene-phytosphingosine 177 (1.9 g, 4.18 mmol) was rendered a anhydrous by co- evaporation with pyridine (3 x 80 mL). The mixture in anhydrous pyridine (80 mL) was treated with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (1.6 g, 8.36 mmol) and heated to 72°C. After 48 h, LCMS indicated complete conversion. The mixture was cooled to rt and then concentrated to dryness. The resulting residue was dissolved in 9/1 (v/v) methylene chloride/methanol and washed with water (80 mL). The organic phase was concentrated to dryness and the resulting solid dissolved in methanol (30 mL) and treated dropwise with acetonitrile (60 mL). After stirring for 8h, the slurry was filtered and the collected white solid was washed with acetonitrile (2 x 40 mL) and then dried under high vacuum for 12h at 35°C to yield conjugate 178 (1.54 g, 50%).

1H NMR (300 MHz, Methanol-^) δ 8.42 (s, 1H), 8.27 (s, 1H), 4.46 (dd, J= 14.1, 1.7 Hz, 1H), 4.26 (dd, J = 14.5, 6.4 Hz, 1H), 4.14 - 4.04 (m, 2H), 3.99 (m, 3H), 3.78 (dd, J = 12.9, 9.5 Hz, 1H), 3.73 - 3.68 (m, 1H), 3.61 (dd, J = 12.9, 9.1 Hz, 1H), 1.51 (s, 3H), 1.42 (s, 9H), 1.35 (s, 3H), 1.28 (s, 27H), 1.15 (d, J = 6.2 Hz, 3H), 0.89 (t, J= 6.5 Hz, 3H). 31

P NMR (121 MHz, Methanol-^) δ 16.88.

MS CssHesNeOsP [M+H⁺]; calculated: 727.4, found: 727.4.

Phytosphingosine-tenofovir conjugate 179

A solution of l-N-tert-butyloxycarbonyl-3,4-0-isopropylidene-phytosphingosine- tenofovir conjugate 178 (1.25 g, 1.72 mmol) in cold (4°C) 70% aqueous trifluoroacetic acid (20 mL) was stirred with gradual warming to rt. After 4h, the mixture was concentrated to dryness and then co-evaporated with methanol (5 x 40 mL) to remove residual trifluoroacetic acid. The resulting white solid was purified by flash chromatography over silica gel (19 mm x 170 mm) using a solvent gradient from 5 to 20% methanol in chloroform with 2% (v/v) ammonium hydroxide and then by reverse-phase column chromatography (19 mm x 150 mm) over Amberchrom CG161M resin (Rohm & Haas) using solvent gradient from 10 to 100% methanol in water. Fractions containing pure product were concentrated and the resulting solid re- dissolved in water (50 mL) and lyophilized to give conjugate 179 (500 mg, 50%>) as a white solid.

1H NMR (400 MHz, Methanol-^) δ 8.27 (s, 1H), 8.19 (s, 1H), 4.36 (dd, J= 14.5, 3.3 Hz, 1H), 4.21 (dd, J= 14.6, 6.9 Hz, 1H), 4.15 (td, J= 7.7, 7.3, 3.8 Hz, 1H), 4.05 (dt, J= 11.8, 7.9 Hz, 1H), 3.86 (td, J= 6.5, 3.3 Hz, 1H), 3.72 (dd, J= 12.9, 9.6 Hz, 1H), 3.55 - 3.45 (m, 4H), 1.76 (t, J = 10.0 Hz, 1H), 1.52 (s, 2H), 1.26 (s, 24H), 1.16 (d, J= 6.2 Hz, 3H), 0.88 (t, J= 6.7 Hz, 3H).

³¹P NMR (121 MHz, Methanol-^) δ 17.25. MS CzvHsiNeOeP [M+H⁺]; calculated: 587.4, found: 587.3.

3,4-O-Isopropylidene-enigmol-tenofovir conjugate 180

A mixture of tenofovir (191mg, 0.66 mmol) and 3,4-O-isopropylidene-enigmol (250 mg, 0.73 mmol) was rendered ahydrous by co-evaporation with pyridine (3 x 20 mL). The mixture in anhydrous pyridine (6 mL) was treated with ^-(S-dimethylaminopropy^-N'-ethylcarbodiimide hydrochloride (255mg, 1.33 mmol) and heated to 65°C. After 16 h, tic (25% methanol in chloroform with 2% (v/v) ammonium hydroxide) indicated complete conversion. The mixture was cooled to rt and then concentrated to dryness. The resulting residue was purified by flash chromatography (19 mm x 225 mm) over silica gel using a gradient from 10 to 25% methanol in chloroform with 1% ammonium hydroxide to give 180 (200 mg, 49%).

1H NMR (400 MHz, Methanol-^) δ 8.37 (s, 1H), 8.19 (s, 1H), 4.38 (dd, J= 14.3, 3.5 Hz, 1H), 4.26 (dd, J= 14.4, 5.5 Hz, 1H), 3.89 (tt, J = 5.9, 3.0 Hz, 1H), 3.84 - 3.72 (m, 3H), 3.64 (dd, J = 12.2, 8.8 Hz, 1H), 3.44 (dd, J = 12.2, 9.9 Hz, 1H), 1.37 (s, 3H), 1.32 (m, 2H), 1.29 (s, 3H), 1.28 (s, 25H), 1.10 (d, J= 7.2 Hz, 3H), 0.89 (t, J= 6.5 Hz, 3H).

³¹P NMR (121 MHz, Methanol-^) δ 17.23.

MS C₃oH₅₅N₆0₅P [M-H⁺]; calculated: 609.4 found: 609.4.

Enigmol-tenofovir conjugate 181 A solution of 3,4-O-Isopropylidene-enigmol-tenofovir conjugate 180 (70 mg, 0.115 mmol) in 70% trifluoroacetic acid (1.5 mL) was allowed to stir at rt. After 4h, the mixture was concentrated to dryness and the resulting residue was co-evaporated with methanol (3 x 10 mL) to remove residual TFA. The resulting solid was purified by flash column chromatography (11 mm x 180 mm) over silica gel using 25% methanol in chloroform with 2% ammonium hydroxide. Fractions containing pure product were concentrated and the resulting residue re- dissolved in water (12 mL) and lyophilized to give 181 (18 mg, 28%) as a white solid.

1H NMR (400 MHz, Methanol-^) δ 8.45 (s, 1H), 8.32 (s, 1H), 4.47 (dd, J= 14.5, 3.2 Hz, 1H), 4.40 - 4.33 (m, 1H), 4.30 (dd, J = 14.5, 6.1 Hz, 1H), 3.97 (td, J = 6.2, 3.2 Hz, 1H), 3.89 - 3.80 (m, 1H), 3.75 (dd, J = 12.9, 9.4 Hz, 1H), 3.57 (dd, J = 12.9, 10.0 Hz, 1H), 3.53 - 3.43 (m, 1H), 1.82 (ddd, J = 14.9, 6.3, 3.0 Hz, 1H), 1.69 (ddd, J = 14.3, 9.1, 4.3 Hz, 1H), 1.33 (d, J = 6.8 Hz, 3H), 1.27 (m, 21H), 1.16 (d, J= 6.2 Hz, 3H), 0.90 (t, J= 6.6 Hz, 3H).

³¹P NMR (121 MHz, Methanol-^) δ 17.89.

MS C₂7H₅iN₆0₅P [M+H⁺]; calculated: 569.4, found: 569.4. Example 22.

Synthesis of Cyclic Phosphate Prodrugs

b

Reagents and conditions: a) i. RiOP(Nz^'Pr₂)₂, DO, ii. mCPBA; b) i. chlorophosphoramidate, imidazole, ii. t-BuOK; c) i. imidazole, ii. t-BuOK

References:

1) J. Med. Chem. (2010), 53, 6825-6837.

2) J. Med. Chem. (2007), 50, 5765-5772.

3) Bioorganic and Medicinal Chemistry (2010), 18, 3606-3617.

4) Antimicrobial Agents and Chemotherapy (2008), 52(2), 655-665.

5) J. Med. Chem. (2005), 48, 2867-2875.

6) JOC (1995), 60, 2563-2569.

7) Bioorganic and Medicinal Chemistry Letters (2012), 22, 5924-5929.

8) European Journal of Medicinal Chemistry (2009), 44, 3765-3770.

9) Bioorganic and Medicinal Chemistry Letters (2012), 22, 4497-4501.

Example 23.

Synthesis of 2'-Fluoro-2'-Methyl-5-Fluorouridine Nucleoside Analog

188 189 90

Reagents and conditions: a) i. HMDS, (NH₄)₂S0₄, ii. SnCl₄, chlorobenzene; b) 80% AcOH; c) NH₃, MeOH

188 In a 50 mL pear-shaped flask charged with N-(5-fluoro-2-oxo-l,2-dihydropyrimidin-4- yl)benzamide (0.185 g, 0.793 mmol) was added bis(trimethylsilyl)amine (1.77 ml, 8.45 mmol) and ammonium sulfate (2.6 mg, 0.02 mmol) under N₂. This was heated for refluxing for 2 h, after cooling to rt, solvent was removed in vacuo and further dried under high vacuum for 1 h. The residue was dissolved in dry chlorobenzene (10 ml) and (2R,3R,4R,5R)-5-acetoxy-2- ((benzoyloxy)methyl)-4-fluoro-4-methyl-tetrahydrofuran-3-yl benzoate (0.22 g, 0.53 mmol) was added. Then SnCl₄ (0.27 ml, 2.3 mmol) was added dropwise. After stirring at rt for 1 h, this was heated to 60 °C overnight. After cooling to 0 °C, solid sodium bicarbonate (0.85 g) was added, followed by EtOAc (5 mL). This was allowed to stir for 15 min and then water (0.5 mL) was added slowly. The insoluble material was filtered off and washed wtih more EtOAc (2.5 mL). The filtrate was washed with water once, bine once, dried (Na₂S0₄) and concentrated in vacuo. The crude material was purified by Si0₂ column chromatography eluting from 10% to 35% EtOAc in hexanes to afford (2R,3R,4R,5R)-5-(4-benzamido-5-fluoro-2-oxopyrimidin-l(2H)-yl)- 2-((benzoyloxy)methyl)-4-fluoro-4-methyltetrahydrofuran-3-yl benzoate (30 mg, 9.6 % yield) as a white solid.

1H NMR (400 MHz, CDC1₃) δ 8.29 (d, J = 7.2 Hz 1H), 8.14 - 8.09 (m, 4H), 7.83 (d, J = 5.6 Hz, 1H), 7.65 - 7.47 (m, 10H), 6.36 (d, J = 21.2 Hz, 1H), 5.51 (dd, J = 22.0, 9.6 Hz, 1H), 4.86 - 4.64 (m, 3H), 1.52 (d, J= 22.0 Hz, 3H).

LCMS C₃iH₂6F₂N₃0₇ [M+H⁺]; calculated: 589.2, found 590.1.

189

A solution of (2R,3R,4R,5R)-5-(4-benzamido-5-fluoro-2-oxopyrimidin-l(2H)-yl)-2- ((benzoyloxy)methyl)-4-fluoro-4-methyltetrahydrofuran-3-yl benzoate (600 mg, 1.02 mmol) in 80%) aqueous AcOH (30 mL) was heated under reflux for 16 h. Then solvent was removed in vacuo and dried under high vacuum. The white solid was triturated with ether, filtered off and washed with more ether. (2R,3R,4R,5R)-3-(Benzoyloxy)-4-fluoro-5-(5-fluoro-2,4-dioxo-3,4- dihydropyrimidin-l(2H)-yl)-4-methyltetrahydrofuran-2-yl)methyl benzoate (400 mg, 81% yield) was obtained as a white solid.

1H NMR (400 MHz, CDC1₃) δ 8.11 - 8.04 (m, 4H), 7.69 (d, J = 5.6 Hz, 1H), 7.67 - 7.45 (m, 6H), 6.28 (d, J = 18.4 Hz, 1H), 5.49 (dd, J = 21.2, 9.6 Hz, 1H), 4.83 (d, J = 10.4 Hz, 1H), 4.68 - 4.62 (m, 2H), 1.50 (d, J= 22.4 Hz, 3H).

LCMS C24H19F2N2O7 [M-H⁺]; calculated: 486.1, found 485.1.

