WO2013036846A2 - N4 derivatives of deoxycytidine prodrugs - Google Patents

Abstract

The present invention provides hydrophobic prodrugs of nucleic acid bases, nucleosides, and nucleotides. The compounds of the invention have a structure according to Formula I. Methods for the hypomethylation of oligonucleotides, treatment of cancer, and treatment of HIV are also provided.

Description

N4 DERIVATIVES OF DEOXYCYTIDINE PRODRUGS

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 61/532,975, filed September 9, 201 1 , which is incorporated in its entirety herein for all purposes.

BACKGROUND OF THE INVENTION

[0002] RNA viral diseases are responsible for the vast majority of viral morbidity and mortality of viral diseases of mankind, including AIDS, hepatitis, rhinovirus infections of the respiratory tract, flu, measles, polio and others. There are a number of other chronic persistent diseases caused by RNA or DNA viruses that replicate through an RNA intermediate which are difficult to treat, such as hepatitis B and C, and T-cell human leukemia. A number of common human diseases are caused by RNA viruses that are replicated by a viral encoded RNA replicase. Included in this group are influenza (Zurcher, et at, J. Gen. Virol. 77: 1745 (1996), dengue fever (Becker, Virus-Genes 9:33 (1994), and rhinovirus infections (Horsnell, et al, J. Gen. Virol, 76:2549 (1995). Important RNA viral diseases of animals include feline leukemia and immunodeficiency, Visna maedi of sheep, bovine viral diarrhea, bovine mucosal disease, and bovine leukemia.

[0003] Chain terminating nucleoside analogs have been used extensively for the treatment of infections by DNA viruses and retroviruses. These analogs have been designed to be incorporated into DNA by DNA polymerases or reverse transcriptases. Once incorporated, they cannot be further extended and thus terminate DNA synthesis. Unfortunately, there is immediate selective pressure for the development of resistance against such chain terminating analogs that results in development of mutations in the viral polymerase that prevent incorporation of the nucleoside analog.

[0004] An alternative strategy is to utilize mutagenic deoxyribonucleosides (MDRN) or mutagenic ribonuclcosides (MRN) that are preferentially incorporated into a viral genome. MDRN are incorporated into DNA by viral reverse transcriptase or by a DNA polymerase enzyme. MRN are incorporated into viral RNAs by viral RNA replicases. As a result, the mutations in the viral genome are perpetuated and accumulated with each viral replication cycle. With each cycle of viral infection, there ensues a chain like increase in the number of mutations in the viral genome. Eventually the number of mutations in each viral genome is so large that no active virally encoded proteins are produced. [0005] 5-aza-2'-deoxycytidine (5-aza-dC) 5-aza-2'-cytidine (5-aza-C) are antineoplastic agents currently prescribed for the treatment of myelodysplasia syndrome. They are thought to act predominantly by demethylating DNA. Methylation is thought to silence tumor growth suppressor and differentiation genes. Interestingly deamination of 5-aza-dC to 5-aza-2'- deoxyuridine (5-aza-dU) has been shown to result in loss of antineoplastic activity (see e.g. , Momparler, et al, Leukemia. 1 1 : 1-6 ( 1997)).

[0006] Also, both 5-aza-C and 5-aza-dC were shown to inhibit HIV replication in vitro, although the mechanism of action was not determined (see e.g. , Bouchard et al, Antimicrob. Agents Chemother. 34:206-209 (2000)). More recently, 5-aza-C has been shown to be mutagenic to foot-and-mouth disease virus (see e.g., Sierra et al., J. Virol. 74(18):8316-8323 (2000)). Both 5-aza-C and 5-aza-dC are unstable compounds. 5-aza-dC has been shown to be rapidly degraded upon reconstitution. At pH 7.0, a 10% degradation occurs at temperatures of 25°C and 50°C after 5 and 0.5 hours, respectively (see e.g., Van Groeningen et al., Cancer Res. 46:4831 -4836 (1986)). Thus, therapeutic use of 5-aza-C and 5-aza-dC is limited. The present invention solves this and other problems by providing prodrug compounds of nucleoside analogs that possess the therapeutic benefits of 5-aza-c and 5-aza-dC as well as improved

pharmacokinetic properties.

BRIEF SUMMARY OF THE INVENTION

[0007] The present invention provides hydrophobic prodrugs of bases, nucleosides, and nucleotides as well as methods of using the prodrugs as antiviral and anti-cancer

chemotherapeutic agents. Thus, in one embodiment, the compounds of the invention have a structure according to Formula I:

In Formula I, X and Y are independently selected from O, S, and N(R^a); and Z is selected from CHR^a, O, S, and N(R^a). Each R^a is independently selected from H and C_U6 alkyl. R¹ is Ci-20 alkyl, and R² and R^J are independently selected from H and Ci_₆ alkyl. R⁴, R⁵, and R⁶ are independently selected from H, OH, and Ci.6 alkoxy. When X is NH, Y is O, and Z is CH₂, then R¹ is Ci.io alkyl. When X is NH, Y is O and Z is O, then R¹ is Ci_₆ alkyl.

[0008] In a second embodiment, the invention provides a method of hypomethylating an oligonucleotide by contacting the oligonucleotide with a compound of Formula I. In a third embodiment, the invention provides a method of treating cancer including the administration of a compound of Formula I to a patient in need thereof. In a fourth embodiment, the invention provides a method of treating HIV including the administration of a compound of Formula I to a patient in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 depicts the EC₅₀ values for 5-Aza-dC, DHAdC and 5-Me-DHAdC against wild-type HIV virus. The experiments were carried out in MT-2 cells infected with HIV strain

LAI. DETAILED DESCRIPTION OF THE INVENTION

I. General

[0010] The invention is directed to compounds which inhibit viral replication and the growth of cancerous cells. These compounds are hydrophobic prodrugs of bases, nucleosides, and nucleotides. The compounds of the invention are useful for inhibiting viral replication in cell culture as well as in antiviral therapy for animals and humans. In one embodiment, the compounds and methods of the invention are advantageous when used to target RNA viruses (viruses with a RNA genome), and retroviruses or other viruses otherwise replicated by a RNA intermediate. The compounds of the invention are also useful for inhibiting the growth of cancer cells in cell culture as well as in treating cancer in animals and humans.

II. Definitions

[0011] As used herein, the term "alkyl" refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated. For example, C_\ -C alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Other alkyl groups include, but are not limited to heptyl, octyl, nonyl, decyl, etc. Alkyl can include any number of carbons, such as 1 -2, 1 -3, 1 -4, 1 -5, 1 -6, 1 -7, 1 -8, 1 - 9, 1 -1 0, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6 and 5-6. The alkyl group is typically monovalent, but can be divalent, such as when the alkyl group links two moieties together.

[0012] As used herein, the term "alkoxy" refers to an alkyl group having an oxygen atom that connects the alkyl group to the point of attachment. Alkoxy groups include, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc. The alkoxy groups can be further substituted with a variety of substituents described within. For example, the alkoxy groups can be substituted with halogens to form a "halo-alkoxy" group. [0013] As used herein, the term "prodrug" refers to covalently bonded carriers which are capable of releasing the active agent of the methods of the present invention, when the prodrug is administered to a mammalian subject. Release of the active ingredient occurs in vivo. Prodrugs can be prepared by techniques known to one skilled in the art. These techniques generally modify appropriate functional groups in a given compound. These modified functional groups however regenerate original functional groups by routine manipulation or in vivo. Prodrugs of the active agents of the present invention include active agents wherein a hydroxy, amidino, guanidino, amino, carboxylic or a similar group is modified.

[0014] As used herein, the term "salt" refers to acid or base salts of the compounds used in the methods of the present invention. Illustrative examples of pharmaceutically acceptable salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, and the like) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid and the like) salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts. It is understood that the

pharmaceutically acceptable salts are non-toxic. Additional information on suitable

pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, which is incorporated herein by reference.

[0015] As used herein, the term "pharmaceutically acceptable excipient" refers to a substance that aids the administration of an active agent to and absorption by a subject. Pharmaceutical excipients useful in the present invention include, but are not limited to, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors and colors. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0016] The terms "nucleic acid," "oligonucleotide," and "polynucleotide" refer to

deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. , degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 ( 1991); Ohtsuka et al, J. Biol. Chem. 260:2605-2608 ( 1 85); and Rossolini et al, Mol. Cell. Probes 8:91 -98 ( 1994)). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene. [0017] As used herein, the term "hypomethylating" refers to bringing about a level of nucleic acid methylation that is lower than the level observed in a reference nucleic acid sample. In mammalian subjects, DNA methylation is mediated by DNA methyltransferases (DNMTs), which carry out the covalent addition of a methyl group to position 5 of cytosine within cytosine- guanine (CpG) dinucleotides. In certain instances, the reference sample can consist of DNA derived from healthy cells or tissues. The reference sample can also consist of DNA derived from diseased cells or tissues, such as cancerous cells or tissues.

