US20100087388A1

US20100087388A1 - Odcase inhibitors as anti-virals and antibiotics

Info

Publication number: US20100087388A1
Application number: US12/089,100
Authority: US
Inventors: Lakshmi P. Kotra; Emil F. Pai
Original assignee: Individual
Current assignee: Individual
Priority date: 2005-10-03
Filing date: 2006-10-03
Publication date: 2010-04-08
Also published as: WO2007038860A2; US20090221524A1; CN101321775A; WO2007038860A8; AP2870A; AP2008004436A0; EP1931691A4; WO2007038859A1; CA2665370A1; EP1931691B1; CN101321775B; EP1931691A1; US8067391B2; WO2007038860A3; ES2436404T3

Abstract

The present invention includes the utility of anti-viral and/or antibacterial effective amounts of 6-substituted nucleoside derivatives of formula (I) (e.g. 6-iodouridine and 6-iodouridine monophosphate) in the treatment or prevention of viral infections (e.g. Flavivridae, Bunyaviridae, or Togaviridae, or viral infections of hepatitis C, hepatitis B, herpes, influenza, HIV, polio, Coxsackie A/B, rhino, small pox, Ebola, West Nile, or corona virus) and/or bacterial infections (e.g. H. pylori, S. Aureus, B. anthracis, Mycobacterial tuberculosis, M. leprae, M. avium, P. aueruginosa, Streptococcal species, and Pneumocystis carinii).

Description

FIELD OF THE INVENTION

The present invention relates to methods of using certain 6-substituted uridine compounds for the treatment and prevention of viral and bacterial infections.