190 (2R,3R,4R,5R)-3-(Benzoyloxy)-4-fluoro-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)- 4-methyl-tetrahydrofuran-2-yl)methyl benzoate (350 mg, 0.72 mmol) in 7N ammonia in MeOH (15 mL) and MeOH (15 mL) was stirred in a sealed tube for 16 h. Then solvent was removed in vacuo and the residue was triturated with ether to remove the benzamide byproduct. The residue was further purified by S1O₂ column chromatography eluting from 1% to 6% MeOH in DCM to afford 5-fluoro- 1 -((2R,3R,4R,5R)-3-fiuoro-4-hydroxy-5-(hydroxymethyl)-3- methyltetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione (40 mg, 20% yield) as a white solid.

1H NMR (400 MHz, CD₃OD) δ 8.46 (d, J = 7.2 Hz, 1H), 6.09 (d, J

- 3.93 (m, 3H), 3.81 (dd, J= 12.8, 2.0 Hz, 1H), 1.38 (d, J= 22.4 Hz, 3H).

LCMS CioHi₃F₂N₂0₅ [M+H⁺]; calculated: 278.1, found 279.0.

191 (2R,3R,4R,5R)-5-(4-benzamido-5-fluoro-2-oxopyrimidin-l(2H)-yl)-2-((benzoyloxy)methyl)-4- fluoro-4-methyltetrahydrofuran-3-yl benzoate (0.15 g, 0.25 mmol) was stirred with 7 N ammonia in MeOH at rt for 15.5 h. Then solvent was removed and the crude material was purified by Si02 column chromatography eluting from 5% to 15% MeOH in DCM to afford 4-amino-5-fluoro-l- ((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)-3-methyltetrahydrofuran-2- yl)pyrimidin-2(lH)-one (65 mg, 92% ) as a white solid.

1H NMR (400 MHz, CD₃OD) δ 8.40 (d, J= 6.8 Hz 1H), 6.15 (d, J= 18.0 Hz, 1H), 4.04 - 3.93 (m, 3H), 3.81 (d, J= 12.8 Hz, 1H), 1.33 (d, J= 22.4 Hz, 3H).

LCMS CioHi₄F₂N₃04 [M+H⁺]; calculated: 278.1, found 278.0.

Example 24.

Synthesis of 2'-Fluoro-2'-Methylguanosine Nucleoside Analogs and Prodrugs:

Following a literature procedure (Ref: J. Org. Chem. 2011, 76, 3782-3790.), (2R,3R,4R,5R)-5-(2-amino-6-chloro-9H-purin-9-yl)-2-((benzoyloxy)methyl)-4-fluoro-4- methyltetrahydrofuran-3-yl benzoate was obtained as a white foamy solid in 63% yield. 1H NMR (400 MHz, CDC1₃) δ 8.05 (dd, J = 8.4, 1.6 Hz 2H), 7.95 (dd, J = 6.0, 1.6 Hz,

2H), 7.89 (s, 1H), 7.63 - 7.59 (m, 1H), 7.53 - 7.43 (m, 3H), 7.36 - 7.32 (m, 2H), 6.45 (dd, J = 22.4, 9.2 Hz, 1H), 6.13 (d, J = 18.4 Hz, 1H), 5.32 (bs, 2H), 5.02 (dd, J= 11.6, 4.4 Hz, 1H), 4.79 - 4.74 (m, 1H), 4.60 (dd, J= 12.0, 5.6 Hz, 1H), 1.35 (d, J= 22.4 Hz, 3H).

LCMS C₂5H₂₂C1FN₅0₅[M+H⁺]; calculated: 526.1, found 526.1.

193

To a 500 mL round-bottomed flask charged with (2R,3R,4R,5R)-5-(2-amino-6-chloro- 9H-purin-9-yl)-2-((benzoyloxy)methyl)-4-fluoro-4-methyltetrahydrofuran-3-yl benzoate (2.0 g, 3.8 mmol) was added 7 N ammonia in MeOH (70 mL), after stirring at rt for 3.5 h, solvent was removed in vacuo and the crude material was purified by ISCO column chromatography eluting from 1% to 20% MeOH in DCM to afford the product which was further purified by recrystallization from MeOH to afford (2R,3R,4R,5R)-5-(2-amino-6-chloro-9H-purin-9-yl)-4- fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol (0.9 g, 75% yield) as a white solid.

1H NMR (400 MHz, ₆-DMSO) δ 8.44 (s, 1H), 7.10 (bs, 2H), 6.07 (d, J = 17.2 Hz, 1H), 5.69 (d, J = 6.8 Hz, 1H), 5.27 (t, J = 4.8 Hz, 1H), 4.25 - 4.15 (m, 1H), 3.94 - 3.83 (m, 2H), 3.73 - 3.67 (m, 1H), 1.12 (d, J= 22.4 Hz, 3H).

LCMS C_{I I}H_I4C1FN₅0₃ [M-H⁺]; calculated: 318.1, found 318.0.

To a 10 mL oven-dried pear-shaped flask charged with (2R,3R,4R,5R)-5-(2-amino-6- chloro-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol (51 mg, 0.16 mmol) was added dry DCM (0.8 mL) under argon. Then Et₃N (0.089 ml, 0.64 mmol) was added dropwise. This was cooled to -20 °C and then isopropyl phosphorodichloridate (0.043 ml, 0.32 mmol) was added dropwise. After stirring for 15 min, 1-methylimidazole (0.051 ml, 0.64 mmol) was added dropwise. The reaction was allowed to stir overnight with the temperature warm up gradually to rt. After 22.5 h, solvent was removed in vacuo and the crude material was purified directly by ISCO column chromatography eluting from 40% to 90% EtOAc in hexanes to afford (2R,4aR,6R,7R,7aR)-6-(2-amino-6-chloro-9H-purin-9-yl)-7-fluoro-2-isopropoxy-7- methyltetrahydro-4H-furo[3,2-d][l,3,2]dioxaphosphinine 2-oxide (34.7 mg, 51% yield) as a white solid.

1H NMR (400 MHz, CDC1₃) δ 7.77 (s, 1H), 6.07 (d, J= 19.2 Hz, 1H), 5.11 (bs, 3H), 4.94 - 4.85 (m, 1H), 4.70 - 4.61 (m, 1H), 4.47 - 4.39 (m, 2H), 1.52 - 1.42 (m, 6H), 1.37 (d, J = 22.0 Hz, 3H).

LCMS CI₄HI₉C1FN₅0₅P [M+H⁺]; calculated: 422.1, found 422.1.

195

Following a literature procedure (Ref: J. Org. Chem. 2011, 76, 3782-3790.), (2R,3R,4R,5R)-5-(2-amino-6-ethoxy-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)-4- methyltetrahydrofuran-3-ol was obtained as an off-white solid in 93% yield.

1H NMR (400 MHz, d₆-DMSO) δ 8.16 (s, 1H), 6.54 (bs, 2H), 6.04 (d, J = 18.0 Hz, 1H), 5.65 (d, J = 7.6 Hz, 1H), 5.23 (t, J = 4.8 Hz, 1H), 4.47 - 4.42 (m, 2H), 4.23 - 4.13 (m, 1H), 3.92 - 3.83 (m, 2H), 3.72 - 3.66 (m, 1H), 1.36 (t, J= 7.2 Hz, 3H), 1.06 (d, J= 22.4 Hz, 3H).

LCMS Ci₃Hi₉FN₄ [M+H⁺]; calculated: 328.1, found 328.1.

Following a literature procedure (Ref: J. Org. Chem. 2011, 76, 3782-3790.), (2R,4aR,6R,7R,7aR)-6-(2-amino-6-ethoxy-9H-purin-9-yl)-7-fluoro-2-isopropoxy-7- methyltetrahydro-4H-furo[3,2-d][l,3,2]dioxaphosphinine 2-oxide was obtained as a white solid in 38% yield.

1H NMR (400 MHz, CDC1₃) δ 7.59 (s, 1H), 6.02 (d, J= 19.2 Hz, 1H), 5.42 (bs, 1H), 4.94 - 4.84 (m, 1H), 4.85 (bs, 2H), 4.68 - 4.48 (m, 4H), 4.43 - 4.37 (m, 1H), 1.50 - 1.45 (m, 9H), 1.35 (d, J= 22.0 Hz, 3H).

LCMS Ci₆H₂₄FN₅0₆P [M+H⁺]; calculated: 432.1, found 432.1.

197 To a 100 mL pear-shaped flask charged with (2R,3R,4R,5R)-5-(2-amino-6-chloro-9H- purin-9-yl)-4-fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol (0.22 g, 0.69 mmol) was added dry MeOH (4 mL) to give a white suspension. This was vacuumed and charged with N₂ and then 2-mercaptanol (0.24 ml, 3.46 mmol) was added, followed by NaOMe (8.73 ml, 4.36 mmol, 0.5 M) dropwisely. This was heated to 75 °C. After 15 h, the reaction was neutralized to pH = 7 by dropwsie addition of glacial acetic acid. The solvent was removed in vacuo and the residue was purified by Si0₂ column chromatography eluting from 0 to 15% MeOH in DCM and then repurified by Amberchrom CG161M resin column to afford 2-amino-9-((2R,3R,4R,5R)-3- fluoro-4-hydroxy-5 -(hydroxymethyl)-3 -methyltetrahydrofuran-2-yl)- 1 H-purin-6(9H)-one (0.13 g, 63% yield) as a white solid.

1H NMR (400 MHz, d₆-DMSO) δ 10.65 (bs, 1H), 8.01 (s, 1H), 6.57 (bs, 2H), 5.95 (dd, J = 18.0, 1.6 Hz, 1H), 5.65 (d, J = 7.6 Hz, 1H), 5.22 (t, J = 4.8 Hz, 1H), 4.19 - 4.07 (m, 1H), 3.90 - 3.82 (m, 2H), 3.69 - 3.61 (m, 1H), 1.07 (d, J = 22.4 Hz, 3H).

LCMS CiiHi₅FN₅0₄ [M+H⁺]; calculated: 300.1, found 300.1.

To a 50 mL oven-dried round-bottomed flask charged with (2R,3R,4R,5R)-5-(2-amino-6- chloro-9H-purin-9-yl)-2-((benzoyloxy)methyl)-4-fluoro-4-methyltetrahydrofuran-3-yl benzoate (0.5 g, 0.95 mmol) was added dry BnOH (8 mL). To another 50 mL round-bottomed flask charged with NaH (0.091 g, 3.8 mmol, 60% in mineral oil) was added dry DMF (2.5 mL). The suspension was stirred at 0 °C in an ice-water bath and dry BnOH (1.7 mL) was added dropwise. A solution was slowly formed and it was transferred to the nucleoside suspension quickly under N₂ at rt. The mixture was heated to 50 °C for 3 h and cooled to rt. The reaction mixture was neutralized by the addition of 4 N HC1 to pH = 7. The solution was concentrated in vacuo and the residue was diluted with DCM and washed with water three times, brine once, dried (Na₂S0₄) and concentrated in vacuo. The crude material was purified by Si0₂ column chromatography eluting from 0% to 8% MeOH in DCM to afford (2R,3R,4R,5R)-5-(2-amino-6-(benzyloxy)-9H- purin-9-yl)-4-fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol (0.14 g, 38% yield) as a white foamy solid.

1H NMR (400 MHz, CDC1₃) δ 7.69 (s, 1H), 7.53 - 7.48 (m, 2H), 7.39 - 7.31 (m, 3H), 6.00 (dd, J= 20.0 Hz, 1H), 5.56 (s, 2H), 5.20 (bs, 1H), 5.00 (bs, 2H), 4.77 (bd, J= 22.0 Hz, 1H), 4.19 (dd, J = 12.8, 1.6 Hz, 1H), 4.09 (dd, J = 9.2, 2.0 Hz, 1H), 3.96 (d, J

(bs, 1H), 1.14 (d, J= 22.8 Hz, 3H).

LCMS Ci₈H₂iFN₅0₄ [M+H⁺]; calculated: 390.1, found 390.1.