[0018] As used herein, the term "contacting" refers to the process of bringing into contact at least two distinct species such that they can react. It should be appreciated, however, the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture.

[0019] As used herein, a "patient" refers to any warm-blooded animal, preferably a human.

[0020] As used herein, "cancer" includes solid tumors and hematological malignancies, as well as a collection of hematological medical conditions defined by an ineffective production of the myeloid class of blood cells collectively called myelodysplastic syndromes. The former includes cancers such as breast, colon, and ovarian cancers. The latter includes hematopoietic malignancies such as leukemias, lymphomas and myelomas. This invention provides new effective methods and compositions for treatment and/or prevention of various types of cancer.

[0021] As used herein, "HIV" means the human immunodeficiency virus. HIV includes but is not limited to extracellular viral particles and all forms of HIV associated with HIV-infected cells.

[0022] As used herein, the terms "treat", "treating" and "treatment" refers to any indicia of success in the treatment or amelioration of an injury, pathology, condition, or symptom (e.g., HIV), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the symptom, injury, pathology or condition more tolerable to the patient; decreasing the frequency or duration of the symptom or condition; or, in some situations, preventing the onset of the symptom or condition. The treatment or amelioration of symptoms can be based on any objective or subjective parameter; including, e.g., the result of a physical examination. III. Compounds

[0023] In some embodiments, the present invention provides compounds of Formula I:

In the compounds of Formula I, X and Y are each O, S, or N(R^a). Z is CHR^a, O, S, or N(R^a). R is C] .2o alkyl. R² and R³ are each independently H or Ci_-6 alkyl. R⁴, R⁵ and R⁵ are each independently H, OH or Ci„₆ alkoxy. Each R^a is independently H or C]_6 alkyl. When X is NH, Y is O, and Z is CH₂, then R¹ is C_M0 alkyl. When X is NH, Y is O, and Z is O, then R¹ is Ci_6 alkyl. The invention also provides salts and isomers of the compounds of Formula I.

[0024] In some embodiments, X is NH; Y is O or S; Z is CH₂, O, S or NH; R², R³ and R⁴ are each H; and R⁵ and R⁶ are each OH. In some embodiments, Y is O and Z is CH₂. In some embodiments, Y is O and Z is O. In some embodiments, Y is O and Z is S. In some

embodiments, Y is O and Z is NH. In some embodiments, Y is S and Z is CH₂. In some embodiments, Y is S and Z is O. In some embodiments, Y is S and Z is S. In some

embodiments, Y is S and Z is NH. [0025] In some embodiments, X is NH; Y is O; and Z is CH₂, O, S or NH. In some embodiments, X is NH; Y is S; Z is CH₂, O, S or NH. In some embodiments, X is NH; Y is O or S; Z is CH₂, S or NH.

[0026] In some embodiments, Y is NH and Z is NH. In some embodiments, Y is NH and Z is CH₂. [0027] In some embodiments, the compound is l -heptyl-3-[5-((2R,4S,5R)-4-hydroxy-5- hydroxymethyl-tetrahydrofuran-2-yl)-4-oxo-l ,4,5,6-tetrahydro-[l ,3,5]triazin-2-yl]-thiourea; 1 - heptyl-3-[5-((2R,4S,5R)-4-hydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-4-oxo-l , 4,5,6- tetrahydro-[l,3,5]triazin-2-yl]-urea; octanoic acid [5-((2R,4S,5R)-4-hydroxy-5-hydroxymethyl- tetrahydrofuran-2-yl)-4-oxo-l,4,5,6-tetrahydro-[l ,3,5]triazin-2-yl]-amide; [5-((2R,4S,5R)-4- hydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-4-oxo-l ,4^

thiocarbamic acid heptyl ester; or phenyl [5-((2R,4S,5R)-4-hydroxy-5-hydroxymethyl- tetrahydrofuran-2-yl)-4-oxo-l ,4,5,6-tetrahydro-[l ,3,5]triazin-2-yl]-carbamodithioate.

[0028] Pharmaceutically acceptable salts of the acidic compounds of the present invention are salts formed with bases, namely cationic salts such as alkali and alkaline earth metal salts, such as sodium, lithium, potassium, calcium, magnesium, as well as ammonium salts, such as ammonium, trimethyl-ammonium, diethylammonium, and

tris-(hydroxymethyl)-methyl-ammonium salts.

[0029] Similarly acid addition salts, such as of mineral acids, organic carboxylic and organic sulfonic acids, e.g., hydrochloric acid, methanesulfonic acid, maleic acid, are also possible provided a basic group, such as pyridyl, constitutes part of the structure.

[0030] The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.

[0031] Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are all intended to be encompassed within the scope of the present invention. [0032] The compounds of the present invention can be prepared by a variety of methods known to one of skill in the art. Synthetic transformations useful for making the compounds of the present invention can be found in Comprehensive Organic Transformations, Robert C. Larock, Wiley-VCH, 2d edition (November 3, 1999). The exemplary schemes 1 -7 below illustrate methods of preparing the compounds of the invention. These methods are not limited to producing the compounds listed, but can be used to prepare other compounds as well. The compounds of the invention can also be produced by methods not explicitly illustrated in the schemes. The compounds can be prepared using readily available starting materials or known intermediates. [0033] The compounds of the invention are prodrugs of DHAdC:

DHAdC is also effective against virus with mutations conferring resistance to many of the drugs currently approved for treatment of HIV/AIDS. In preclinical cell culture studies, DHAdC demonstrated an efficacy-dependent increase in random transitional mutations in the HIV genome without host cell toxicity. The prodrug compounds of the invention are metabolized and activated to form DHAdC-triphosphate, the active metabolite and substrate for viral reverse transcriptase (RT). The compounds are eventually incorporated into HIV DNA as DHAdC- monophosphate.

[0034] The carbonyl of compound 1 can be protected according to the method of Scheme 1.

Scheme 1

(1) (NH₄)₂S0₄ (2)

[0035] In this scheme, a trimethylsilyl protecting group is added to the carbonyl of commercially available 5-azacytosine (CAS #: 931 -86-2, Sigma Chemical Company).

[0036] Compound 2 can be attached to a protected saccharide according to the method of

Scheme 2. Scheme 2

[0037] In Scheme 2, compound 2 is reacted with compound 3 in dichloromethane to produce compound 4. Compound 4 represents a mixture of approximately 70% β-anomer 4a and 30% a- anomer 4b.

[0038] The double bond at the 5-position in compound 4 can be reduced with sodium borohydride according to the method of Scheme 3.

Scheme 3

[0039] In Scheme 3. compound 4 is reacted with sodium borohydride in acetic acid to produce compound 5. Compound 5 represents a mixture of approximately 70% β-anomer 5a and 30% a- anomer 5b.

[0040] The pure β-anomer 5a is separated from the a-anomer 5b through recrystallization in methanol. The protecting groups are removed from compound 5a through the method according to Scheme 4.

Scheme 4

[0041] Adding sodium methoxide to compound 5a in methanol produces the deprotected compound 6.

[0042] A mixture of exo and endo N-acylated products can be produced according to the method of Scheme 5.

Scheme 5

[0043] In Scheme 5, the hydroxyl groups of compound 6 are first protected with TMS chloride to form compound 7. Subsequently, the treatment of 7 with one equivalent of the appropriate chloroformate produces a mixture of exo-N-acylated product (8a) and endo-N-acylated product (8b). The main exo-N-acylated product is separated from the endo-N-acylated isomer by flash chromatography on silica gel.

[0044] Pure endo-N-acylated isomer can be obtained by the method of Scheme 6.

Scheme 6

[0045] In Scheme 6, compounds 8a and 8b are first treated with an excess of a chloroformate to create the bis-N-acylated compound 9. Subsequent deprotection of the exo-N-acylated moiety from compound 9 with triethylamine in methanol produces the endo-N-acylated isomer 10 in a high overall yield.

[0046] Compound ] 0 is treated with t-butyldimethylsilyl chloride and imidazole to protect the 5' and 3' hydroxyl groups. Subsequent hydrolysis of the carbamate moiety with sodium hydroxide in methanol/water solution yields a free amino group at the 4-position of the cytidine base. This amine can be reacted with electrophiles including, but not limited to, isocyanates, isothiocyanates, and acid chlorides to produce the compounds of Formula I.