BACKGROUND OF THE INVENTION

ODCase (EC 4.1.1.23) plays a central role in the de novo synthesis of uridine-5′-O-monophosphate (UMP). UMP is a building block, synthesized de novo from aspartic acid, for the synthesis of other pyrimidine nucleotides such as uridine-5′-O-triphosphate (UTP), cytidine-5′-O-triphosphate (CTP), thymidine-5′-O-triphosphate (TMP) and 2′-deoxy-cytidine-5′-O-triphosphate (dCTP) (FIG. 1). Pyrimidine nucleotides are the building blocks for the synthesis of RNA and DNA, the essential molecules for cell replication and survival. Due to its important role in the cell's de novo nucleic acid synthesis, ODCase is present in bacteria, archea, parasites and in humans, i.e. almost every species except in viruses. This enzyme catalyzes the decarboxylation of orotidine monophosphate (OMP) to uridine monophosphate (UMP) ( compounds 1 and 2 in the final step in FIG. 1). This enzyme is particularly interesting for enzymologists because it exhibits an extraordinary level of catalytic rate enhancement of over 17 orders of magnitude compared to the uncatalyzed decarboxylation reaction in water at neutral pH 7.0, 25° C.^i,iiAn uncatalyzed decarboxylation of OMP takes about 78 million years, and the enzymic decarboxylation occurs at a millisecond time scale. Thus, ODCase is one of the most proficient members of the enzymic world.^ii,iii,iv,v
Interestingly, decarboxylases found in Nature use either a cofactor or covalent intermediates during the catalysis of decarboxylation reactions.^vi,viiFor example, thiamin diphosphate-dependent, indole pyruvate decarboxylase (IPDC) uses thiamin as a cofactor and there are covalent intermediates formed with the cofactor during the decarboxylation process. ODCase is thought to be quite unusual in catalyzing decarboxylation with such proficiency without the help of any co-factors, metals, or covalent-intermediates.^i,ii,iiiOne interesting difference when one looks at this enzyme across species is that in certain higher level organisms such as human or mouse, ODCase is a part of the bifunctional enzyme, UMP synthase.^viiiIn pathogenic organisms such as bacteria, fungi and parasites, ODCase is a monofunctional enzyme.^ix,xIn all species, ODCase (whether monofunctional or bifunctional) is active as a dimeric unit.
In general, investigations targeting ODCase focused on malaria, cancer and few antiviral investigations. In the past two decades, several analogs of OMP were investigated extensively to understand the catalytic mechanism of ODCase.^iii,xiAmong these analogs, 6-aza-UMP (3) and 6-hydroxy-UMP (or BMP, 4), pyrazofurin, xanthosine-5′-monophosphate (XMP, 12) and 6-thiocarboxamido-UMP (13) are some of the potent inhibitors that were studied against ODCase (FIG. 2).^xii,xiii,xivHowever, the development of inhibitor candidates has been limited due to their toxicities and lack of specificity.^xiiThere is also very limited or non-existent structure-activity relationship investigations and inhibitor design against ODCase. Thus, ODCase has not gained much traction in 1980s and 1990s as a drug target.
Aside from its obvious pharmacological interest, ODCase has been a favorite enzyme for biochemists and structural biologists due to its unusual catalytic properties. A number of mechanisms were proposed prior to and after the availability of X-ray crystal structures for several ODCases in 2000.^{xv,xvi,xvii,xviii}Although ideas of covalent catalysis were discussed, none of the mechanisms presented included a covalent species formation as a key step during the decarboxylation by ODCase. An analysis of the catalytic site of ODCase from Methanobacterium thermoautotrophicum (Mt) revealed two aspartate residues (Asp70 and Asp75B, the latter contributed by the second subunit of the dimeric ODCase) and two lysine residues (Lys42 and Lys72) that are held via a strong network of hydrogen bonds (FIG. 3). Analyses of several co-crystal structures of ODCase with a variety of ligands confirm that these residues are held tightly in their respective positions in the active site and there is less than 0.5 Å movement in the positions of the side chains of these residues. Existing evidence does not support any active site residue forming a covalent bond either to the substrate during catalysis or to any known inhibitor.^{iii,xvi,xix,xx,xxi}The above four residues are proposed to exert strong steric and electrostatic stress onto the C-6 carboxylate group of OMP and eliminate the carboxyl group.^xvi
The x-ray crystal structures of ODCase from ten different species are known today. In 2000, four x-ray crystal structures of ODCase brought insights into the catalytic mechanism of this enzyme. Based on the structure of S. cerevisiae ODCase complexed with the transition-state analogue BMP (4), a transition-state stabilization mechanism of OMP decarboxylation was proposed.^xviiiA similar proposal was also suggested by Appleby et al. based on the crystal structure of ODCase (Bacillus subtilis) complexed with the product, UMP.^xviiThese authors suggested that the decarboxylation reaction proceeds via an electrophilic substitution in which C-6 is protonated by Lys62 as the carbon dioxide molecule is released.^xviiThe structure of the ODCase enzyme from E. coli co-crystallized with BMP was the basis of the proposal submitted by Harris et al.^xxiiBased on the proximity of the carboxylate moiety on OMP (1) and Asp71 residue in the active site of ODCase, it was proposed that OMP decarboxylation depends on the existence of a shared proton between Asp71 and the carboxyl group of the substrate.^xxiiA similar mechanism involving electrostatic repulsion was put forward by Wu et al.^xviThis mechanism of OMP decarboxylation is based on the principles of the Circe effect described by Jencks in 1975.^xxiiiThe Circe effect states that only the reactive group of the substrate needs to be destabilized. The strong interaction between the unreactive part of the substrate and the enzyme active site provides the energy to directly destabilize the reactive group of the substrate.^xxiiiThe electrostatic repulsion mechanism points to the active site aspartate residue. In four different species the location and function of this residue is highly conserved. The catalytic residues, Asp70 and Lys72 are located near the reaction center C-6 of the pyrimidine ring of the substrate OMP and Asp70 (M. thermoautotrophicum) was postulated to provide the electrostatic destabilization of the enzyme-substrate complex. Lys72 in the active site furnishes the proton to neutralize the carbanion developed after the departure of the carboxylate.^xviDespite several x-ray structures and in-depth enzymology in the past two decades, ODCase continues to challenge biochemists with still-unresolved mechanism and new twists (vide infra).
In the active site of ODCase, the monophosphate group of OMP is proposed to bind first and this group contributes the largest energy required for the binding of the substrate to ODCase.^xxivThe removal of phosphate from the molecule of substrate resulted in a significantly lower catalytic efficiency measured as the second-order rate constant (k_cat/K_M) for the catalysis of substrate to product.^xxivIn an interesting experiment, the binding of phosphite dianion (HPO₃ ²⁻) to ODCase (from S. cerevisiae) resulted in an 80,000 fold increase in the second order rate constant for the decarboxylation of the truncated substrate lacking a phosphate moiety.^xxvThus, the phosphate group is an important component for ODCase binding. Thus, in order for nucleoside drugs (correct terminology is prodrugs) to be active against ODCase in vivo, the nucleoside compound has to be converted into its monophosphate form inside the cell by any nucleoside kinase and then inhibit ODCase (whether in a pathogen or human cell). This is very similar to other nucleoside drugs such as AZT, 3TC, gemcitabine among several nucleoside drugs that are clinically used, thus there is a good possibility for the “nucleoside forms” of ODCase inhibitors to function as drugs.
The market size for antimicrobial agents is more than US$25 billion per year and the emergence of bacterial resistance worldwide is a limiting factor to current drugs. In the US alone, more than 80 million people are infected with H. pylori. Worldwide, 25-75% of people are infected with H. pylori depending on the region and will eventually develop gastritic diseases, many leading to cancer. Staphylococcus aureus has become a serious issue in hospitals and has recently become a community-based infection with resistance against the most effective drugs, including the widely-used methicillin, and the drug of last resort, vancomycin. New and effective drugs against these two infectious diseases, especially against the new ODCase target, have the potential to achieve significant market penetration.
The cellular machinery for the synthesis of pyrimidine nucleotides varies in different bacterial species. For example, an analysis of the complete genome of H. pylori reveals the metabolic pathways for purine and pyrimidine biosynthesis.^xxvi,xxviiInterestingly, there are no identifiable genes for the enzymes in the pyrimidine salvage pathway (except one) in H. pylori genome and thus, this organism is completely dependent on the de novo synthesis of pyrimidine nucleotides.^xxvii,xxviiiThis clearly suggests that inhibition of ODCase will have a fatal effect on the survival of H. pylori. This rationale provides a strong basis to design inhibitors against ODCase, as antibacterial agents. S. aureus based on its complete genome analysis (strain MRSA A252), appears to lack five of the eight enzymes in the pyrimidine salvage pathway.
ODCase has also been identified as a target for drugs directed against RNA viruses like pox and flavi viruses; the former causing increasing concern as a potential bio-terrorist weapon.^{xxix,xxx,xxxi,xxxii}ODCase inhibitors have also been effective against West Nile virus, a recent threat to humans and birds in the US and Canada.^xxxiii
There remains a need for new inhibitors of ODCase as therapeutic agents, for example, for the prevention and treatment of viral and bacterial infections.