To a 50 mL pear-shaped flask charged with (2R,3R,4R,5R)-5-(2-amino-6-(benzyloxy)- 9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol (0.14 g, 0.36 mmol) was added dry DCM (2 mL) under N₂ to give a colorless solution. Then Et₃N (0.20 ml, 1.44 mmol) was added dropwsie and this was cooled to -20 °C and isopropyl phosphorodichloridate (0.096 ml, 0.719 mmol) was added dropwise. After 15 min, 1-methylimidazole (0.12 ml, 1.44 mmol) was added dropwise. This was allowed to warm up to rt gradually overnight. The next day, solvent was removed in vacuo and the residue was purified by Si0₂ column chromatography eluting from 30% to 70% EtOAc in hexanes to afford (2R,4aR,6R,7R,7aR)-6- (2-amino-6-(benzyloxy)-9H-purin-9-yl)-7-fluoro-2-isopropoxy-7-methyltetrahydro-4H-furo[3,2- d][l,3,2]dioxaphosphinine 2-oxide (0.11 g, 62 %> yield) as a white solid.

1H NMR (400 MHz, CDC1₃) δ 7.60 (s, 1H), 7.51 (d, J= 7.2 Hz, 2H), 7.40 - 7.32 (m, 3H), 6.02 (d, J = 19.6 Hz, 1H), 5.57 (s, 2H), 5.42 (bs, 1H), 4.94 - 4.86 (m, 3H), 4.63 (ddd, J = 13.6, 8.8, 4.4 Hz, 1H), 4.50 (t, J = 10.4 Hz, 1H), 4.43 - 4.37 (m, 1H), 1.47 (dd, J = 9.6, 6.4 Hz, 6H), 1.36 (d, J = 22.4 Hz, 3H).

LCMS C₂iH₂₆FN₅0₆P [M+H⁺]; calculated: 494.1, found 494.1.

To a 10 mL pear-shaped flask charged with (2R,4aR,6R,7R,7aR)-6-(2-amino-6- (benzyloxy)-9H-purin-9-yl)-7-fluoro-2-isopropoxy-7-methyltetrahydro-4H-furo[3,2- d][l,3,2]dioxaphosphinine 2-oxide (0.1 g, 0.2 mmol) was added MeOH (2 mL), followed by 10% Pd/C (0.02 g, 0.2 mmol) under N₂. Then the flask was vacuumed and charged with a H₂ balloon. After reacting for 1 h, solvent was removed in vacuo and the crude material was purified by Si0₂ column chromatography eluting from 0% to 10% MeOH in DCM to afford (2R,4aR,6R,7R,7aR)-6-(2-amino-6-hydroxy-9H-purin-9-yl)-7-f uoro-2-isopropoxy-7-methyl- tetrahydro-4H-furo[3,2-d][l,3,2]dioxaphosphinine 2-oxide (0.055 g, 67 % yield) as a white solid.

1H NMR (400 MHz, d₆-DMSO) δ 10.78 (bs, 1H), 8.03 (bs, 1H), 6.51 (bs, 2H), 6.19 (d, J = 21.2 Hz, 1H), 4.73 - 4.61 (m, 4H), 4.26 - 4.20 (m, 1H), 1.38 - 1.29 (m, 6H), 1.21 (d, J = 22.8

Hz, 3H).

LCMS Ci₄H₂oFN₅0₆P [M+H⁺]; calculated: 404.1, found 404.1.

Example 25.

Synthetic Routes to Base Substituted Cytidine Nucleoside Analogs

201 202 203

Reagents and conditions: a) i. tosyl chloride, triethylamine, ii. methylamine; b) TBAF, THF A stirred suspension of uridine (340mg, .716mmol) in MeCN (lOmL) in a sealed tube was cooled to 0°C and charged sequentially with triethyl amine (399uL, 2.86mmol), tosyl chloride (273mg, 1.4mmol), and DMAP(87mg, .716mmol) and was stirred at 0°C. After 2h tosylation was complete by tic and reaction was charged with methyl amine (20mL 40% in water) and was capped and heated to 50°C. After 2 days reaction was cooled, concentrated, and purified by silica gel chromatography 20-80% ethyl acetate in hexane to afford 170mg product 49%.

Protected nucleoside (170mg, .349mmol) was then dissolved in THF (1.7mL) and was charged with tbaf( 0.767mL, 1M in THF). After 16H reaction was concentrated and purified by silica gel chromatography (2-7% methanol in dcm) to provide product 60mg, 66%.

Reagents and conditions: a) i. tosyl chloride, triethylamine, ii. dimethylamine; b) TBAF, THF

A stirred suspension of uridine (340mg, .716mmol) in MeCN (lOmL) in a sealed tube was cooled to 0°C and charged sequentially with triethyl amine (399uL, 2.86mmol), tosyl chloride (273mg, 1.4mmol), and DMAP(87mg, .716mmol) and was stirred at 0°C. After 2h tosylation was complete by tic and reaction was charged with dimethyl amine (20mL 40% in water) and was capped and heated to 50°C. After 2 days reaction was cooled, concentrated, and purified by silica gel chromatography 20-80% ethyl acetate in hexane to afford 226mg product 63%.

Protected nucleoside (226mg, .450mmol) was then dissolved in THF (2.3mL) and was charged with tbaf( 0.99mL, 1M in THF). After 16H reaction was concentrated and purified by silica gel chromatography (2-7% methanol in dcm) to provide product 121mg, 98%.

Reagents and conditions: a) i. tosyl chloride, triethylamine, ii. ammonium hydroxide; b) TBAF, THF A stirred suspension of thymadine (650mg, 1.3mmol) in MeCN (19mL) in a sealed tube was cooled to 0°C and charged sequentially with triethyl amine (741uL, 5.3mmol), tosyl chloride (507mg, 2.66mmol), and DMAP(162mg, 1.3mmol) and was stirred at 0°C. After 2h tosylation was complete by tic and reaction was charged with ammonium hydroxide (30mL) and was capped and heated to 50°C. After 2 days reaction was cooled, concentrated, and purified by silica gel chromatography 20-80% ethyl acetate in hexane to afford 468mg product 72%.

Protected nucleoside (468mg, .959mmol) was then dissolved in THF (4.8mL) and was charged with tbaf( 2.1mL, 1M in THF). After 16H reaction was concentrated and purified by silica gel chromatography (2-7% methanol in dcm) to provide product 80mg, 32%.

Reagents and conditions: a) i. benzyl hydroxyl uridine, MH₄CH0₂, HMDS, ii. 227, TMSOTf, DCE; b) i. tosyl chloride, triethylamine, ii. ammonium hydroxide; c) BC1₃, DCM

210

A stirred suspension of benzyl hydroxymethyl uridine (1.26g, 5.43mmol) and ammonium formate ( lOmg, .077mmol) in HMDS (15mL, 72.3mmol) was heated to 140°C for 4H, until the reaction became a clear solution. Reaction was then cooled to room temperature and concentrated in vacuo. The resulting solid was then suspended in 12mL dichloroethane and was charged with acetate (1.35g, 3.62mmol) as a solution in 12mL dichloroethane. Resulting suspension was cooled to 0°C charged with trimethylsilane triflate (1.14mL, 6.33mmol) and was stirred 18h reaction at room temperature was then poured into sodium bicarbonate (lOOmL sat aq.) and stirred with dichloromethane (20mL) for 20 min. The resulting colloidal suspension was filtered through a fritted funnel and then extracted with dichloromethane 3xl00mL. The combined organics were dried with sodium sulfate and concentrated and purified by silica gel chromatography 10-50% ethyl aceate in hexanes to afford 490mg 25% of beta nucleoside.

211

A stirred suspension of thymadine (490mg, .896mmol) in MeCN (13mL) in a sealed tube was cooled to 0°C and charged sequentially with triethyl amine (500uL, 3.6mmol), tosyl chloride (342mg, 1.8mmol), and DMAP(11 lmg, .896mmol) and was stirred at 0°C. After 2h tosylation was complete by tic and reaction was charged with ammonium hydroxide (30mL) and was capped and heated to 50°C. After 2 days reaction was cooled, concentrated, and purified by silica gel chromatography 20-80% ethyl acetate in hexane to afford 489mg product 99%.

212

A stirred solution of benzyl ether (489mg, .896mmol) in dichloromethane (4.5 mL) was cooled to -78°C and charged with boron trichloride (5.4mL, 1M solution in dichloromethane) via addition funnel over 20 min. After 1.5H reaction was complete by tic and reaction was quenched with the careful addition of ammonia in methanol (3mL, 7M in methanol). Reaction was then concentrated and purified by silica gel chromatography 10 - 25% methanol in dichloromethane to afford 88mg 36%.

Example 26.

Synthesis of 2',3'-Dideoxy-2'-Fluorouridine

Reagents and conditions: a) i. HMDS, (NH₄)₂S0₄, uracil, reflux, ii. #, TMSOTf, DCE; b) TBAF,

THF

213 Following a literature procedure (Ref: J. Org. Chem. 1998, 63, 2161-2167), 1- ((2R,3R,5S)-5-(((tert-butyldiphenylsilyl)oxy)m

2,4(lH,3H)-dione was obtained as a crude in 77% yield.

A solution of l-((3R,5S)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-3- fluorotetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione (5.837 g, 12.46 mmol) in THF (50 ml) was cooled to 0-5 °C. Tetrabutylammonium fluoride (14.95 ml, 14.95 mmol) was added over a 5-10 min. period and the mixture was stirred at 0-5°C for lh. The mixture was then concentrated and purified on flash chromatography using Si02 with a linear gradient of 0- 5%MeOH in EtOAc to obtain the product as a mixture of diastereomers. The product was recrystallized in methanol to obtain required distereomer (l-((2R,3R,5S)-3-fiuoro-5- (hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione) as a while solid (yield 29.6%).

1H NMR (CD30D, 300 MHz) δ: 8.09 (d, J = 9.0 Hz, 1H), 5.94 (d, J= 18.0 Hz, 1H), 5.64 (d, J = 9.0 Hz, 1H), 5.31 (d, J = 6.0 Hz, 0.5H), 5.14 (m, 0.5H), 4.43 (m, 1H), 3.98 (dd, J = 12.0 and 3.0 Hz, 1H), 3.69 (dd, J= 15.0 and 3.0 Hz, 1H), 2.44 (m, 2H).

HRMS calcd for [M+H]⁺ C₉H₁₂FN₂O₄ 231.07756, found 231.07752. Example 27.

General Procedure for Preparation of 5'-Triphosphates:

Nucleoside analogue was dried under high vacuum at 50°C for 18h and then dissolved in anhydrous trimethylphosphate (0.3 M). After addition of proton-sponge® (1.5 molar equiv), the mixture was cooled to 0°C and treated dropwise with phosphoryl chloride (1.3 molar equiv) via microsyringe over a 15 min period. The mixture continued stirring at 0°C for 4 to 6 h while being monitored by tic (7:2: 1 isopropanol: cone. NH₄OH: water). Once greater than 85% conversion to the monophosphate, the reaction mixture was treated with a mixture of bis(tri-n-butylammonium pyrophosphate) (3 molar equiv) and tributylamine (6 molar equiv) in anhydrous DMF (1 mL). After 20 min at 0°C with monitoring by tic (11 :7:2 ΝΗ₄ΟΗ: isopropanol: water), the mixture was treated with 20 mL of a 100 mM solution of triethylammonium bicarbonate (TEAB), stirred for lh at rt and then extracted with ether (3 x 15 mL). The aqueous phase was then purified by anion-exchange chromatography over DEAE Sephadex® A-25 resin (11 x 200 mm) using a buffer gradient from 50 mM (400 mL) to 600 mM (400 mL) TEAB. Fractions of 10 mL were analyzed by tic (11 :7:2 NH₄OH: isopropanol: water). Triphosphate (eluted @ 500 mM TEAB) containing fractions were combined and concentrated by rotary evaporator (bath < 25°C). The resulting solid was reconstituted in DI water (10 mL) and concentrated by lyophilization.

215

Following the General Procedure for making triphosphate, (2R,3R,4R,5R)-4-fluoro-5-(5-fluoro- 2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3-hydroxy-4-methyltetrahydrofuran-2-yl)methyl triphosphate (19 mg, 16% yield) was obtained as tris-triethylammonium salt as a white solid.