IV. Formulation and Administration

[0047] The present invention provides pharmaceutical compositions which inhibit the replication of viruses and the growth of cancer cells. These pharmaceutical compositions comprise a prodrug of a base, nucleoside, or nucleotide and a pharmaceutically acceptable carrier. In some embodiments, the invention provides a pharmaceutical composition including a compound of Formula I as defined above and a pharmaceutically acceptable excipient.

[0048] A pharmaceutical composition of the invention, or pharmaceutically acceptable addition salt or hydrate thereof, can be delivered to a patient using a wide variety of routes or modes of administration. Suitable routes of administration include, but are not limited to, oral, transdermal, transmucosal (such as intranasal or intravaginal), and parenteral administration, including intramuscular, subcutaneous and intravenous injections.

[0049] For oral administration, the compounds can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by combining the composition with a suitable solid phase excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, for example, calcium carbonate, calcium phosphate, polymers such as poly(ethylene oxide), fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0050] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, poly(ethylene oxide), and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0051] Pharmaceutical preparations, which can be used orally, include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration. [0052] The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal

administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. [0053] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0054] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated can be used in the formulation. Such penetrants are generally known in the art, and include, e.g., for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents can be used to facilitate permeation. Transmucosal administration can be through nasal sprays, for example, or using suppositories. [0055] For topical administration, the agents are formulated into ointments, creams, salves, powders and gels. In one embodiment, the transdermal delivery agent can be DMSO. In another embodiment, the transdermal delivery agent can be a transdermal patch. The compounds may be formulated, for example, with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. [0056] Examples of aqueous solutions that can be used in formulations for transmucosal drug delivery include, e.g., water, saline, phosphate buffered saline, Hank's solution, Ringer's solution, dextrose/saline, glucose solutions and the like. The formulations can contain pharmaceutically acceptable auxiliary substances to enhance stability, deliverability or solubility, such as buffering agents, tonicity adjusting agents, wetting agents, detergents and the like. Additives can also include additional active ingredients such as bactericidal agents, or stabilizers. For example, the solution can contain sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate or triethanolamine oleate. These compositions can be sterilized by conventional, well-known sterilization techniques, or can be sterile filtered. The resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.

[0057] The choice of therapeutic agents that can be co-administered with the compounds of the invention will depend, in part, on the condition being treated. For example, when administered to a patient undergoing cancer treatment, the compounds may be administered in cocktails containing other bioactive agents, such as anti-cancer agents and/or supplementary potentiating agents. The compounds may also be administered in cocktails containing agents that treat the side-effects of radiation therapy, such as anti-emetics, radiation protectants, etc.

[0058] Other suitable bioactive agents include, for example, antineoplastic agents, such as platinum compounds (e.g., spiroplatin, cisplatin, and carboplatin), methotrexate, adriamycin, taxol, mitomycin, ansamitocin, bleomycin, cytosine arabinoside, arabinosyl adenine, mercaptopoly lysine, vincristine, busulfan, chlorambucil, melphalan (e.g., PAM, L-PAM or phenylalanine mustard), mercaptopurine, mitotane, procarbazine hydrochloride dactinomycin (actinomycin D), daunorubicin hydrochloride, doxorubicin hydrochloride, mitomycin, plicamycin (mithramycin), aminoglutethimide, estramustine phosphate sodium, flutamide, leuprolide acetate, megestrol acetate, tamoxifen citrate, testolactone, trilostane, amsacrine (m- AMSA), asparaginase (L-asparaginase) Erwina asparaginase, etoposide (VP- 16), interferon a- 2a, interferon a-2b, teniposide (VM-26), vinblastine sulfate (VLB), vincristine sulfate, bleomycin, bleomycin sulfate, methotrexate, adriamycin, and arabinosyl; blood products such as parenteral iron, hemin, hematoporphyrins and their derivatives; biological response modifiers such as muramyldipeptide, muramyltripeptide, microbial cell wall components, lymphokines (e.g., bacterial endotoxin such as lipopoly-saccharide, macrophage activation factor), sub-units of bacteria (such as Mycobacteria and Corynebacteria), the synthetic dipeptide N-acetyl- muramyl-L-alanyl-D-isoglutamine; anti-fungal agents such as ketoconazole, nystatin, griseofulvin, flucytosine (5-fc), miconazole, amphotericin B, ricin, and β-lactam antibiotics (e.g., sulfazecin); hormones and steroids such as growth hormone, melanocyte stimulating hormone, estradiol, beclomethasone dipropionate, betamethasone, betamethasone acetate and

betamethasone sodium phosphate, vetamethasone disodium phosphate, vetamethasone sodium phosphate, cortisone acetate, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, flunsolide, hydrocortisone, hydrocortisone acetate, hydrocortisone cypionate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, paramethasone acetate, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebutate, prednisone, triamcinolone, triamcinolone acetonide, triamcinolone diacetate, triamcinolone hexacetonide and fludrocortisone acetate; vitamins such as cyanocobalamin neinoic acid, retinoids and derivatives such as retinol palmitate, and a-tocopherol; peptides, such as manganese super oxide dimutase; enzymes such as alkaline phosphatase; anti-allergic agents such as amelexanox; anti-coagulation agents such as phenprocoumon and heparin; circulatory drugs such as propranolol; metabolic potentiators such as glutathione; antituberculars such as para-aminosalicylic acid, isoniazid, capreomycin sulfate cycloserine, ethambutol hydrochloride ethionamide, pyrazinamide, rifampin, and streptomycin sulfate; antivirals such as acyclovir, amantadine azidothymidine (AZT or Zidovudine), ribavirin, amantadine, vidarabine, and vidarabine monohydrate (adenine arabinoside, ara-A); antianginals such as diltiazem, nifedipine, verapamil , erythrityl tetranitrate, isosorbide dinitrate, nitroglycerin (glyceryl trinitrate) and pentaerythritol tetranitrate; anticoagulants such as phenprocoumon and heparin; antibiotics such as dapsone, chloramphen icol, neomycin, cefaclor, cefadroxil, cephalexin, cephradine, erythromycin, clindamycin, lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin, dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, ticarcillin rifampin and tetracycline; antiinflammatories such as diffinisal, ibuprofen, indomethacin, meclofenamate, mefenamic acid, naproxen, oxyphenbutazone, phenylbutazone, piroxicam, sulindac, tolmetin, aspirin and salicylates; antiprotozoans such as chloroquine, hydroxychloroquine, metronidazole, quinine and meglumine antimonate; antirheumatics such as penicillamine; narcotics such as paregoric; opiates such as codeine, heroin, methadone, morphine and opium; cardiac glycosides such as deslanoside, digitoxin, digoxin, digitalin and digitalis; neuromuscular blockers such as atracurium besylate, gallamine triethiodide, hexafluorenium bromide, metocurine iodide, pancuronium bromide, succinylcholine chloride (suxamethonium chloride), tubocurarine chloride and vecuronium bromide; sedatives (hypnotics) such as amobarbital, amobarbital sodium, aprobarbital, butabarbital sodium, chloral hydrate, ethchlorvynol, ethinamate, flurazepam hydrochloride, glutethimide, methotrimeprazine hydrochloride, methyprylon, midazolam hydrochloride, paraldehyde, pentobarbital, pentobarbital sodium, phenobarbital sodium, secobarbital sodium, talbutal, temazepam and triazolam; local anesthetics such as bupivacaine hydrochloride, chloroprocaine hydrochloride, etidocaine hydrochloride, lidocaine hydrochloride, mepivacaine hydrochloride, procaine hydrochloride and tetracaine hydrochloride; general anesthetics such as droperidol, etomidate, fentanyl citrate with droperidol, ketamine hydrochloride, methohexital sodium and thiopental sodium; and radioactive particles or ions such as strontium, iodide rhenium and yttrium. In certain preferred

embodiments, the bioactive agent is a monoclonal antibody, such as a monoclonal antibody capable of binding to a melanoma antigen.

[0059] Frequency of administration of the therapeutic compositions described herein, as well as dosage, will vary from individual to individual, and may be readily established using standard techniques. Preferably, between 1 -100 doses may be administered over a 52-week period.

When treating a viral disease, a suitable dose is an amount of a compound that, when administered as described above, is capable of killing or limiting the infectivity of a virus. When treating cancer, a suitable dose is an amount of a compound that, when administered as described above, is capable of killing or slowing the growth of cancers or cancer cells. Those of skill in the art are aware of the routine experimentation that will produce an appropriate dosage range for a patient in need of treatment by oral administration or any other method of administration of a drug, e.g. , intravenous administration or parenteral administration, for example. Those of skill are also aware that results provided by in vitro or in vivo experimental models can be used to extrapolate approximate dosages for a patient in need of treatment.