SUMMARY OF THE INVENTION

Several C6 derivatives of uridine were prepared and found to be noncovalent (competitive) and covalent (irreversible) inhibitors against ODCase. These compounds also exhibited potent anti-viral and antibiotic activity.
Accordingly, the present invention includes a method of treating or preventing viral and/or bacterial infections comprising administering to a subject in need thereof a an anti-viral effective amount and/or an antibacterial effective amount of a compound selected from a compound of Formula I, tautomers thereof and pharmaceutically acceptable salts, solvates, and prodrugs thereof:
wherein,

R¹is selected from I, Br, C₁, N₃and NO₂;
R²is selected from H, halo, C₁-C₆alkyl, C₁-C₆alkoxy, fluoro-substituted-C₁-C₆alkyl, fluoro-substituted-C₁-C₆alkoxy, N₃, NH₂and CN;
R³is selected from OH, NH₂, H and NHC(O)C₁-C₆alkyl;
Z is selected from:

wherein,

R⁴is selected from H, C_j—C₆alkyl and hydroxy-substituted-C₁-C₆alkyl;
One of R⁵and R⁶is hydrogen and the other is selected from H, OH and F and one of R^5′ and R^6′ is hydrogen and the other is selected from H, OH and F or R⁵and R⁶or R^5′ and R^6′ together may be ═O or ═CH₂;
R⁷is selected from H, F and OH;
R⁸is selected from H, C(O)C₁-C₆alkyl, P(O)(OH)₂, P(O)(OC₁-C₆alkyl)₂and P(O)(OC₁-C₆alkyl)OH;
R⁹is selected from H, N₃, CN, C₁-C₆alkyl; and
X is selected from —CH₂—O—, O—CH₂— and —S—CH₂—.