1H NMR (400 MHz, D₂0) δ 8.09 (d, J= 6.4 Hz, 1H), 6.24 (d, J= 18.8 Hz, 1H), 4.44 (dd, J = 12.0, 3.2 Hz, 1H), 4.35 (dd, J= 11.6, 6.4 Hz, 1H), 4.29 - 4.21 (m, 2H), 3.22 (q, J= 7.2 Hz, 18 H), 1.43 (d, J = 23.2 Hz, 3H), 1.29 (t, J = 7.6 Hz, 27H).

216 Following the General Procedure for making triphosphate, sodium ((2R,3R,4R,5R)-5-(2-amino- 6-oxo- 1 H-purin-9(6H)-yl)-4-fluoro-3 -hydroxy-4-methyltetrahydrofuran-2-yl)methyl

triphosphate (29 mg, 36% yield) was obtained the tetra-sodium salt as a white solid.

1H NMR (400 MHz, D₂0) δ 8.07 (s, 1H), 6.21 (d, J

1H), 4.41 - 4.26 (m, 3H), 1.25 (d, J= 23.2 Hz, 3H).

LCMS CiiHieFNsOisPs [M-H⁺]; calculated: 538.0, found 537.9.

217

Following the General Procedure for making triphosphate, triethylammonium ((2S,4R,5R)-5- (2,4-dioxo-3 ,4-dihydropyrimidin- 1 (2H)-yl)-4-fluorotetrahydrofuran-2-yl)methyl triphosphate (15 mg, 18.52% yield) was obtained as a white solid.

1H NMR (300 MHz, D₂0) δ: 7.95 (d, J = 6.0 Hz, 1H), 6.0 (d, J = 9.0 Hz, 1H), 5.88 (d, J = 6.0 Hz, 1H), 5.33 (dd, J = 27.0 and 3.0 Hz, 1H), 4.66 (m, 1H), 4.27 (m, 2H), 3.16 (q, J= 6.0 and 3.0 Hz, 2H), 2.27 (m, 4H), 1.24 (t, J= 3.0 Hz,3H).

HRMS calcd for [M-H]⁺ C₉Hi₃FN₂0i3P3 468.96149, found 468.96169.

218

The triphosphate was prepared by following the general procedure as its free acid. ³¹P NMR (162 MHz, Methanol-^) δ -12.54 (d, J= 25.1 Hz), -13.33 (d, J= 25.0 Hz), -24.84 (t, J = 25.1 Hz). HRMS CiiHi₅FN₄0i2P3 [M-H⁺ alculated: 506.98835, found: 506.98888.

219

Following the General Procedure for making triphosphates, ((2R,3R,4R,5R)-5-(4-amino-5- fluoro-2-oxopyrimidin- 1 (2H)-yl)-4-fluoro-3 -hydroxy-4-methyltetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate (23 mg, 17% ) was obtained as a white solid as a tris- triethylammonium salt.

1H NMR (400 MHz, D₂0) δ 8.04 (d, J = 6.0 Hz, 1H), 6.24 (d, J = 18.8 Hz, 1H), 4.46 - 4.15 (m, 4H), 3.20 (q, J= 7.2 Hz, 18H), 1.36 (d, J= 23.2 Hz, 3H), 1.28 (t, J= 7.2 Hz, 27 H).

P NMR (121 MHz, D₂0) δ -9.59 (d, J= 19.8 Hz, γΡ), -10.66 (d, J= 20.2 Hz, P), -22.24 (t, J

19.8 Hz, βΡ).

LCMS C₂₄Hi9F₂N₂0₇ [M-H⁺]; calculated: 486.1, found 485.1.

Following the General Procedure for making triphosphates, triethylammonium ((2S,4R,5R)-5- (2,4-dioxo-3 ,4-dihydropyrimidin- 1 (2H)-yl)-4-fluoro-4-methyltetrahydrofuran-2-yl)methyl dihydrogen triphosphate (14 mg, 25% yield) was obtained as its bis-triethylammonium salt. 1H NMR (CD₃OD, 400 MHz) δ 7.69 - 7.54 (m, 1H), 6.12 (d, J = 19.2 Hz, 1H), 5.67 (d, J = 8.1 Hz, 1H), 4.71 (m, 1H), 4.28 - 3.97 (m, 2H), 3.13 (q, J= 7.2 Hz, 12H), 2.46 - 2.21 (m, 2H), 1.51 (d, J= 21.7 Hz, 3H), 1.36 - 1.18 (t, J= 7.2 Hz, 18H).

LC-MS: [M-H]⁺ = 482.8.

221

Following the General Procedure for making triphosphates, 60 mg of the triphosphate as triethylamine salt was made, which was dissolved in 1.3 mL HPLC grade MeOH and it was transferred to centrifuge vials containing 1 M Nal solution in acetone. After centrifuge, the supernatant was decanted and the residue was repeatedly washed with acetone three more times. Then the white precipitate was dissolved in water, concentrated in vacuo by rotovap and further lyophilized to afford ((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-4-fluoro-3- hydroxy-4-methyltetrahydrofuran-2-yl)methyl triphosphate (39 mg, 11% ) as a white solid as four sodium salt.

1H NMR (400 MHz, D₂0) δ 7.95 (d, J = 8.0 Hz, 1H), 6.25 (d, J = 19.2 Hz, 1H), 5.99 (d, J = 8.0 Hz, 1H), 4.38 - 4.17 (m, 4H), 1.41 (d, J= 23.2 Hz, 3H).

³¹P NMR (121 MHz, D₂0) δ -7.42 (d, J= 18.8 Hz, γΡ), -10.46 (d, J= 20.3 Hz, P), -21.66 (t, J = 20.8 Hz, βΡ). Example 28.

Synthesis of (R)-2,2,2-trifluoro-N-(l-hydroxyoctadecan-2-yl)acetamide

wo s eps 225

Phytosphingosine (15.75 mmol) was dissolved in EtOH (0.5M) and ethyl trifluoroacetate (15.75 mmol) was added dropwise. NEt₃ (24.41 mmol) was added next the reaction mixture stirred overnight. The solvent was removed in vacuo and the residue was taken up in EtOAc and brine, washed, dried and concentrated. The crude material that was a white powder was good enough to use in the next step without further purification. Characterization matched literature: Synthesis, 2011, 867.

_H

The primary alcohol (15.75 mmol), DMAP (1.575 mmol) and NEt₃ (39.4 mmol) were dissolved in CH₂C1₂ and DMF (0.18M) mixture and cooled to 0°C. TBDPSC1 (19.69 mmol) was added dropwise then the solution was allowed to warm to room temperature and stirred overnight. NH₄CI solution was added to quench. The reaction mixture was extracted with EtOAc and the combined organic layers were washed with water (x2) to remove DMF. It was then dried and concentrated. A column was run to purify the mixture. 10-20% EtO Ac/Hex. Characterization matched literature: Synthesis, 2011, 867.

The diol (12.58 mmol), triphenylphosphine (50.3 mmol) and imidazole (50.03 mmol) were dissolved in toluene and reheated to reflux. The iodine (37.7 mmol) was then added slowly and the reaction mixture continued to be stirred at reflux. After three hours it was cooled to room temperature and 1 equivalent of iodine (12.58 mmol) was added followed by 8 equivalents of 1.5M NaOH (100.64 mmol). The reaction mixture was stirred until all the solids dissolved. The aqueous layer was removed in a separatory funnel and the organic layer was washed with Na₂S₂0₃ solution then NaHC0₃ solution then brine. It was dried and concentrated. A column was run to purify the mixture 0-20% EtO Ac/Hex and a mixture of cis and trans was obtained but carried on to the next step. δ 1H NMR (400 MHz, Chloroform-^ ) δ 7.64 (ddt, J = 7.8, 3.8, 1.7 Hz, 4H), 7.51 - 7.35 (m, 6H), 6.68 (dd, J = 16.0, 8.2 Hz, 1H), 5.6 - 5.40 (m, 2H), 4.57 - 4.46 (m, 1H), 3.84 - 3.62 (m, 2H), 2.04 (q, J= 7.0 Hz, 1H), 1.28-1.21 (m, 24H), 1.15 - 0.98 (m, 9H), 0.90 (t, J= 6.8 Hz, 3H).

HRMS : 617.38759.

The alkene (2.91 mmol) was dissolved in MeOH (0.1M) and Pd(OH)2_/C (0.146 mmol) was added. A Parr Hydrogenator was used at 40 psi. The palladium catalyst was carefully filtered off through celite and rinsed with EtOAc. The crude material was used in the next step and provided quantitative yield.

The silyl ether was dissolved in THF and cooled to 0°C then TBAF was added dropwise. After stirring for 1 hour it was warmed to room temperature. After two hours NH4C1 solution was added and it was extracted with EtOAc, washed with brine and dried and concentrated. A column was run 10-50% EtO Ac/Hex.

1H NMR (400 MHz, Chloroform-;/) δ 7.60 (tt, J = 7.0, 1.5 Hz, 2H), 7.48 - 7.33 (m, 4H), 3.73 - 3.61 (m, 1H), 1.24 (d, J= 3.5 Hz, 18H), 1.05 (s, 6H), 0.86 (t, J= 6.8 Hz, 3H). HRMS : 381.28546.

Example 29.

Synthesis of 2-Amino-Octadecyl-Tenofovir-5'-Monophosphate Conjugates

Tenofovir (0.149 mmol) and the alcohol (0.149 mmol) were dissolved in pyridine (0.05 M) and trisyl chloride (0.448 mmol) was added. It was stirred at 50°C overnight. The solvent was removed in vacuo and the crude material was purified by column using 5%-50% MeOH/NH₄OH/CHCl₃.

1H NMR (400 MHz, Methanol-^) δ 8.3 (s, 1H), 8.2 (s, 1H), 4.4-3.2 (m, 8H), 1.6 (m, 2H), 1.4- 1.1 (m, 31H), 0.9 (t, 3H).

HRMS: 650.35324.

The phosphate was dissolved in MeOH (0.05M) and NH₄OH in a pressure tube and stirred at 40 °C overnight. This reaction was very stubborn and on more than one occasion the reaction was not complete after 24 hours. In those cases it was resubjected for another 24 hours.

1H NMR (300 MHz, Methanol-^) δ 8.31 (d, J = 5.4 Hz, 1H), 8.21 (d, J

(m, 10H), 1.63 - 1.52 (m, 2H), 1.4-1.0 (m, 31H), 0.87 (t, J= 6.3 Hz, 3H).

HRMS : 554.37094.

Example 30.

Synthes '-Fluo -2'-Methyluridine-5'- -Monophosphate Conjugate

Compound 228 was prepared following literature procedure (Bioorganic & Medicinal Chemistry 20 (2012) 3658-3665).

Compound 229: To a solution of IH-tetrazole (0.415 g, 5.92 mmol) in 25 ml ether and 10 ml acetonitrile diisopropylamine (0.93 ml, 0.726 g, 7.03 mmol) was added. The precipitate was filtered off, washed with ether and dried under vacuum to give diisopropylammonium tetrazolide.

Compound 230: 3-(hexadecyloxy)propan-l-ol (0.301 g, 1.00 mmol) and diisopropylammonium tetrazolide (0.115 g, 0.67 mmol) were coevaporated with DCM-AcCN mixture(10: 10) 3 times. Dried mixture was dissolved in 7 ml DCM and added 3- ((bis(diisopropylamino)phosphino)oxy)propanenitrile (0.673 ml, 2.120 mmol). After 1 hour stirring at room temperature 1 ml methanol was added and stirred for 15 minutes. Then reaction was concentrated under vacuum; diluted with 10%TEA solution in EtOAc (100 ml) and washed with 10%NaHCO3 solution (2 x 50 ml) and water (2 x 50 ml); dried over anhydrous MgS04; filtered off and evaporated. The crude product was purified with column chromatography using Hexanes : EtO Ac : TEA (10:4:0.5). 1H-NMR: 3.89-3.54 (m, 6H); 3.49 (t, 2H, J=6.4 Hz); 3.39 (t, 2H, J=6.4Hz); 2.63 (dt, 2H, J=1.6, 6.4 Hz); 1.89-1.83 (m, 1H); 1.57-1.51 (m, 1H); 1.24 (s, 24H); 1.19-1.16 (m, 16H); 0.87 (t, 3H, J=6.4 Hz). Compound 231, 232, 233: The amidophosphite (compound 3) (0.114 g, 0.228 mmol) and 1- ((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)-3-methyltetrahydrofuran-2- yl)pyrimidine-2,4(lH,3H)-dione (0.065 g, 0.248 mmol) were dried by coevaporating with anhydrous DCM (4 x 10 ml), dissolved in 4 ml DCM and a solution of 3% lH-tetrazole in acetonitrile (0.75 ml, 0.320 mmol) was added. After 1 hour stirring at room temperature 5.5 M solution of tert-butyl peroxide (0.25 ml, 1.359 mmol) was added. After 40 minutes the solvents were evaporated and reaction mixture was dissolved in toluene and 1 ml TEA was added. It was stirred for 5 hours. All the solvents were evaporated and coevaporated with toluene (2 x 2 ml). Crude material was purified with column chromatography starting with CHC13 and increased the polarity slowly with CHC13:MeOH:NH40H (75:25:5) to give compound 319.