[0060] In general, an appropriate dosage and treatment regimen provides the pharmaceutical composition in an amount sufficient to provide therapeutic and/or prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome (e.g., longer viral disease-free survival or, for cancer patients, more frequent remissions or complete, partial, or longer disease-free survival) in treated patients as compared to non-treated patients.

V. Method for hypomethylation of oligonucleotides [00 1] Epigenetic events, i.e. changes to nucleic acid structure other than changes in nucleotide sequence, govern changes in gene expression in normal and abnormal cells, affecting processes including cellular differentiation and disease development. A common epigenetic event in the progression of cancer cells toward malignancy, for example, involves transcriptional silencing of nonmutated genes such as tumor suppressor genes. A primary transcriptional silencing mechanism is the methylation of cytosine-guanine (CpG) dinucleotide islands located in gene promoter regions of cancer cells. In mammals this process is mediated by DNA methyltransferases (DNMTs), which carry out the covalent addition of a methyl group to position 5 of cytosine within CpG dinucleotides. Recent studies suggest that some methylation patterns are discernible in certain diseases. Reversing the silencing of genes implicated in the prevention or reversal of cancer development has become a new therapeutic target. Depending on the particular gene, this may prevent the development of cancer or slow disease progression. CpG methylation has also been implicated in the progress of viral diseases including HIV.

Methylation of the HIV- 1 promoter, for example has been shown to stabilize HIV-1 latency, conferring resistance to antiretroviral therapy. [0062] In some embodiments, the invention provides a method of hypomethylating an oligonucleotide, comprising contacting the oligonucleotide and a compound of Formula I as defined above or metabolites thereof, thereby hypomethylating the oligonucleotide. Without being bound to a particular theory, it is believed that the prodrugs of Formula 1 are incorporated into nucleic acid polymers in vivo. The resulting nucleic acid sequences contain a cytosine analog with an sp3-hybridized (CH2) at position 6 and an NH group at position 5, which mimics the non-aromatic character of the transiently formed cysteine-linked dihydrocytosine intermediate during methylation by DNMTs. The cytosine analog is believed to occupy the active site of DNMT as a transition state mimic, acting as a potent inhibitor of methylation because of the high degree of affinity of its interaction with the enzyme. [0063] Those of skill in the art are aware of methods to test the effectiveness of compounds in inducing hypomethylation of nucleic acids. For example, cells can be cultured in the presence of nucleoside analogs including the compounds of Formula I prior to isolation of genomic DNA and detection of methylated gene markers using methylation-specific PCR (Matousova, et al., Epigenetics. 6:769-776 (201 1 )). Total levels of methylated cytidine monophosphate in hydrolyzed DNA can also be assessed by HPLC or other techniques. Alternatively, target oligonucleotides incorporating a compound of interest can be synthesized chemically or enzymatically (Bouchard, et al., Mol Pharmacol. 24: 109-1 14 (1983)) or isolated from cell culture (Creusot, et al., J Biol Chem. 257:2041 -2048 (1982)) in order to assess oligonucleotide methylation by DNMTs with isotopically-labeled methyl donors in vitro.

VI. Method for treatment of cancer [0064] The compounds, pharmaceutical compositions, and methods of the invention are useful in the treatment of cancer. The invention provides a method of treating cancer including the administration of a compound according to Formula I as defined above to a patient in need of such treatment. The cancer can be a leukemia, lymphoma, or other cancers as defined above.

[0065] Leukemias are generally neoplastic disorders of hematopoietic stem cells, and include adult and pediatric acute myeloid leukemias (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia and secondary leukemia. Myeloid leukemias are characterized by infiltration of the blood, bone marrow, and other tissues by neoplastic cells of the hematopoietic system. CLL is characterized by the accumulation of mature-appearing lymphocytes in the peripheral blood and the infiltration of these mature-appearing lymphocytes into the bone marrow, spleen and lymph nodes.

[0066] Specific leukemias include acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, aleukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, and undifferentiated cell leukemia.

[0067] Lymphomas are generally neoplastic transformations of cells that reside primarily in lymphoid tissue. Among lymphomas, there are two major distinct groups: non-Hodgkin's lymphoma (NHL) and Hodgkin's disease. Lymphomas are tumors of the immune system and generally involve both T- and B-cells. Lymphomas are typically found in bone marrow, lymph nodes, the spleen and the circulatory system. Treatment protocols include removal of bone marrow from the patient, purging the bone marrow of tumor cells (often using antibodies directed against antigens present on the tumor cell type), followed by storage of the bone marrow. After the patient receives a toxic dose of radiation or chemotherapy, the purged bone marrow is reinfused in order to repopulate the patient's hematopoietic system.

[0068] Other hematological malignancies include myelodysplastic syndromes (MDS), myeloproliferative syndromes (MPS) and myelomas, such as multiple myeloma and solitary myeloma. Multiple myeloma (also called plasma cell myeloma) affects the skeletal system and is characterized by multiple tumorous masses of neoplastic plasma cells scattered throughout the system. It may also spread to lymph nodes and other sites such as the skin. Solitary myeloma involves solitary lesions that tend to occur in the same locations as multiple myeloma.

[0069] The compounds of the invention are also directed against other cancers. Such cancers include those characterized by solid tumors. Examples of other cancers of concern are skin cancers, including melanomas, basal cell carcinomas, and squamous cell carcinomas. Epithelial carcinomas of the head and neck are also encompassed by the present invention. These cancers typically arise from mucosal surfaces of the head and neck and include salivary gland tumors.

[0070] The present invention also encompasses cancers of the lung. Lung cancers include squamous or epidermoid carcinoma, small cell carcinoma, adenocarcinoma, and large cell carcinoma. Breast cancer is also included.

[0071] The present invention also encompasses gastrointestinal tract cancers. Gastrointestinal tract cancers include esophageal cancers, gastric adenocarcinoma, primary gastric lymphoma, colorectal cancer, small bowel tumors and cancers of the anus. Pancreatic cancer and cancers that affect the liver are also of concern, including hepatocellular cancer. The present invention also includes treatment of bladder cancer and renal cell carcinoma. [0072] The present invention also encompasses prostatic carcinoma and testicular cancer.

[0073] Gynecologic malignancies are also encompassed by the present invention and include ovarian cancer, carcinoma of the fallopian tube, uterine cancer, and cervical cancer.

[0074] Treatment of sarcomas of the bone and soft tissue are encompassed by the present invention. Bone sarcomas include osteosarcoma, chondrosarcoma, and Ewing's sarcoma.

[0075] The present invention also encompasses malignant tumors of the thyroid, including papillary, follicular, and anaplastic carcinomas.

[0076] In an embodiment for the treatment of cancer, the compounds of the invention are efficiently delivered into the bloodstream of a patient, such as a mouse, rat, dog or human, and subsequently incorporated into a polynucleotide sequence (either DNA or RNA) of a cancerous cell. The compounds of the invention can have phosphodiester linkages or can acquire phosphodiester linkages, allowing them to be incorporated into the genome of a cancer cell by a polymerase. The compounds of the invention can have altered base-pairing properties and are incorporated into the cancer cell genome. Incorporation subsequently increases the number of mutations in the cancer cell. Mutations can be incorporated into transcription products, e.g., mRNA molecules that encode proteins or tRNA molecules useful for protein translation. The mutated transcription products possess altered amino acid sequences which often result in inactive proteins. Regardless of the method of introduction, an increase in the number of mutations in the cancer cell causes reduced population growth rates, decreased viability of progeny cells, diminished ability to proliferate or metastasize, and cancer cell death.

[0077] Those of skill in the art are aware of methods to test the effectiveness of compounds in treating cancer. For example, cancer cells of interest can be grown in culture and incubated in the presence of varying concentrations of the compounds of the present invention. Frequently, the uptake of viral dyes, such as MTT, is used to determine cell viability and cell proliferation. When inhibition of cell proliferation is seen, the IC50 of the compound can be determined. Those of skill in the art will also know to test the compounds of the present invention in animal models. For example, the compounds of the invention are injected into nude mice with transformed cancer cells. The data gathered in tissue culture models and animal models can be extrapolated by those of skill in the art for use in human patients. VII. Method for treatment of HIV

[0078] The compounds of the invention possess activity against viruses. Some of these viruses are able to integrate their viral genome into the genome of a cell. Examples of viruses which have this ability include, but are not limited to, retroviruses. In an exemplary embodiment, the virus is HIV and its variants, such as HIV-1 , HIV-2, HTLV- 1 , HTLV-I1, and SIV. In another embodiment, the virus is a DNA virus such as hepatitis B virus, herpesviruses (e.g., Herpes Simplex Virus, CytoMegaloVirus (CMV), Epstein-Barr Virus, (EBV)), smallpox virus, or human papilloma virus (e.g. , HPV). Alternatively, the viral genome can be episomal. These include many human and animal pathogens: flaviviruses, such as dengue fever, West Nile, and yellow fever; pestiviruses, such as bovine viral diarrhea (BVD), and hepaciviruses, such as hepatitis C; filoviruses such as ebola; parainfluenza viruses, including respiratory syncytial; rubulaviruses, such as mumps; morbillivirus, such as measles; picornaviruses, including the echoviruses; the coxsackieviruses; the polioviruses; the togaviruses, including encephalitis; coronaviruses, including Severe Acute Respiratory Syndrome (SARS); rubella; bunyaviruses; reoviruses, including rotaviruses; rhabdoviruses; arenaviruses, such as lymphocytic

choriomeningitis, as well as other RNA viruses of man and animal.