In further embodiments, the present invention includes a use of a compound selected from a compound of Formula I as defined above, and pharmaceutically acceptable salts, solvates, and prodrugs thereof, for the prevention or treatment of viral infections and/or bacterial infections, as well as a use of a compound selected from a compound of Formula I as defined above, and pharmaceutically acceptable salts, solvates, and prodrugs thereof, for the preparation of a medicament for the prevention or treatment of viral infections and/or bacterial infections.
The present invention further includes a method of preventing or treating an infection of one or more bacteria and/or one or more virues in a subject comprising administering to the subject an anti-viral effective amount and/or an antibacterial effective amount of a compound selected from a compound of Formula I as defined above, and pharmaceutically acceptable salts, solvates, and prodrugs thereof.
The present invention also includes a use of a compound of Formula I as defined above, and pharmaceutically acceptable salts, solvates, and prodrugs thereof, to prevent or treat an infection of a virus and/or bacterium in a subject as well as a use of a compound of Formula I as defined above, and pharmaceutically acceptable salts, solvates, and prodrugs thereof, to prepare a medicament to prevent or treat an infection of a virus and/or bacterium in a subject
According to another aspect of the present invention, there is included a pharmaceutical composition for the treatment or prevention of viral infections and/or bacterial infections comprising a anti-viral effective amount and/or a antibacterial effective amount of a compound selected from a compound of Formula I as defined above, and pharmaceutically acceptable salts, solvates, and prodrugs thereof, and a pharmaceutically acceptable carrier therefore.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will know be described in great detail with reference to the drawings in which:

FIG. 1 is a schematic showing the de novo synthesis of uridine monophosphate from aspartic acid (prior art).

FIG. 2 is a schematic showing the chemical structures of analogs of orotidine monophosphate (OMP) that are known as inhibitors of ODCase.

FIG. 3 shows the X-ray structure of the catalytic site of ODCase from Methanobacterium thermoautotrophicum.

FIG. 4 shows bargraphs of the results of an antiviral assay in MDCK cell lines against influenza A/WSN/33. Panels A and B reflect the results of two separate experiments. Numbers on top of each bar represent the % protection due to the inhibitor in comparison to the control.

FIG. 5 shows a bargraph of the results of an antiviral assay in L2 cell likes against MHV-1 (mouse SARS-like CoV).