1H-NMR: (CDC13-CD30D, 3: 1) 1.024 (t, 3H, 6.4 Hz); 1.35-1.49 (m, 27H); 1.55 (d, 2H, 6.0 Hz); 1.63-1.70 (m, 2H); 2.011-2.067 (m, 2H); 3.47-3.49 (m, 2H); 3.54 (q; 2H, J=4.8 Hz); 3.65- 3.70 (m, 2H); 4.07-4.20 (m, 3H); 4.23-4.42 (m, 2H); 5.99 (d, 1H, J=8.4 Hz); 6.32 (dd, 1H, J=19.2, 5.2 Hz); 8.125 (t, 1H, J=8.0 Hz).

31 P-NMR: (CDC13-CD30D, 3: 1) 17.95 ppm.

HRMS: 623.34722. Example 31.

Synthesis of Cyclobutyl Phosphonate Analogs

234 235 236 237

236

A 2.4 eq portion of N-butyllithium (1.6 M in hexane, 243 mL) was added dropwise to a solution containing 2.4 eq of methyl methylsulfinyl methylsulfide (41 mL, 0.39 mmol) in 400 mL of tetrahydrofuran at -10 °C. The reaction mixture was stirred at -10 °C for 2 hours and then cooled to -78 °C. The yellow reaction mixture was maintained at -78 °C as a 1 eq portion of the ((1,3- dibromopropan-2-yloxy)methyl)benzene (50 g, 0.16 mmol) in 85 mL of tetrahydrofuran was added dropwise. The reaction mixture was allowed to warm to room temperature overnight. The reaction mixture was added to brine and extracted twice with ethyl acetate. The combined organic layers were subject to the usual work up to provide 60 mL of dark red-brown liquid. This mixture of syn- and anti-dithioketal S-oxide intermediates was purified in three portions via silica gel column chromatography. Less polar impurities were eluted first with 3:7 ethyl acetate: hexane followed by elution of product with pure ethyl acetate. A total of 23.8 g of intermediate was obtained in this manner. The syn- and anti-dithioketal S-oxide intermediates (23.8 g, 0.09 mol) were dissolved in 600 mL of diethyl ether and treated with 34 mL of 35% perchloric acid. After overnight stirring, the reaction mix was neutralized with sodium bicarbonate followed by purification via silica gel column chromatography (15:85 ethyl acetate: hexane) which provided the ketone 367 (11.8 g, 41% yield) as an orange-yellow liquid:

1H NMR (400MHz, CDC1₃) δ 3.29 (m, 4H), 4.35-4.42 (m, 1H), 4.53 (s, 2H), 7.30-7.40 (m, 5H).

Under argon a solution of lithium tri-sec-butylboranuide (1.0 M in THF, 23.5 mL, 0.023 mmol) in THF was added dropwise over a period of 10 minutes to a stirred solution of butanone (4.0 g, 0.02 mmol) in dry THF (20 mL) at -78 °C. The product was then allowed to warm up to r.t. and saturated NaHC0₃ (8 mL) was added over a period of 2 minutes. The resulting solution was then cooled and 30% aqueous hydrogen peroxide (3 mL) was added dropwise at such a rate so as to maintain the temperature at 25 - 30 °C. Finally, water and ethyl acetate were added. The organic layer was separated, washed with water, dried and evaporated under reduced pressure to give the crude alcohol, which was purified by flash column chromatography (ethyl acetate: hexane = 1 :4). The appropriate fractions were combined, and the solvent was removed in vacuo to give product 7 (3.8 g, 95% yield) as colorless oil. 1H NMR (400 MHz, CDC1₃) δ 1.90 - 1.98 (m, 2H), 2.68 - 2.75 (m, 2H), 2.63 (dt, 1H, J = 6.8 Hz), 3.90 (m, 1H), 4.42 (s, 2H), 7.20 - 7.34 (s, 5H).

To a 100 ml flask with czs-3-(benzyloxy)cyclobutanol (1.65 g, 9.47 mmol), 4-nitrobenzoic acid (3.16 g, 18.90 mmol), Ph₃P (5.21 g, 19.90 mmol) and dry THF 25 mL were added under argon. Then the reaction mixture was cooled to 0 °C and DIAD (3.9 mL, 20 mmol) was added drop by drop to give a yellow solution. This was allowed to warm up to room temperature gradually and was left stirring for 77 hours, after which time the solvent was removed and applied directly to a silica gel column (hexane: ethyl acetate = 20: 1) to give the desired product with a little impurity. The product was redissolved in 1,4-dioxane (6.6 mL) and was treated with aqueous NaOH (0.4 mol/L, 4.3 mL, 1.70 mmol) at room temperature. After 30 minutes, 0.07 mL of AcOH was added and the products were concentrated to small volume under reduced pressure. The residue was partitioned between EtOAc and saturated NaHC0₃. The organic phase was dried over MgS04, and the solvent was removed under reduced pressure to provide the crude product. This material was subjected to silica gel flash chromatography (Hexane: EtOAc = 3: 1) to give the product (1.04 g, 52%).

IR (cm ¹) 3000 (br), 1336 (s).

1H NMR (400 MHz, CDC1₃) δ 2.18 (m, 2H), 2.37 (m, 2H), 4.29 (m, 1H), 4.21 (s, 2H), 4.56 (m, 1H), 7.26 - 7.35 (m, 5H).

¹³C NMR (100 MHz CDC1₃) δ 39.7, 65.2, 70.5, 70.7, 127.9, 128.0, 128.6, 138.3.

HRMS (FAB) m/z 179.1064, calcd for CnHi₅0₂ 179.1067 (M⁺H).

237 To a 50 mL flask with trans-3-(benzyloxymethyl)cyclobutanol (66 mg, 0.37 mmol) inside, dry CH₂C1₂ (20 mL) was added to give a clear solution followed by the addition of Et₃N (0.26 mL, 1.9 mmol). After 10 minutes, the reaction mixture was cooled to 0 °C, and MsCl (0.04 mL, 0.5 mmol) was added dropwise. The resulting solution was allowed to stir and warm gradually to room temperature. After 3 hourrs, the reaction was quenched by adding H₂0. The organic phase was separated, washed with brine once, and dried over MgS0₄. Removal of the solvent afforded the crude product which was purified by flash column chromatography (ethyl acetate: hexane = 1 :4). The appropriate fractions were combined, and the solvent was removed in vacuo to give product (3.8 g, 95%) as colorless oil. IR Ccrn ¹) 1344 (s), 1171 (s).

1H NMR (400 MHz, CDC1₃) δ 2.54 (t, 4H, J = 5.6, 6.0 Hz), 2.99 (s, 3H), 4.31 (dt, 1H, J = 5.6, 5.0 Hz), 4.42 (s, 2H), 5.22 (m, 1H), 7.26 - 7.37 (m, 5H); ¹³C NMR (100 MHz, CDC1₃) δ 38.0, 69.9, 70.9, 73.8, 128.0, 128.1, 128.7, 137.7.

HRMS (FAB) m/z 257.0841, calcd for Ci₂Hi₇0₄Si 257.0842 (M⁺H).

238

To adenine (0.54 g, 4.00 mmol) was added under argon 10 mL of dry DMF followed by the mesylate (0.85 g, 3.32 mmol), 18-crown-6 (1.05 g, 4.00 mmol) and K₂C0₃ (0.55 g, 4.00 mmol). After stirring at 120 °C overnight, the mixture was cooled and extracted by EtOAc three times. After washing with water, the organic solvent was evaporated. The residue was then purified by flash column chromatography (CH₂C1₂: MeOH = 9: 1). The appropriate fractions were combined, and the solvent was removed in vacuo to give product (0.45 g, 46% yield) as white solid. Melting point: 161 - 162 °C.

IR (cm^"1) 3325 (br), 2472 (br), 2070, 1617, 1119, 972.

1H NMR (400 MHz, CD₃OD) δ 2.44-2.53 (m, 2H), 2.78-2.87 (m, 2H), 3.93 (m, 1H), 4.39 (s, 2H), 4.53 (m, 1H), 7.08 - 7.25 (m, 5H), 8.11 (s, 1H), 8.21 (s, 1H).

1³C NMR (100 MHz, CD₃OD) δ 39.4, 43.1, 44.4, 68.0, 71.8, 95.2, 77.8, 95.2, 103.2, 129.0, 129.3, 129.6, 142.2.

HRMS (FAB) m/z 296.1506, calcd for Ci₆Hi₈N₅0296.1508 (M⁺H).

239

In a 25 mL flask under argon containing 9-(czs-3-(benzyloxy)cyclobutyl)-9H-purin-6-amine (1.00 g, 3.40 mmol) was added anhydrous CH₂CI₂ (10 mL) resulting in a white emulsion. This flask was then cooled to -78 °C and after 10 mins, BC1₃ (1.0 M in CH₂CI₂, 17 mL, 17 mmol) was added dropwise. The reaction mixture was allowed to stir at -78 C for 6 hours and was then quenched by adding 7N NH₃ in MeOH (5 mL) dropwise. The mixture was concentrated under reduced pressure and was purified by silica gel flash chromatography (CH₂CI₂: MeOH = 5: 1) to give the desired product (500 mg, 75%) as white solid.

Melting point: 228 - 230 °C.

IR (cm^"1) 3325 (br), 2467, 2362, 2070, 1618, 1120, 957.

1H NMR (400 MHz, CD₃OD) δ 2.49-2.54 (m, 2H), 2.94-2.99 (m, 2H), 4.17 (m, 1H), 4.56 (m, 1H), 8.17 (s, 1H), 8.23 (s, 1H). ^1JC NMR (100 MHz, CD₃OD) δ 29.3, 41.1, 59.5, 70.0, 119.2, 140.1, 149.6, 152.1, 155.8.

HRMS (FAB) m/z 206.1034, calcd for C₉Hi₂0iN₅206.1036 (M⁺H).

Anal. Calcd for C₉HnOiN₅: C, 52.67; H, 5.40; N, 34.13. Found: C, 52.60; H, 5.50; N, 34.10.

240

To the cz^'5-3-(6-amino-9H-purin-9-yl) cyclobutanol (0.30 g, 1.46 mmol) under argon was added 10 mL of anhydrous DMF followed by l,l-diethoxy-N,N-dimethylmethanamine (0.66 g, 4.38 mmol). After stirring at r.t. overnight, the mixture was concentrated, and the residue was purified by flash column chromatography (CH₂C1₂: MeOH = 95:5). The appropriate fractions were combined, and the solvent was removed in vacuo to give product (430 mg, 94% yield) as white solid.

Melting point: 137 - 138 °C.

IR ^m ¹) 3329 (br), 3189 (br), 2920, 1648, 1601, 1100.

1H NMR (400MHz, CDC1₃) δ 2.51-2.60 (m, 2H), 2.93-3.10 (m, 2H), 3.10 (s, 3H), 3.13 (s, 1H), 4.20 (dt, 1H, J = 6.8, 7.2 Hz), 4.52 (dt, 1H, J = 8.0, 8.4 Hz), 7.89 (s, 1H), 8.42 (s, 1H), 8.85 (s, 1H).

¹³C NMR (100MHz, CDC1₃) δ 35.3, 41.1, 41.5, 42.2, 61.2, 70.6, 126.6, 140.8, 151.5, 152.3, 158.5, 159.9.

HRMS (FAB) m/z 257.0841, calcd for C₁₂H₁₇O₄S₁ 257.0842 (M⁺H).