[0079] Retroviruses that can be targeted include HTLV viruses such as HTLV-1 and HTLV-2, adult T-cell leukemia (ATL), HIV- 1 and HIV-2 and SIV. In some embodiments, the HIV virus is resistant to non-nucleoside reverse transcriptase inhibitors. In certain embodiments, the virus is hepatitis A or hepatitis B. See, Knipe et al. FIELDS VIROLOGY, 4th ed. Lippincott, Williams, and Wilkins (2001 ). Further information regarding viral diseases and their replication can be found in White and Fenner, MEDICAL VIROLOGY, 4th ed. Academic Press ( 1994) and in Zuckerman, Banatvala and Pattison (ed.), PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY, John Wiley and Sons (1994). [0080] The compounds, pharmaceutical compositions, and methods of the present invention are useful in the treatment of viral diseases. The invention provides a method of treating HIV including the administration of a compound according to Formula I as defined above to a patient in need of such treatment.

[0081] In some embodiments, the compounds of the invention are efficiently delivered into the bloodstream of a patient, such as a mouse, rat, dog or human, and subsequently incorporated into the genome of the HIV. The compounds of the invention can have phosphodiester linkages or acquire phosphodiester linkages, enabling them to be incorporated into the viral genome by a polymerase. The compounds of the invention can have altered base-pairing properties which allow the incorporation of mutations into the viral genome, thereby increasing the total number of mutations. Increases in the total number of mutations result in reduced viral population growth rates, as well as decreased viability of progeny virus.

[0082] The compounds of the present invention are particularly well-suited to treat HIV strains that are resistant to chain-terminating nucleosides. HIV strains resistant to chain-terminating nucleosides are known and mutations in the reverse transcriptase (RT) enzyme responsible for the resistance have been analyzed. Two mechanisms of viral resistance toward chain- terminating nucleosides have been described. In the first mechanism, the virus discriminates between a chain-terminating nucleoside and a naturally occurring nucleoside, thus preventing the chain-terminating nucleoside's incorporation into the viral genome. For example, chain- terminating nucleoside-resistant viral strains contain a version of HIV-RT which recognizes the absence of a 3'-OH group, a feature present in some chain-terminating nucleosides {see, e.g., Sluis-Cremer et al, Cell. Mol. Life Sci. 57: 1408-1422 (2000)). In the second mechanism, the virus excises the chain-terminating nucleoside after its incorporation into the viral genome via pyrophosphorolysis in the presence of nucleotides (see, e.g., Isel et al., J. Biol. Chem.

276:48725-48732 (2001 )). In pyrophosphorolysis, also known as reverse nucleotide

polymerization, pyrophosphate acts as an acceptor molecule for the removal of the chain- terminating nucleoside. Removal of the chain-terminating nucleoside frees RT to incorporate the natural nucleotide substrate and maintain accurate viral replication. ATP has also been proposed as an acceptor molecule for the removal of chain-terminating nucleosides and is referred to as primer unblocking {see, e.g. , Naeger et al., Nucleosides Nucleotides Nucleic Acids 20:635-639 (2001 )). [0083] The compounds of the invention can reduce viral resistance through the first mechanism mentioned above. Because the compounds of the invention comprise sugars with hydroxyls at the 3' position, it is believed that HIV-RT should be unable to differentiate between them and natural nucleosides.

[0084] In general, the compounds of the invention will reduce viral resistance compared to treatment with chain-terminating nucleosides. Currently approved chain-terminating nucleosides target one aspect of the viral growth cycle, replication, and immediately attempt to stop it through chain termination. Since the antiviral's effect is narrowly targeted and abrupt, there is great selective pressure for the development of resistant viral strains. The compounds of the invention act by a different method. The compounds act through the gradual accumulation of random mutations in the viral genome. This corresponds to the gradual inactivation of potentially any of the viral proteins. Since the effect of the compounds of the invention is broadly targeted and gradual, there is less selective pressure for the emergence of resistant viral strains.

[0085] Cross resistance between chain-terminating nucleosides and the compounds of the invention can be tested by determining the EC₅₀ for a prodrug in a wild-type HIV strain and in a HIV strain resistant to one or more chain-terminating nucleosides. If the EC₅₀ for the prodrug is higher in the chain-terminating nucleoside resistant strain than in the wild-type strain, then cross resistance has occurred. Experiments have demonstrated that cross resistance is unlikely to develop between chain-terminating nucleosides and compounds of the invention.

VIII. Examples

GENERAL

[0086] In the examples below, unless otherwise stated, temperatures are given in degrees Celsius (°C); operations were carried out at room or ambient temperature, "rt," or "RT," (typically a range of from about 18-25 °C); evaporation of solvent was carried out using a rotary evaporator under reduced pressure (typically, 4.5-30 mm Hg) with a bath temperature of up to 60 °C; the course of reactions was typically followed by TLC or LC/MS and reaction times are provided for illustration only; melting points are uncorrected; products exhibited satisfactory Ή- NMR and/or microanalytical data; yields are provided for illustration only; and the following conventional abbreviations are also used: mp (melting point), L (liter(s)), mL (milliliters), mmol (millimoles), g (grams), mg (milligrams), min (minutes), and h (hours). Unless otherwise specified, all solvents (HPLC grade) and reagents were purchased from suppliers and used without further purification. HPLC was conducted using a Poroshell 120 column (100 mm x 4.6 mm, 2.7-micron particle size) and a flow rate of 1 .2 mL/min at 40 ^CC while monitoring elution by UV/visible absorption spectroscopy at a wavelength of 210 nm. Mobile Phase A consisted of 0.1 % phosphoric acid and Mobile Phase B consisted of acetonitrile. A chromatography gradient was employed as outlined in the following table:

Example 1. Synthesis of l-Heptyl-3-[5-((2R,4S,5R)-4-hvdroxy-5-hvdroxymethyi- tetrahvdrofuran-2-yl)-4-oxo-l,4,5,6-tetrahvdro-H,3,51triazin-2-yll-thiourea (Compound A)

[0087] Preparation of Compound 1 1 :

1 1 [0088] N4-Heptyloxycarbonyl-a-2'-deoxy-5,6-dihydro-5-azacytidine (25.0 g, 0.067 moles) (prepared as described above), t-butyldimethylsilyl chloride (21.2 g, 0.141 moles), imidazole (9.6 g, 0.141 moles) in dimethylformamide (250 ml) were combined and stirred at room temperature. The reaction was complete by LC/MS after - 16 hours and stripped to dryness. The residue was dissolved in dichloromethane and washed with water (2x 100 ml), dried over magnesium sulfate, filtered and concentrated under reduced pressure to yield compound 1 1 (44.8 g). Ή NMR (400 MHz, DMSO-c ₆) d ppm -0.01 - 0.10 (1 1 H, m), 0.82 - 0.91 (17 H, m), 1.73 (1 H, ddd, 7=13.06, 6.08, 2.93 Hz), 2.02 - 2.1 1 (1 H, m), 3.31 (2 H, br. s.), 3.53 - 3.63 (3 H, m), 4.23 - 4.28 (1 H, m), 4.33 - 4.43 (2 H, m), 6.08 (1 H, dd, J=8.47, 6.17 Hz). Purified by silica gel chromatography with dichloromethane: methanol gradient ( 100: 0) to 85 : 1 5, with visualization of TLC with potassium permanganate and heating. Rf=0.42 in Dichloromethane:methanol (9:1 ).