242

A mixture of compound 373 (100 mg, 0.35 mmol) in anhydrous DMF (30 mL) was cooled to 0 °C and treated with sodium hydride (60%, 41.7 mg, 1.05 mmol). After 10 minutes, diethyl phosphonomethyl tosylate (270 mg, 1.05 mmol) was added. The reaction mixture was stirred at r.t. for 3 days and quenched by the addition of methanol and acetic acid. After the solvent was evaporated, the residue was purified by column chromatography on silica gel (EtOAc/MeOH 9: 1) to afford an intermediate nucleotide (260 mg, 58% yield) as white solid.

1H NMR (400MHz, CDC1₃) δ 1.34 (m, 6H), 2.58 (m, 2H), 2.05 (m, 2H), 3.75 (d, 2H, J = 9.0), 3.07 (m, 1H), 4.17 (m, 4H), 4.67 (m, 1H), 8.0 (s, 1H), 8.51 (s, 1H), 8.96 (s, 1H).

A mixture of the intermediate (45 mg, 0.13 mmol) and 60 mL 7 N ammonia in methanol were added to a sealed flask and stirred at 65 °C overnight. After the reaction mixture was cooled to r.t., the solvent was removed by reduced pressure to afford the title compound as a white solid (41 mg, 91% yield).

Melting point: 130-131 °C.

IR (cm^"1) 3334 (br), 3171 (br), 2978, 1643, 1596, 1240, 1018.

1H NMR (400 MHz, CDC1₃) δ 1.17 (m, 6H), 2.41-2.44 (m, 2H), 2.83-2.87 (m, 2H), 3.61 (d, 2H, J = 9.2 Hz), 3.88 (dt, 1H, J = 6.8, 7.2 Hz), 4.00 (m, 4H), 4.83 (m, 1H), 6.86 (s, 2H), 7.83 (s, 1H), 8.12 (s, 1H); ¹³C NMR (100 MHz, CDC1₃) δ 16.56, 16.61, 38.2, 40.6, 61.7, 62.7, 62.8, 63.3, 69.3, 69.4, 119.8, 138.6, 149.9, 152.9, 156.3, 164.1; HRMS (FAB) m/z 356.1480, calcd for Ci₄H₂₃0₄N₅P 356.1482 (M⁺H).

The above compound was dissolved in 10 mL anhydrous acetonitrile followed by the dropwise addition of trimethylsilyl bromide (0.17 mL, 1.3 mmol). After the mixture was stirred at r.t. overnight, ammonia hydroxide was added to quench the reaction. After evaporating the solvent, the residue was diluted by water and methanol, and separated by preparative HPLC with C-18 reverse phase column (99% water in methanol- 50% water in methanol 60 mins, retention time was 18 mins) to give the desired product (30 mg, 70%>).

Melting point: 130 - 131 °C.

IR (cm^"1) 3340, 3206, 1753, 1677, 1146, 957.

1H NMR (400 MHz, CDC1₃) δ 1.20 (s, 18H), 2.01-2.09 (m, 2H), 2.79-2.85 (m, 2H), 3.75 (d, 2H,

J = 8.8 Hz), 3.90 (dt, 1H, J = 6.8, 7.2 Hz), 4.57 (m, 1H), 5.69 (m, 6H), 7.49 (d, 2H, J = 6.0 Hz).

¹³C NMR (100 MHz, CDC1₃) δ 27.0, 37.4, 38.9, 43.4, 62.1, 63.8, 69.3, 69.5, 81.9, 81.9, 125.8,

126.1, 135.6, 138.1, 154.9, 157.7, 157.8, 177.1.

HRMS (FAB) m/z 300.0855, calcd for Ci₀Hi₅O₄N₅Pi 300.0856 (M⁺H).

Anal. Calcd for Ci₀Hi₅O₄N₅Pi + 0.3 H₂0: C, 39.43; H, 4.83; N, 22.99. Found: C, 39.24; H, 4.85;

N, 23.12.

To a solution of 28 (61 mg, 0.22 mmol) in a 4 ml of anhydrous DMF were added N,N- dicyclohexyl-4-morpholine carboxamidine (0.20 mg, 0.66 mmol) and chloromethyl pivalate (0.16 mL, 1.10 mmol). The heterogeneous mixture became homogeneous after 15 mins and was stirred at r.t. for 36 hours. After evaporating solvent, the residue was purified by silica gel chromatography eluting with 15% methanol in dichloromethane to give the product 4 (43 mg, 42% yield).

Melting point: 148 - 149 °C.

IR (cm^"1) 3329, 3165, 1747, 1642, 1590, 1251, 965.

1H NMR (400 MHz, CDC1₃) δ 1.23 (s, 12H), 2.60 (m, 2H), 3.02 (m, 2H), 3.83 (d, 2H, J = 8.8 Hz), 4.08 (m, 1H), 4.65 (m, 1H), 5.72 (dt, 1H, J = 5.2, 7.2 Hz), 5.90 (br, 2H), 8.00 (s, 1H), 8.32 (s, 1H).

¹³C NMR (100 MHz, CDC1₃) δ 27.1, 38.5, 39.0, 40.6, 62.2, 63.8, 69.5, 69.6, 81.9, 82.0, 120.1, 139.0, 150.3, 152.7, 155.4, 177.1.

HRMS (FAB) m/z 528.2210, calcd for C₂2H₃₅0₈N₅Pi 528.2218 (M⁺H).

Anal. Calcd for C22H34O8 5P1 : C, 50.09; H, 6.50; N, 13.28. Found: C, 50.01; H, 6.57; N, 13.33.

O O

HO OH

EtO AA OEt EtO I OEt

(CH₂)₁₅CH₃

(CH₂)i5CH₃

247

¾eagents and Conditions: (a) hexadecyl bromide, NaOEt, EtOH; (b) LAH, THF; (c) POCl₃, Et₃N, DCM; (d) Et₃N, MeCN, dioxane.

To 33.4 g sodium ethoxide solution (21% wt) in ethanol, diethyl malonate(15g) and then 1- bromohexadecane (31.5g) were added dropwise. After reflux for 8 hrs, ethanol was evaporated in vacuo. The remaining suspension was mixed with ice-water( 200 ml) and extracted with diethyl ether (3 X 200ml). the combined organic layers were dried over MgS04, filtered and the filtrate was evaporated in vacou to yield a viscous oil residue . This residue was purified by column chromatography(silica: 500 g) using hexane/diethyl ether( 12: 1) as mobile phase to yield the main compound.

2⁴⁸

In a 250 mL round-bottomed flask was aluminum lithium hydride (2.503 g, 66.0 mmol) in Diethyl ether (90 ml) to give a suspension. To this suspension was added diethyl 2- hexadecylmalonate (18.12 g, 47.1 mmol) dropwise and the reeaction was refluxed for 6 h. The reaction was followed up by TLC using PMA and H2S04 as drying agents. The excess lithium aluminium hydride was destroyed by 200ml of ice-water. 150 ml of 10 % H2S04 was added to dissolve aluminium hydrate. Th reaction mixture was extracted by diethyl ether (100 ml X 3). The organic layer including undissolved product was filtered. The collect solids were washed with ethyl acetate. The filtrate was dried over MgS04, filtered and concentrated under reduced pressure. The product was purified on silica (lOOg) column eluting with Hexane:EtOAC(3: l) to (1 : 1).

249

To a solution of 2-hexadecylpropane-l,3-diol (7.04 g, 23.43 mmol) in 100 ml of DCM was added dropwise phosphorous trichloride (3.59 g, 23.43 mmol) dissolved in 20 ml of DCM followed by triethylamine (6.53 ml, 46.9 mmol). The reaction was refluxed for one hour. TLC analysis showed that the starting paterial was consumed and two new spots formed. The mixture was concentrated to dryness, dissolved in dry diethyl ether and filtered. The filtrate was concentrated to yied the crude product (8.85 g) which was used in the next step without further purification.

A mixture of cyclobutyl phosphonate and lipid phosphochloridate in anhydrous acetonitrile/dioxane was treated with triethylamine. The reaction was allowed to stir at room temperature for 18h and then concentrated. The crude mixture was purified by column chromatography over silica gel (19 mm x 175 mm). Prior to loading the crude material, the column was washed with 5% methanol in DCM with 3% TEA followed by 5% methanol in DCM without 3% TEA. The crude material was loaded onto the column and eluted with a gradient from 5% to 10% methanol in DCM. Pooled pure containing fractions and concentrated. The obtained white solid was co-evaporated with acetone (3x) and then dried under high vacuum.

Example 32.

Synthesis of a Cyclobutyl Diphosphophosphonate Analog

250 252

Compound 383 (30.0 mg, 0.10 mmol) and tributylamine in water (10 mL) were mixed and the solvent was coevaporated under reduced pressure twice. The residue was repeatedly evaporated with anhydrous ethanol and toluene. To the resulting powder were added DMF (4 mL) and 1,1 '- carbonyldiimidazole (107 mg, 0.66 mmol). After stirring at r.t. for 3.5 hours, the reaction mixture was quenched by addition of methanol (40 mL). Bis(tri-n-butyl ammonium)- pyrophosphate (264.2 mg, 0.58 mmol) was added and the stirring was continued for 16 hours. The reaction was quenched by the addition of 5 mL 0.1 M TEAB, and this mixture was directly applied to the DEAE-sephadex dianion exchange column (11 mm X 220 mm, eluent from 0.1 M TEAB to 0.7 M TEAB). After analyzing the fractions by HPLC with C-18 reverse phase column (250 mm X 4.6 mm), all the products were collected and lyophilized to give the triethylammonium salt of 4'-0-diphosphate of (cis -3-(6-amino-9H-purin-9- yl)cyclobutoxy)methylphosphonate 385 ( 345 mg, 40% yield) as a white solid.

1H NMR (400MHz, CDC1₃) δ 1.06 (m, 44H), 2.32 (m, 2H), 2.95 (m, 30H), 3.61 (d, 2H, J = 8.8 Hz), 4.00 (dt, 1H, J = 6.8, 7.2 Hz), 4.34 (dt, 1H, J = 7.6, 9.2 Hz), 7.90 (s, 1H), 8.10 (s, 1H).

3¹P NMR (D₂0, 162 MHz): δ 8.4 (αΡ, d, IP, J = 48.9 Hz), -10.4 (γΡ, dd, IP, J = 22.0, 199.1 Hz), -2 (βΡ, dt, IP, J = 21.4, 20.1, 5.5 Hz).

HRMS (FAB) m/z 458.0033, calcd for Ci₀Hi₅Oi₀N₅P₃ 458.0026 (ΜΉ).

Example 33.

Assay Protocols

(1) Screening Assays for DENV, JEV, POWV, WNV, YFV, PTV, RVFV, CHIKV, EEEV, VEEV, WEEV, TCRV, PCV, JUNV, MPRLV

Primary cytopathic effect (CPE) reduction assay. Four-concentration CPE inhibition assays are performed. Confluent or near-confluent cell culture monolayers in 96-well disposable microplates are prepared. Cells are maintained in MEM or DMEM supplemented with FBS as required for each cell line. For antiviral assays the same medium is used but with FBS reduced to 2% or less and supplemented with 50 μg/ml gentamicin. The test compound is prepared at four logio final concentrations, usually 0.1, 1.0, 10, and 100 μg/ml or μΜ. The virus control and cell control wells are on every microplate. In parallel, a known active drug is tested as a positive control drug using the same method as is applied for test compounds. The positive control is tested with each test run. The assay is set up by first removing growth media from the 96-well plates of cells. Then the test compound is applied in 0.1 ml volume to wells at 2X concentration. Virus, normally at <100 50% cell culture infectious doses (CCID₅₀) in 0.1 ml volume, is placed in those wells designated for virus infection. Medium devoid of virus is placed in toxicity control wells and cell control wells. Virus control wells are treated similarly with virus. Plates are incubated at 37°C with 5% C0₂ until maximum CPE is observed in virus control wells. The plates are then stained with 0.011% neutral red for approximately two hours at 37°C in a 5% C0₂ incubator. The neutral red medium is removed by complete aspiration, and the cells may be rinsed IX with phosphate buffered solution (PBS) to remove residual dye. The PBS is completely removed and the incorporated neutral red is eluted with 50% Sorensen's citrate buffer/50% ethanol (pH 4.2) for at least 30 minutes. Neutral red dye penetrates into living cells, thus, the more intense the red color, the larger the number of viable cells present in the wells. The dye content in each well is quantified using a 96-well spectrophotometer at 540 nm wavelength. The dye content in each set of wells is converted to a percentage of dye present in untreated control wells using a Microsoft Excel computer-based spreadsheet. The 50%> effective (EC₅₀, virus-inhibitory) concentrations and 50% cytotoxic (CC₅₀, cell-inhibitory) concentrations are then calculated by linear regression analysis. The quotient of CC₅₀ divided by EC₅₀ gives the selectivity index (SI) value.