[0089] Preparation of Compound 12:

[0090] Compound 1 1 (44.4 g, 0.074 moles), water (26.7 ml, 1 .484 moles) and methanol (444 ml) were combined. Sodium hydroxide (6.20 g, 0.155 moles) was added and stirred at room temperature. After stirring for ~16 hours LC/MS indicated the reaction was complete and the mixture concentrated under reduced pressure. The residue was dissolved in water (200 ml) and extracted with ethyl acetate (3x100 ml), dried over magnesium sulfate, filtered and concentrated under reduced pressure to a clear yellow oil (35.5 g) to give compound 12. LC/MS (M+ l ) =

459. Ή NMR (400 MHz, DMSO-< ) ΰ ppm 0.03 - 0.08 (8 H, m), 0.83 - 0.89 (15 H, m), 1 .22 - 1 .30 (6 H, m), 1.40 (1 H, t, J=6.56 Hz), 3.31 ( 1 H, s), 3.34 - 3.39 (1 H, m), 3.55 - 3.61 (2 H, m), 4.24 - 4.27 ( 1 H, m), 4.30 (1 H, t, J=5.08 Hz), 4.39 (1 H, br. S.), 5.43 (1 H, br. S.), 6.08 (1 H, dd, J=8.40, 6.20 Hz). Preparation of Compound A

[0092] Compound 12 (2.00 g, 0.0044 moles), 1 -heptyl isothipcyanate (0.83 ml, 0.0048 moles) and tetrahydrofuran (20 ml) were combined and then heated to reflux for 6 hours then heated at 50 °C for ~ 3 days. The mixture was concentrated under reduced pressure and then redissolved in dichloromethane (30 m l) and washed with water (2x25 ml), dried over magnesium sulfate, filtered and concentrated under reduced pressure to an oil. The oil was dissolved in

tetrahydrofuran (1 3 ml) deprotected with tetrabutylammonium fluoride in THF (13 ml of 1 .0 M). After stirring for 2 hours at room temperature the reaction was complete by LC/MS and dichloromethane (30 ml) was added. The mixture was washed with saturated ammonium chloride (2x30ml), dried over magnesium sulfate, filtered and concentrated under reduced pressure to a clear oil ( 1 .80 g). The oil was purified by silica gel chromatography using a gradient of 100% acetonitri le for 6 minutes then an increase to 2.5% methanol : aceton itrile over 31 minutes. The fractions were checked by TLC (5% MeOH:acetonitrile) and stained with potassium permanganate stain and heated to visualize. The pure fractions were combined and concentrated under reduced pressure to a white solid (0.34 g, 20% yield) which was identified as A. HPLC purity AUC 96 %. LC/MS (M+l ) = 346. Ή NMR (400 MHz, DMSO- ₆) d ppm 0.84 - 0.88 (3 H, m), 1 .26 (8 H, br. s.), 1 .53 ( 1 H, d, J=5.71 Hz), 1 .71 - 1 .90 (1 H, m), 2.05 ( 1 H, dd, JH 4.18, 6.52 Hz), 3.43 (2 H, br. s.), 3.50 ( 1 H, d, J=5.91 Hz), 3.57 - 3.66 ( 1 H, m), 4. 1 1 (1 H, br. s.), 4.53 - 4.72 (2 H, m), 4.73 - 4.85 (1 H, m), 5.1 1 ( 1 H, br. s.), 5.97 - 6.1 1 ( 1 H, m), 1 1 .1 1 ( 1 H, br. s.)

Example 2. Synthesis of l-Heptyl-3-[5-((2R,4S,5R)-4-hvdroxy-5-hvdroxymethyl- tetrahvdrofuran-2-yl)-4-oxo-l,4,5,6-tetrahydro-[l,3,51triazin-2-yll-urea (Compound B)

B

[0093] Compound 12 (2.00 g, 0.0044 moles), 1 -heptyl isocyanate (0.77 ml, 0.0048 moles) and dichloromethane (20 ml) were combined and stirred at room temperature under nitrogen. After -16 hours the reaction was complete by LC/MS. The mixture was washed with water (2x25 ml), dried over magnesium sulfate, filtered and concentrated under reduced pressure to a clear oil (2.40 g). The oil was dissolved in tetrahydrofuran (13 ml) deprotected with tetrabutylammonium fluoride in THF (13 ml of 1 .0 M). After stirring for 2 hours at room temperature the reaction was complete by LC/MS and dichloromethane (30 ml) was added. The mixture was washed with saturated ammonium chloride (2x30ml), dried over magnesium sulfate, filtered and concentrated under reduced pressure to a clear oil (2.19 g). The oil was purified by silica gel chromatography gradient (dichloromethane: methanol; 0 to 10 % over 19 minutes then dichloromethane: methanol; 90: 10 to 35 minutes). All the fractions that contained the major product were combined and concentrated under reduced pressure to a white solid (0.25 g. 15.4 % yield) which was identified as compound B. HPLC purity AUC 99 %. LC/MS (M+l ) = 372. Ή NMR (400 MHz, DMSO-i ₆) 9 ppm 0.78 - 0.90 (3 H, m), 1 .25 (6 H, br. s.), 1.40 (2 H, t, .7=6.91 Hz), 1.79 (1 H, d, J=9.57 Hz), 1 .95 - 2.1 1 (1 H, m), 3.04 (2 H, br. s.), 3.42 (2 H, t, J=5.17 Hz), 3.55 - 3.65 (1 H, m), 4.04 - 4.16 (1 H, m), 4.56 (2 H, br. s.), 4.76 (1 H, t, .7=5.52 Hz), 5.1 1 (1 H, d, J=4.10 Hz), 5.97 - 6.09 (1 H, m)

Example 3. Synthesis of Octanoic acid [5-((2R,4S,5R)-4-hvdroxy-5-hYdroxymethyl- tetrahydrofuran-2-yl)-4-oxo-l,4,5,6-tetrahydro-[l,3,51triazin-2-yl1-amide (Compound C)

C

[0094] Compound 12 (2.0 g, 0.0044 moles), diisopropylethylamine 0.62 g, 0.0048 moles) and dichloromethane (20 ml) were combined and octanoyl chloride (0.82 ml, 0.0048 moles) added dropwise. The mixture was stirred under nitrogen overnight. After ~16 hr at room temperature, the reaction was washed with water (2x25 ml), dried over magnesium sulfate, filtered and concentrated under reduced pressure to a clear and colorless oil (2.36 g). The oil was dissolved in tetrahydrofuran (28 ml) and tetrabutylammonium fluoride (1 M in THF, 13 ml) was added. The reaction was complete by LC/MS after 2 hours at room temperature. The reaction was concentrated under reduced pressure and redissolved in dichloromethane (30 ml), washed with water (2x25 ml), dried over magnesium sulfate filtered and concentrated under reduced pressure to a clear yellow oil. The oil was purified by silica gel chromatography using a gradient of 100% acetonitrile for 6 minutes then an increase to 2.5% methanol : acetonitrile over 31 minutes. The fractions were checked by TLC (5% MeOH:acetonitrile) and stained with potassium

permanganate stain and heated to visualize. The pure fractions were combined and concentrated under reduced pressure to a white solid (0.58 g, 37% yield) which was identified as Compound C. HPLC purity AUC 99 %. LC/MS (M+l) = 357. Ή NMR (400 MHz, DMSO- ₆) d ppm 0.83 - 0.88 (3 H, m), 1 .20 - 1.30 (7 H, m), 1.47 - 1.55 (2 H, m), 1 .78 ( 1 H, ddd, J=\ 3.23, 6.30, 2.98 Hz), 2.01 - 2.08 (1 H, m), 2.29 (2 H, t, J=7.39 Hz), 3.17 (1 H, d, .7=5.22 Hz), 3.40 - 3.44 (2 H, m), 3.58 - 3.62 (1 H, m), 4.03 - 4.14 (1 H, m), 4.54 - 4.70 (2 H, m), 4.76 (1 H, t, J=5.52 Hz), 5.10 ( 1 H, d, J=4.20 Hz), 6.06 (1 H, dd, J=8.22, 6.32 Hz), 10.01 (1 H, br. s.) Example 4. Synthesis of fS-((2R,4S,5R)-4-Hvdroxy-5-hvdroxyinethyl-tetrahvdrofuran-2- yl)-4-oxo-l,4,5,6-tetrahvdro-[l,3,51triazin-2-yn-thiocarbamic acid heptyl ester

(Compound D)

[0095] Preparation of imidazole-l -carbothioic acid O-heptyl ester:

[0096] Ι , Γ-Thiocarbonyldiimidazole (4.44 g, 0.0249 moles), 1 -heptanol 1.76 ml, 0.0125) and tetrahydrofuran (125 ml) were combined and heated at reflux. After -16 hours TLC (1 : 1 ;

Heptane: Ethyl Acetate) showed a new product with an Rf=0.74, The reaction mixture was concentrated under reduced pressure. The residue was purified on silica gel with Heptane: Ethyl Acetate (7:3). The fractions with the major product were combined and concentrated under reduced pressure to a clear and colorless liquid (2.33 g, 82.6% yield) which was identified as imidazole-l -carbothioic acid O-heptyl ester. LC/ S (M+l ) = 227. Ή NMR (400 MHz, CHLOROFORM -if) d ppm 0.79 - 0.98 (3 H, m), 1 .26 - 1 .51 (8 H, m), 1.83 - 1 .94 (2 H, m), 4.66 (2 H, t, J=6.66 Hz), 7.03 - 7.05 ( 1 H, m), 7.64 (1 H, t, J=l .42 Hz), 8.35 (1 I I, s). [0097] Preparation of Compound D:

D

[0098] Compound 12 (3.0 g, 0.0065 moles), imidazole- l -carbothioic acid O-heptyl ester (1 .48 g, 0.0065 moles) and benzene ( 105 ml) were combined and heated to 50 °C for -48 hours. The reaction was washed with water (2x25 ml), dried over magnesium sulfate, filtered and concentrated under reduced pressure to a yellow oil (4.02 g). The oil was dissolved in tetrahydrofuran (28 ml) and tetrabutylammonium fluoride (1 in THF, 13 ml) was added. The reaction was complete by LC/MS after 2 hours at room temperature. The reaction was concentrated under reduced pressure and redissolved in dichloromethane (30 ml), washed with water (2x25 ml), dried over magnesium sulfate filtered and concentrated under reduced pressure to a clear yellow oil. The oil was purified by silica gel chromatography using a gradient of 100% acetonitrile for 6 minutes then an increase to 2.5% methanol : acetonitrile over 31 minutes. The fractions were checked by TLC (5% MeOH:acetonitrile) and stained with potassium

permanganate stain and heated to visualize. The pure fractions were combined and concentrated under reduced pressure to a white solid (1.08 g, 42% yield) which was identified as D. HPLC purity AUC 95 %. LC/MS (M+l) = 389. Ή NMR (400 MHz, DMSO-i¾) d ppm 0.84 - 0.88 (3 H, m), 1.22 - 1.34 (8 H, m), 1.62 (2 H, t, J=6.96 Hz), 1.86 (1 H, ddd, J=13.26, 6.21 , 3.03 Hz), 2.04 (1 H, ddd, J=13.54, 7.83, 6.15 Hz), 3.43 - 3.46 (2 H, m), 3.62 - 3.65 (1 H, m), 4.12 (1 H, dd, .7=6.42, 3.15 Hz), 4.23 (2 H, t, J=6.59 Hz), 4.70 (1 H, s), 4.75 ( 1 H, s), 4.77 - 4.82 (1 H, m), 5.15 (1 H, d, J=4.25 Hz), 6.02 ( 1 H, dd, .7=8.05, 6.30 Hz), 10.79 ( 1 H, s), 10.76 (1 H, s).

Example 5. Synthesis of Phenyl [5-((2R,4S,5R)-4-hvdroxy-5-hvdroxymethyl- tetrahvdrofuran-2-yl)-4-oxo-l,4,5,6-tetrahvdro-|l,3,51triazin-2-yll-carbamodithioate (Compound E)

E

[0099] Compound 12, phenyl chlorodithioformate, and benzene are dissolved in benzene and allowed to react for 16 hr at room temperature. The reaction mixture is washed with water, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting crude product is dissolved in tetrahydrofuran, and tetrabutylammonium fluoride is added. After completion of the reaction, as assessed by LC/MS. the product is purified by silica gel chromatography to yield the desired compound E.

Example 6: Treatment of HIV

Viral stocks for test of antiviral activity

[0100] The strains of HIV-1 used for primary drug screening are HIV-1 LAI or the appropriate strain of NRTI resistant HIV for studies of cross-resistance. Virus was propagated on MT-2 cells at an multiplicity of infection (MOI) of 0.01 to generate virus stocks. Briefly, the MT-2 cells were suspended in RPMI 1640 media supplemented with 10% fetal bovine serum, streptomycin and penicillin (cRPMI) and grown in a 37°C incubator containing 5% CO₂. Serial dilution of the virus and infection of MT-2 cells were followed by an ELISA detecting the capsid protein of HIV-1 (p24) and used to determine the titer of the virus stocks (50% tissue culture infectious dose (TCID₅₀)). The ELISA was performed according to the manufacturer's instructions. The MT-2 cells are also used for visualizing the cytopathic effects of HIV-1 growth (e.g. syncytia formation).

Treatment of HIV-1 -infected cells with mutagenic nucleoside analogues

[0101] 0.1 ml of a 3 x 10⁵ cells/ml MT-2 cell suspension were seeded in 96-well plates at 3 x

10⁴ cells/well. The compounds to be assayed were diluted in a separate 96-well at ten times (l Ox) the concentration needed for the screen. 22 μΐ of the lOx compounds was then added to the wells in triplicate except for six control wells, containing uninfected MT-2 cells alone (3 wells) and untreated HIV-infected MT-2 cells (3 wells). This was followed by the addition of HIV-1 at an MOI of 0.01 , except for the three wells serving as the uninfected control. 0.1 ml of cRPMI was added to the uninfected well instead of virus. After the addition of virus, the 96-well plate was centrifuged at 1 ,200 x g for two hours to enhance the adsorption of the virus by the MT-2 cells (see, e.g., O'Doherty, ./. Virol. 74: 10074- 10080 (2000)). After the centrifugation step, the 96-well plate was then incubated in a 37°C incubator containing 5% C02 for three days. At the end of this time period the virus and cells were mixed by gentle pipetting followed by a 1 minute spin at 600 x g to pellet the cells. The supernatant of each well was then serially diluted 1 ,000- fold into new 96-well plates to serve as inoculum for the next passage and assayed by ELISA for the amount of p24 produced. The next passage was performed as described above, except that the virus used to infect the cells was derived from the 1 ,000-fold dilution plate. To generate an EC50 value, half-log concentrations of mutagenic nucleoside analogue capable of eradicating virus in a single passage are tested as above to generate a dose-response curve. [0102] EC50 values were determined for the following compounds: 5-aza-dC, 5-aza-dU, DH- aza-dC, and 5-methyl-5,6-dihydro-5-azadeoxycytidine. 5-aza-dC has an EC5₀ (effective concentration that prevents 50% of viral replication) of 3 nM against the wild-type HIV strain LAI. Results are shown in Figure 1 . The EC₅0 values for the other compounds are 3 μΜ for DHAdC and 10 μΜ for MeDHAdC. Compounds of Formula I are tested in a similar manner with respect to viral replication. Assessment of the frequency of mutations to the viral genome induced by mutagenic nucleoside analogues

[0103] The genomic DNA from cells treated with the deoxyribonucleoside analog 5-aza-dC (30 uM ) was purified using a Qiagen DNeasy® Kit. 1 μg of genomic DNA was used to amplify a 1 kb region of the HIV-1 RT proviral DNA by PCR. The PCR product was then cloned into a TOPO® cloning vector. A Millipore Miniprep Kit was used to purify plasmid containing proviral inserts. About 45 positive clones were sequenced in both directions by a Beckman Coulter CEQ 8000. The sequencing results were analyzed and assembled using the DNASTAR Stagman program. Each mutated base was counted and the mutation rate was calculated over the total number of sequenced nucleotides. The results were compared with the background mutation rate generated from untreated control virus and are shown in Table 1 . Sixty-one mutations were found in 26, 187 bases sequenced in the 5-aza-dC treated cells. Only one mutation in the drug-free control could be confirmed by sequencing in both directions and thus, the rate in the control may be over estimated. Thus, Sequencing of a fragment of the nucleic acid encoding HIV reverse transcriptase has confirmed that 5-aza-dC is mutagenic to the viral genome.

TABLE 1

[0104] Mutation of the viral genome upon treatment with compounds of Formula I is assessed as described above for 5-aza-dC-treated cells.

Assessment of mutagenic nucleoside analogue cytotoxicity

[0105] Compound cytotoxicity was evaluated on MT-2 cells. MT-2 cells were seeded at 3 x 10⁴ cells/well in 96-well plates. The cells were treated with compounds at half-log serial dilutions from 100 μΜ to 0.32 μΜ in triplicate. After 5 days growth in a 37°C incubator containing 5% C02, MTT was added to a final concentration of 0.5 mg/ml and then incubated for four hours at 37°C. 10% SDS in 0.02 N HC1 was added to lyse the cells overnight at 37°C. The plates were read on a Tecan Genius microplate reader at wavelengths of 570 nm/650 nm. The dose response curve was graphed by comparing the treated cells with the untreated control and the IC₅₀ was determined for each compound. For DHAdC, the IC₅o was greater than 1 mM The IC50 for 5-Me-DHAdC was not determined. The IC50 for the compounds of Formula I is determined in a similar matter.