Secondary CPE/Virus yield reduction (VYR) assay. This assay involves similar methodology to what is described in the previous paragraphs using 96-well microplates of cells. The differences are noted in this section. Eight half-logio concentrations of inhibitor are tested for antiviral activity and cytotoxicity. After sufficient virus replication occurs, a sample of supernatant is taken from each infected well (three replicate wells are pooled) and held for the VYR portion of this test, if needed. Alternately, a separate plate may be prepared and the plate may be frozen for the VYR assay. After maximum CPE is observed, the viable plates are stained with neutral red dye. The incorporated dye content is quantified as described above. The data generated from this portion of the test are neutral red EC₅₀, CC₅₀, and SI values. Compounds observed to be active above are further evaluated by VYR assay. The VYR test is a direct determination of how much the test compound inhibits virus replication. Virus that was replicated in the presence of test compound is titrated and compared to virus from untreated, infected controls. Titration of pooled viral samples (collected as described above) is performed by endpoint dilution. This is accomplished by titrating logio dilutions of virus using 3 or 4 microwells per dilution on fresh monolayers of cells by endpoint dilution. Wells are scored for presence or absence of virus after distinct CPE (measured by neutral red uptake) is observed. Plotting the logio of the inhibitor concentration versus logio of virus produced at each concentration allows calculation of the 90% (one logio) effective concentration by linear regression. Dividing EC90 by the CC50 obtained in part 1 of the assay gives the SI value for this test.

(2) Screening Assays for Lassa fever virus (LASV)

Primary Lassa fever virus assay. Confluent or near-confluent cell culture monolayers in 12-well disposable cell culture plates are prepared. Cells are maintained in DMEM supplemented with 10%) FBS. For antiviral assays the same medium is used but with FBS reduced to 2% or less and supplemented with 1% penicillin/streptomycin. The test compound is prepared at four logio final concentrations, usually 0.1, 1.0, 10, and 100 μg/ml or μΜ. The virus control and cell control will be run in parallel with each tested compound. Further, a known active drug is tested as a positive control drug using the same experimental set-up as described for the virus and cell control. The positive control is tested with each test run. The assay is set up by first removing growth media from the 12-well plates of cells, and infecting cells with 0.01 MOI of LASV strain Josiah. Cells will be incubated for 90 min: 500 μΐ inoculum/Ml 2 well, at 37°C, 5% C02 with constant gentle rocking. The inoculums will be removed and cells will be washed 2X with medium. Then the test compound is applied in 1 ml of total volume of media. Tissue culture supernatant (TCS) will be collected at appropriate time points. TCS will then be used to determine the compounds inhibitory effect on virus replication. Virus that was replicated in the presence of test compound is titrated and compared to virus from untreated, infected controls. For titration of TCS, serial ten- fold dilutions will be prepared and used to infect fresh

monolayers of cells. Cells will be overlaid with 1% agarose mixed 1 : 1 with 2X MEM

supplemented with 10%>FBS and l%>penecillin, and the number of plaques determined. Plotting the logio of the inhibitor concentration versus logio of virus produced at each concentration allows calculation of the 90% (one logio) effective concentration by linear regression.

Secondary Lassa fever virus assay. The secondary assay involves similar methodology to what is described in the previous paragraphs using 12-well plates of cells. The differences are noted in this section. Cells are being infected as described above but this time overlaid with 1% agarose diluted 1 :1 with 2X MEM and supplemented with 2% FBS and 1%

penicillin/streptomycin and supplemented with the corresponding drug concentration. Cells will be incubated at 37oC with 5% C02 for 6 days. The overlay is then removed and plates stained with 0.05% crystal violet in 10% buffered formalin for approximately twenty minutes at room temperature. The plates are then washed, dried and the number of plaques counted. The number of plaques is in each set of compound dilution is converted to a percentage relative to the untreated virus control. The 50% effective (EC50, virus-inhibitory) concentrations are then calculated by linear regression analysis.

(3) Screening Assays for Ebola virus (EBOV) and Nipah virus (NIV)

Primary Ebola/Nipah virus assay. Four-concentration plaque reduction assays are performed. Confluent or near-confluent cell culture monolayers in 12-well disposable cell culture plates are prepared. Cells are maintained in DMEM supplemented with 10% FBS. For antiviral assays the same medium is used but with FBS reduced to 2% or less and supplemented with 1% penicillin/streptomycin. The test compound is prepared at four logio final concentrations, usually 0.1, 1.0, 10, and 100 μg/ml or μΜ. The virus control and cell control will be run in parallel with each tested compound. Further, a known active drug is tested as a positive control drug using the same experimental set-up as described for the virus and cell control. The positive control is tested with each test run. The assay is set up by first removing growth media from the 12-well plates of cells. Then the test compound is applied in 0.1 ml volume to wells at 2X concentration. Virus, normally at approximately 200 plaque forming units in 0.1 ml volume, is placed in those wells designated for virus infection. Medium devoid of virus is placed in toxicity control wells and cell control wells. Virus control wells are treated similarly with virus. Plates are incubated at 37°C with 5% C0₂ for one hour. Virus-compound inoculums will be removed, cells washed and overlaid with 1.6% tragacanth diluted 1 : 1 with 2X MEM and supplemented with 2% FBS and 1% penicillin/streptomycin and supplemented with the corresponding drug concentration. Cells will be incubated at 37°C with 5% C0₂ for 10 days. The overlay is then removed and plates stained with 0.05% crystal violet in 10% buffered formalin for approximately twenty minutes at room temperature. The plates are then washed, dried and the number of plaques counted. The number of plaques is in each set of compound dilution is converted to a percentage relative to the untreated virus control. The 50% effective (EC₅₀, virus-inhibitory) concentrations are then calculated by linear regression analysis. Secondary Ebola/NIpah virus assay with VYR component. The secondary assay involves similar methodology to what is described in the previous paragraphs using 12-well plates of cells. The differences are noted in this section. Eight half-logio concentrations of inhibitor are tested for antiviral activity. One positive control drug is tested per batch of compounds evaluated. For this assay, cells are infected with virus. Cells are being infected as described above but this time incubated with DMEM supplemented with 2% FBS and 1%

penicillin/streptomycin and supplemented with the corresponding drug concentration. Cells will be incubated for 10 days at 37°C with 5% C0₂, daily observed under microscope for the number of green fluorescent cells. Aliquots of supernatant from infected cells will be taken daily and the three replicate wells are pooled. The pooled supernatants are then used to determine the compounds inhibitory effect on virus replication. Virus that was replicated in the presence of test compound is titrated and compared to virus from untreated, infected controls. For titration of pooled viral samples, serial ten-fold dilutions will be prepared and used to infect fresh monolayers of cells. Cells are overlaid with tragacanth and the number of plaques determined. Plotting the logio of the inhibitor concentration versus logio of virus produced at each

concentration allows calculation of the 90% (one logio) effective concentration by linear regression.

Anti-Dengue Virus Cytoprotection Assay:

Cell Preparation -BHK21 cells (Syrian golden hamster kidney cells, ATCC catalog #

CCL-I 0) were passaged in DMEM supplemented with 10% FBS, 2 mM L-glutamine,100 U/mL penicillin, and 100 μg/mL streptomycin in T-75 flasks prior to use in the antiviral assay. On the day preceding the assay, the cells were split 1 :2 to assure they were in an exponential growth phase at the time of infection. Total cell and viability quantification was performed using a hemocytometer and Trypan Blue dye exclusion. Cell viability was greater than 95% for the cells to be utilized in the assay. The cells were resuspended at 3 x 10³ cells per well in tissue culture medium and added to flat bottom microtiter plates in a volume of 100 μί. The plates were incubated at 37°C/5%>C0₂ overnight to allow for cell adherence. Monolayers were observed to be approximately 70% confluent.

Virus Preparation-The Dengue virus type 2 New Guinea C strain was obtained from

ATCC (catalog# VR-1584) and was grown in LLC-MK2 (Rhesus monkey kidney cells; catalog #CCL-7.1) cells for the production of stock virus pools. An aliquot of virus pretitered in BHK21 cells was removed from the freezer (-80°C) and allowed to thaw slowly to room temperature in a biological safety cabinet. Virus was resuspended and diluted into assay medium (DMEM supplemented with 2% heat-inactivated FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin) such that the amount of virus added to each well in a volume of 100 was the amount determined to yield 85 to 95% cell killing at 6 days post-infection.

Plate Format-Each plate contains cell control wells (cells only), virus control wells (cells plus virus), triplicate drug toxicity wells per compound (cells plus drug only), as well as triplicate experimental wells (drug plus cells plus virus).

Efficacy and Toxicity XTT-Following incubation at 37°C in a 5% C0₂ incubator, the test plates were stained with the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5- [(phenylamino)carbonyl]-2H-tetrazolium hydroxide). XTT -tetrazolium was metabolized by the mitochondrial enzymes of metabolically active cells to a soluble formazan product, allowing rapid quantitative analysis of the inhibition of virus-induced cell killing by antiviral test substances. XTT solution was prepared daily as a stock of 1 mg/mL in RPMI 1640. Phenazine methosulfate (PMS) solution was prepared at 0.15mg/mL in PBS and stored in the dark at -20°C. XTT/PMS stock was prepared immediately before use by adding 40 of PMS per ml of XTT solution. Fifty microliters ofXTT/PMS was added to each well of the plate and the plate was reincubated for 4 hours at 37°C. Plates were sealed with adhesive plate sealers and shaken gently or inverted several times to mix the soluble formazan product and the plate was read

spectrophotometrically at 450/650 nm with a Molecular Devices Vmax plate reader.

Data Analysis -Raw data was collected from the Softmax Pro 4.6 software and imported into a Microsoft Excel spreadsheet for analysis. The percent reduction in viral cytopathic effect compared to the untreated virus controls was calculated for each compound. The percent cell control value was calculated for each compound comparing the drug treated uninfected cells to the uninfected cells in medium alone.

Anti-RSV Cytoprotection Assay:

Cell Preparation-HEp2 cells (human epithelial cells, A TCC catalog# CCL-23) were passaged in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin 1 mM sodium pyruvate, and 0.1 mM NEAA, T-75 flasks prior to use in the antiviral assay. On the day preceding the assay, the cells were split 1 :2 to assure they were in an exponential growth phase at the time of infection. Total cell and viability quantification was performed using a hemocytometer and Trypan Blue dye exclusion. Cell viability was greater than 95% for the cells to be utilized in the assay. The cells were resuspended at 1 x 10⁴ cells per well in tissue culture medium and added to flat bottom microtiter plates in a volume of 100 μΐ_^. The plates were incubated at 37°C/5% C0₂ overnight to allow for cell adherence.

Virus Preparation -The RSV strain Long and RSV strain 9320 were obtained from ATCC (catalog# VR-26 and catalog #VR-955, respectively) and were grown in HEp2 cells for the production of stock virus pools. A pretitered aliquot of virus was removed from the freezer (- 80°C) and allowed to thaw slowly to room temperature in a biological safety cabinet. Virus was resuspended and diluted into assay medium (DMEMsupplemented with 2% heat-inactivated FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM sodium pyruvate, and 0.1 mM NEAA) such that the amount of virus added to each well in a volume of 100 was the amount determined to yield 85 to 95% cell killing at 6 days post-infection. Efficacy and Toxicity XTT -Plates were stained and analyzed as previously described for the Dengue cytoprotection assay.