In vitro passaging assays of DHAdC

[0106] Passaging experiments were performed for DHAdC (also reffered to as SN1212), to demonstrate that viral eradication is possible in vitro. The experiment was carried out in quadruplicate in the presence of SN1212 at a concentration of 100 nM. Levels of p24 fell permanently below the limit of detection (4 ng/ml) by passage 8. No infectious virus was recovered after passage 12.

DHAdC is a viral mutagen

[0107] Assessment of DHAdC viral mutagenicity was carried out as described above for 5- aza-dC. Mutagenesis of the sense strand of a 0.9 kb fragment of reverse transcriptase of HIV NL4-3 was determined after a single passage in SN 1212 (50 μΜ) and compared to an untreated control. Results are shown in Table 2.

TABLE 2

[0108] The mutation rate induced by 50 μΜ SN 1212 in HIV RT is 1 .4-fold higher than control (0.0015 in DHAdC treated versus 0.001 1 in control). The dominant mutations are C<→T transitions (enhanced 4.6-fold by SN 1212), with a minority of transversions

(pyrimidine-<→purine). In contrast, 5-OH-dC demonstrated only a 1 .14-fold increase in overall mutation rate over background.

DHAdC does not cause significant mutagenesis of cellular DNA

[0109] SN 1212 is a poor substrate for polymerase-a, the cellular polymerase responsible for most DNA synthesis. (Data not shown.) An hgprt assay was also performed to test mutagenesis of cellular DNA by DHAdC. The assay was performed on CHO (Chinese Hamster Ovary) cells and mutants were selected for resistance to 6-thioguanine (6-TG). EMS (ethyl methyl sulfonate), a known mutagen, was used as a positive control. SN 1212 at a concentration of 1 mM did not increase above background the mutation frequency of a cellular gene, hgprt. (Data not shown.) Of note, the EC₅₀ of DHAdC against HIV is in the range of 10 nM, while no significant mutation to cellular DNA is noted at 1 mM, a 10,000-fold difference.

[0110] Mitochondrial toxicity is also a safety concern with nucleoside analogs. SN1212 was also analyzed for mitochondrial toxicity. SN1212 does not demonstrate evidence of mitochondrial toxicity by either an increase in lactate production or inhibition of mitochondrial DNA at the highest dose tested, 320 μΜ. (Data not shown.)

DHAdC is effective against wild-type HIV strains and NRTI resistant HIV strains

[0111] The effectiveness of DHAdC was tested against wild-type HIV strains and NRTI resistant HIV strains as described in Example 2. The following strains were tested: HIV-1 LAI, wild-type; HIV-1 LAI-M 184V-M 184V mutation with resistance to lamivudine (3TC); HIV-1 RTMDR1-74V, 41L, 106A and 215Y mutations with resistance to zidovudine, didanosine, nevirapine and other non-nucleoside reverse transcriptase inhibitors; and HIV-1 RTMC-67N, 70R, 215F and 219Q with resistance to zidovudine. Results are shown in Table 3. TABLE 3

[0112] The EC₅₀'s of SN1212 were the same in wild-type and the three mutant HIV strains, confirming the lack of cross-resistance between SN 1212 and NRTI. Furthermore, based on HIV passaging experiments designed to favor the emergence of resistant strains performed with SN 1212, it appears unlikely that de novo resistance will develop to SN 1212.

DHAdC is effective in treating HIV infections in a mouse model

[0113] SN 1212 was administered at up to 100 mg/kg/day subcutaneously in SCID-Hu Thy/Liv mice for 21 days, without any significant toxicity being demonstrated. After completion of this toxicology experiment, SN 1212 was tested in HIV infected SCID-Hu mice. While SN 1212 did not demonstrate reduction in p24 or HIV RNA, it demonstrated a significant decrease in viral infectivity when compared to untreated animals at a dose of 10 mg/kg (see, e.g., Table 4). The discordance between viral infectivity and conventional surrogate markers of viral load, such as p24 or HIV RNA, is not surprising, as it has also been observed in vitro, and reflects the increased proportion of non-infectious viral particles in the presence of SN 1212. It is also interesting to note that, of the treated groups, the immunologic profile of the SN1212 groups most closely resemble that of the uninfected group. This is compatible with the finding that infection with less "fit" viruses provides a relative clinical benefit by preserving cellular immunity.

TABLE 4

*No drug control. **p<0.05

[0114] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in entirety to the same extent as if each reference was individually incorporated by reference. Where a conflict exists between the instant application and a reference provided herein, the instant application shall dominate.

Claims

WHAT IS CLAIMED IS :

A compound of Formula I:

wherein

X and Y are each independently selected from the group consisting of O, S and N(R^a); Z is selected from the group consisting of CHR^a, O, S, and N(R^a);

R¹ is C i-20 alkyl;

R² and R³ are each independently selected from the group consisting of H and C i_-6 alkyl ; R⁴, R⁵ and R⁶ are each independently selected from the group consisting of H, OH and Ci- alkoxy;

each R^a is independently selected from the group consisting of H and Ci_-6 alkyl;

wherein when X is NH, Y is O, and Z is CH₂, then R¹ is C o alkyl, and when X is NH, Y is O and Z is O, then R¹ is Ci_₆ alkyl;

and salts and isomers thereof.

2. The compound of claim 1 , wherein

X is NH;

Y is O or S;

Z is selected from the group consisting of C¾, O, S and NH;

R², R³ and R⁴ are each H ; and

R⁵ and R⁶ are each OH.

3. The compound of any of claims 1 or 2, wherein Y is O and Z is CH2.

4. The compound of any of claims 1 or 2, wherein Y is O and Z is NH.

5. The compound of any of claims 1 or 2, wherein Y is S and Z is 0.

6. The compound of any of claims 1 or 2, wherein Y is S and Z is NH.

7. The compound of any of claims 1 or 2, wherein Y is S and Z is S.

8 The compound of any of claims 1 or 2, wherein the compound is selected from the group consisting of:

l -heptyl-3-[5-((2R,4S,5R)-4-hydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-4-oxo- l ,4,5,6-tetrahydro-[l ,3,5]triazin-2-yl]-thiourea,

l -heptyl-3-[5-((2R,4S,5R)-4-hydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-4-oxo- l ,4,5,6-tetrahydro-[l ,3,5]triazin-2-yl]-urea,

octanoic acid [5-((2R,4S,5R)-4-hydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-4-oxo-

1 ,4,5,6-tetrahydro-[l ,3,5]triazin-2-yl]-amide,

[5-((2R,4S,5R)-4-hydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-4-oxo-l , 4,5,6- tetrahydro-[l ,3,5]triazin-2-yl]-thiocarbamic acid heptyl ester; and phenyl [5-((2R,4S,5R)-4-hydroxy-5-hydroxymethyI-tetrahydrofuran-2-yl)-4-oxo-l ,4,5,6- tetrahydro-[l ,3,5]triazin-2-yl]-carbamodithioate.

9. A pharmaceutical composition comprising a compound of any of claims 1 to 8 and a pharmaceutically acceptable excipient.

10. A method of hypomethylating an oligonucleotide, comprising contacting the oligonucleotide and a compound of Formula I or metabolites thereof: R¹

wherein

X and Y are each independently selected from the group consisting of O, S and N(R^a);

Z is selected from the group consisting of CHR^d, O, S, and N(R^d); R¹ is Ci.20 alkyl;

R² and R³ are each independently selected from the group consisting of H and C_w alkyl;

R⁴. R⁵ and R⁶ are each independently selected from the group consisting of H, OH and Ci_₆ alkoxy; and

each R^a is independently selected from the group consisting of H and C]-₆ alkyl; thereby hypomethylating the oligonucleotide.

1 1. A method of treating cancer, comprising administering to a patient in need thereof a compound of Formula I:

wherein

X and Y are each independently selected from the group consisting of O, S and N(R^a);

Z is selected from the group consisting of CHR^a, O, S, and N(R ); R¹ is C!_2o alkyl;

R² and R³ are each independently selected from the group consisting of I I and Ci.₆ alkyl;

R⁴, R⁵ and R⁶ are each independently selected from the group consisting of H, OH and Ci_6 alkoxy; and

each R^a is independently selected from the group consisting of H and Cj_6 alkyl; thereby treating the cancer.

12. A method of treating human immunodeficiency virus (HIV), comprising administering to a patient in need thereof a compound of Formula I: R¹

wherein

X and Y are each independently selected from the group consisting of O, S and N(R^a);

Z is selected from the group consisting of CHR^a, O, S, and N(R^a); R¹ is Ci-20 alkyl;

R² and R³ are each independently selected from the group consisting of H and Ci-6 alkyl;

R⁴, R⁵ and R⁶ are each independently selected from the group consisting of H, OH and Ci_6 alkoxy; and

each R^a is independently selected from the group consisting of H and Cj.6 alkyl; thereby treating the HIV.