Anti-Influenza Virus Cytoprotection Assay:

Cell Preparation-MOCK cells (canine kidney cells, ATCC catalog# CCL-34) were passaged in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin 1 mM sodium pyruvate, and 0.1 mM NEAA, T-75 flasks prior to use in the antiviral assay. On the day preceding the assay, the cells were split 1 :2 to assure they were in an exponential growth phase at the time of infection. Total cell and viability quantification was performed using a hemocytometer and Trypan Blue dye exclusion. Cell viability was greater than 95%> for the cells to be utilized in the assay. The cells were resuspended at 1 x 10⁴ cells per well in tissue culture medium and added to flat bottom microtiter plates in a volume of 100 μί. The plates were incubated at 37°C/5% C0₂ overnight to allow for cell adherence.

Virus Preparation-The influenza A/PR/8/34 (A TCC #VR-95), A/CA/05/09

(CDC),A/NY/18/09 (CDC) and A/NWS/33 (ATCC #VR-219) strains were obtained from ATCC or from the Center of Disease Control and were grown in MDCK cells for the production of stock virus pools. A pretitered aliquot ofvirus was removed from the freezer (-80°C)and allowed to thaw slowly to room temperature in a biological safety cabinet. Virus was resuspended and diluted into assay medium (DMEM supplemented with 0.5%BSA, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM sodium pyruvate, 0.1 mM NEAA, and 1 μg/ml TPCK-treated trypsin) such that the amount of virus added to each well in a volume of 100 was the amount determined to yield 85 to 95% cell killing at 4 days post-infection. Efficacy and Toxicity XTT-Plates were stained and analyzed as previously described for the Dengue cytoprotection assay.

Anti-Hepatitis C Virus Assay:

Cell Culture -The reporter cell line Huh-luc/neo-ET was obtained from Dr. Ralf

Bartenschlager (Department of Molecular Virology, Hygiene Institute, University of Heidelberg, Germany) by ImQuest Biosciences through a specific licensing agreement. This cell line harbors the persistently replicating l₃₈₉luc-ubi-neo/NS3-37ET replicon containing the firefly luciferase gene-ubiquitin-neomycin phosphotransferase fusion protein and EMCV IRES driven NS3-5B HCV coding sequences containing the ET tissueculture adaptive mutations (E1202G, T12081, and K1846T). A stock culture of the Huh-luc/neo-ET was expanded by culture in DMEM supplemented with I 0%> FCS, 2mM glutamine, penicillin (100 μυ/mLy streptomycin (100 μg/mL) and I X nonessential amino acids plus 1 mg/mL G418. The cells were split 1 :4 and cultured for two passages in the same media plus 250 μg/mL G418. The cells were treated with trypsin and enumerated by staining with trypan blue and seeded into 96-well tissue culture plates at a cell culture density 7.5 x 10³ cells per well and incubated at 37°C 5% C0₂ for 24 hours.

Following the 24 hour incubation, media was removed and replaced with the same media minus theG418 plus the test compounds in triplicate. Six wells in each plate received media alone as a no-treatment control. The cells were incubated an additional 72 hours at 37°C 5%C0₂ then anti- HCV activity was measured by luciferase endpoint. Duplicate plates were treated and incubated in parallel for assessment of cellular toxicity by XTT staining.

Cellular Viability-The cell culture monloyers from treated cells were stained with the tetrazolium dye XTT to evaluate the cellular viability of the Huh-luc/neo-ET reporter cell line in the presence of the compounds.

Measurement of Virus Replication-HCV replication from the replicon assay system was measured by luciferase activity using the britelite plus luminescence reporter gene kit according to the manufacturer's instructions (Perkin Elmer, Shelton, CT). Briefly, one vial of britelite plus lyophilized substrate was solubilized in 10 mL of britelite reconstitution buffer and mixed gently by inversion. After a 5 minute incubation at room temperature, the britelite plus reagent was added to the 96 well plates at 100 μΐ, per well. The plates were sealed with adhesive film and incubated at room temperature for approximately 10 minutes to lyse the cells. The well contents were transferred to a white 96-well plate and luminescence was measured within 15 minutes using the Wallac 1450Microbeta Trilux liquid scintillation counter. The data were imported into a customized Microsoft Excel 2007 spreadsheet for determination of the 50% virus inhibition concentration (EC₅₀). Anti-Parainfluenza-3 Cytoprotection Assay:

Cell Preparation- HEp2 cells (human epithelial cells, ATCC catalog# CCL-23) were passagedin DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL

penicillin, 100 μg/mL streptomycin 1 mM sodium pyruvate, and 0.1 mM NEAA, T-75 flasksprior to use in the antiviral assay. On the day preceding the assay, the cells were splitl :2 to assure they were in an exponential growth phase at the time of infection.

Total cell and viability quantification was performed using a hemocytometer and Trypan Blue dye exclusion. Cell viability was greater than 95% for the cells to be utilized in the assay. The cells were resuspended at 1 x 10⁴cells per well in tissue culture medium and addedto flat bottom microtiter plates in a volume of 100 μί. The plates were incubated at 37°C/5% C0₂ overnight to allow for cell adherence.

Virus Preparation - The Parainfluenza virus type 3 SF4 strain was obtained from ATCC (catalog# VR-281) and was grown in HEp2 cells for the production of stock virus pools.

A pretitered aliquot of virus was removed from the freezer (-80°C) and allowed to thaw slowlyto room temperature in a biological safety cabinet. Virus was resuspended and dilutedinto assay medium (DMEM supplemented with 2% heat-inactivated FBS, 2 mM L- glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin) such that the amount of virus added to each well in a volume of 100 μΐ, was the amount determined to yield 85 to 95% cell killing at 6 days post-infection.

Plate Format - Each plate contains cell control wells (cells only), virus control wells (cellsplus virus), triplicate drug toxicity wells per compound (cells plus drug only), as well as triplicate experimental wells (drug plus cells plus virus). Efficacy and Toxicity XTT- Following incubation at 37°C in a 5% C0₂incubator, thetest plates were stained with the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H- tetrazolium hydroxide). XTT-tetrazoliumwas metabolized by the mitochondrial enzymes of metabolically active cells to a solubleformazan product, allowing rapid quantitative analysis of the inhibition of virus-inducedcell killing by antiviral test substances. XTT solution was prepared daily as a stock of lmg/mL in RPMI1640. Phenazine methosulfate (PMS) solution was prepared at 0.15mg/mL in PBS and stored in the dark at - 20°C. XTT/PMS stock was prepared

immediatelybefore use by adding 40 ofPMS per ml ofXTT solution. Fiftymicroliters ofXTT/PMS was added to each well of the plate and the plate wasreincubated for 4 hours at 3 7°C. Plates were sealed with adhesive plate sealers andshaken gently or inverted several times to mix the soluble fomlazan product and the platewas read spectrophotometrically at 450/650 nm with a Molecular Devices Vmax platereader.

Data Analysis - Raw data was collected from the Softmax Pro 4.6 software and imported intoa Microsoft Excel spreadsheet for analysis. The percent reduction in viral cytopathic effect compared to the untreated virus controls was calculated for each compound. The percentcell control value was calculated for each compound comparing the drug treated uninfected cells to the uninfected cells in medium alone. Influenza Polymerase Inhibition Assay:

Virus Preparation - Purified influenza virus A/PR/8/34 (1 ml) was obtained from

Advanced Biotechnologies, Inc. (Columbia, MD), thawed and dispensed into five

aliquots for storage at -80°C until use. On the day of assay set up, 20 of 2.5% Triton

N-101 was added to 180 of purified virus. The disrupted virus was diluted 1 :2 in a solution containing 0.25% Triton and PBS. Disruption provided the source of influenza ribonucleoprotein (RNP) containing the influenza RNA-dependent RNA polymerase and template RNA. Samples were stored on ice until use in the assay.

Polymerase reaction - Each 50 polymerase reaction contained the following: 5 μΐ_^ of the disrupted RNP, 100 mM Tris-HCl (pH 8.0), 100 mM KC1, 5 mM MgCl₂. 1 mM

dithiothreitol, 0.25% Triton N- 101 , 5 μ( ϊ of [a-³²P] GTP, 100 μΜ ATP, 50 μΜ each

(CTP, UTP), 1 μΜ GTP, and 200 μΜ adenyl (3'-5') guanosine. For testing the inhibitor, the reactions contained the inhibitor and the same was done for reactions containing the positive control (2'-Deoxy-2'-fluoroguanosine-5'-triphosphate). Other controls included RNP +reaction mixture, and RNP + 1% DMSO. The reaction mixture without the ApG primer and NTPs was incubated at 30°C for 20 minutes. Once the ApG and NTPs were added to the reaction mixture, the samples were incubated at 30°C for 1 hour then

immediately followed by the transfer of the reaction onto glass-fiber filter plates and subsequent precipitation with 10% trichloroacetic acid (TCA ). The plate was then

washed five times with 5% TCA followed by one wash with 95% ethanol. Once the filter had dried, incorporation of [a-³²P] GTP was measured using a liquid scintillation counter (Micro beta).

Plate Format - Each test plate contained triplicate samples of the three compounds (6 concentrations) in addition to triplicate samples of RNP + reaction mixture (RNP alone), RNP + 1% DMSO, and reaction mixture alone (no RNP).

Data Analysis - Raw data was collected from the Micro Beta scintillation counter. The incorporation of radioactive GTP directly correlates with the levels of polymerase

activity. The "percent inhibition values" were obtained by dividing the mean value of each test compound by the RNP + 1% DMSO control. The mean obtained at each

concentration of 2DFGTP was compared to the RNP + reaction control. The data was then imported into Microsoft Excel spreadsheet to calculate the IC₅₀ values by linear regression analysis.

HCV Polymerase Inhibition Assay:

Activity of compounds for inhibition of HCV polymerase was evaluated using methods previously described (Lam eta!. 2010. Antimicrobial Agents and Chemotherapy

54(8):3187-3196). HCV NS5B polymerase assays were performed in 20 volumes in 96 well reaction plates. Each reaction contained 40 ng/μΕ purified recombinant

NS5BA22 genotype-lb polymerase, 20 ng/μΕ of HCV genotype-lb complimentary IRES template, 1 μΜ of each of the four natural ribonucleotides, 1 U/mL Optizyme RNAse inhibitor (Promega, Madison, WI), 1 mM MgCl₂, 0.75 mM MnCl₂, and 2 mM

dithiothreitol (DTT) in 50 mM HEPES buffer (pH 7.5). Reaction mixtures were

assembled on ice in two steps. Step 1 consisted of combining all reaction components except the natural nucleotides and labeled UTP in a polymerase reaction mixture. Ten micro liters(10 μί) of the polymerase mixture was dispensed into individual wells of the 96 well reaction plate on ice. Polymerase reaction mixtures without NS5B polymerase were included as no enzyme controls. Serial half-logarithmic dilutions of test and control compounds, 2^*-0-Methyl-CTP and 2^*-0-Methyl-GTP (Trilink, San Diego, CA), were prepared in water and 5 μΐ, of the serial diluted compounds or water alone (no compound control) were added to the wells containing the polymerase mixture. Five microliters of nucleotide mix (natural nucleotides and labeled UTP) was then added to the reaction plate wells and the plate was incubated at 27°C for 30 minutes. The reactions were quenched with the addition of 80 stop solution (12.5 mM EDTA, 2.25 M NaCl, and 225 mM sodium citrate) and the R A products were applied to a Hybond-N+ membrane (GE Healthcare, Piscataway, N.J) under vacuum pressure using a dot blot apparatus. The membrane was removed from the dot blot apparatus and washed four times with 4X SSC (0.6 M NaCl, and 60 mM sodium citrate), and then rinsed one time with water and once with 100% ethanol. The membrane was air dried and exposed to a phosphoimaging screen and the image captured using a Typhoon 8600 Phospho imager. Following capture of the image, the membrane was placed into a Micro beta cassette along with scintillation fluid and the CPM in each reaction was counted on a Micro beta 1450. CPM data were imported into a custom Excel spreadsheet for determination of compound IC50S.

Additional Antiviral Data:

Rift Valley fever Assay (values reported in μ§/ηιΙ,):

Tacaribe virus (values reported in μ§/ηιΙ,):

Claims

Claim 1. A compound Formula I:

Formula I

or pharmaceutically acceptable salts thereof wherein,

X is O or CH₂;

R¹ is a phosphonate, or polyphosphonate,