FIG. 6 shows graphs of primary cell toxicity data using representative compounds of Formula I.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The term “C_1-nalkyl” as used herein means straight and/or branched chain, saturated alkyl radicals containing from one to “n” carbon atoms and includes (depending on the identity of n) methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, 2,2-dimethylbutyl, n-pentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, n-hexyl and the like, where the variable n is an integer representing the largest number of carbon atoms in the alkyl radical.
The term “fluoro-substituted C_1-nalkyl” as used herein means straight and/or branched chain, saturated alkyl radicals containing from one to n carbon atoms in which one or all of the hydrogen atoms have been replaced with a fluorine, and includes (depending on the identity of “n”) trifluoromethyl, pentafluoroethyl, fluoromethyl and the like, where the variable n is an integer representing the largest number of carbon atoms in the alkyl radical.
The term “hydroxy-substituted C_1-nalkyl” as used herein means straight and/or branched chain, saturated alkyl radicals containing from one to n carbon atoms in which one or two of the hydrogen atoms have been replaced with a hydroxyl group, and includes (depending on the identity of “n”) CH₂OH, CHOHCH₂CH₃, CH₂CHOHCH₂CH₂OH and the like, where the variable n is an integer representing the largest number of carbon atoms in the alkyl radical.
The term “halo” as used herein means halogen and includes chloro, fluoro, bromo and iodo.
The term “tautomer” as used herein refers to compounds that are interconvertible by a formal migration of a hydrogen atom or proton, accompanied by a switch of a single bond and an adjacent double bond. In solutions where tautomerization is possible, a chemical equilibrium of the tautomers will be reached. The exact ratio of the tautomers depends on several factors, including temperature, solvent and pH.
The term “solvate” as used herein means a compound of Formula I, or a salt of a compound of Formula I, wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent is physiologically tolerable at the dosage administered. Examples of suitable solvents are ethanol, water and the like. When water is the solvent, the molecule is referred to as a “hydrate”.
The term “compound(s) of the invention” as used herein means compound(s) of Formula I, and salts, solvates and prodrugs thereof.
The term “pharmaceutically acceptable salt” means an acid addition salt or a basic addition salt which is suitable for or compatible with the treatment of patients.
The term “pharmaceutically acceptable acid addition salt” as used herein means any non-toxic organic or inorganic salt of any base compound of the invention, or any of its intermediates. Basic compounds of the invention that may form an acid addition salt include, for example, where the R²and/or R³is NH₂and NHC_1-6alkyl. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of the compounds of the invention are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable acid addition salts, e.g. oxalates, may be used, for example, in the isolation of the compounds of the invention, for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.
The term “pharmaceutically acceptable basic addition salt” as used herein means any non-toxic organic or inorganic base addition salt of any acid compound of the invention, or any of its intermediates. Acidic compounds of the invention that may form a basic addition salt include, for example, where R⁸is a phosphate. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium or barium hydroxide. Illustrative organic bases which form suitable salts include aliphatic, alicyclic or aromatic organic amines such as methylamine, trimethylamine and picoline, alkylammonias or ammonia. The selection of the appropriate salt will be known to a person skilled in the art. Other non-pharmaceutically acceptable basic addition salts, may be used, for example, in the isolation of the compounds of the invention, for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.
The term “viral infection” as used herein refers to the detrimental colonization of a host organism by one or more foreign viruses. The infecting virus seeks to utilize the host's resources in order to multiply (usually at the expense of the host) and interferes with the normal functioning of the host and can lead to chronic wounds, gangrene, loss of an infected limb, disease and even death. Examples of viral infections include, but are not limited to, RNA viral infections, including infections by the following virus families: Flaviviridae (yellow fever [YF], Dengue type 4 and Japanese encephalitis [JE] viruses), Bunyaviridae (Punta Toro [PT] and sandfly fever [SF] viruses) and Togaviridae (Venezuelan equine encephalomyelitis [VEE] virus), as well as hepatitis C virus (HCV), hepatitis B virus (HBV), herpes viruses, influenza virus, human immuno virus (HIV), polio virus, Coxsackie A and B viruses, Rhino virus, small pox virus, Ebola virus, West Nile virus and coronavirus (such as severe acute respiratory syndrome or SARS).
The term “bacterial infection” as used herein refers to the detrimental colonization of a host organism by one or more foreign bacteria. The infecting bacteria seeks to utilize the host's resources in order to multiply (usually at the expense of the host) and interferes with the normal functioning of the host and can lead to chronic wounds, gangrene, loss of an infected limb, disease and even death. Examples of bacterial infections include all bacterial species in which ODCase is an important component of pyrimidine synthetic pathway, for example, but are not limited to, H. pylori, S. aureus, B. anthracis, Mycobacterial species such as M. tuberculosis, M. leprae, M. avium, P. aueruginosa, Streptococcal species, Pneumocystis carinii and the like.
The term a “therapeutically effective amount”, “effective amount” or a “sufficient amount” of a compound of the present invention is a quantity sufficient to, when administered to the subject, including a mammal, for example a human, effect beneficial or desired results, including clinical results, and, as such, an “effective amount” or synonym thereto depends upon the context in which it is being applied. For example, in the context of inhibiting ODCase, for example, it is an amount of the compound sufficient to achieve such an inhibition in ODCase activity as compared to the response obtained without administration of the compound. In the context of disease, therapeutically effective amounts of the compounds of the present invention are used to treat, modulate, attenuate, reverse, or effect bacterial and/or viral infections in a mammal. An “effective amount” is intended to mean that amount of a compound that is sufficient to treat, prevent or inhibit viral and/or bacterial infection or a disease associated with viral and/or bacterial infection. In some suitable embodiments, viral and/or bacterial infection or the disease or disorder associated with viral and/or bacterial infection is caused by an RNA virus (as defined above) and/or bacteria, thus it is the amount sufficient to, when administered to the subject, including a mammal, e.g., a human, to treat, prevent or inhibit viral and/or bacterial infection or a disorder associated with viral and/or bacterial infection. The amount of a given compound of the present invention that will correspond to such an amount will vary depending upon various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art. Also, as used herein, a “therapeutically effective amount” of a compound of the present invention is an amount which prevents, inhibits, suppresses or reduces viral and/or bacterial infection (e.g., as determined by clinical symptoms or the amount of virues and/or bacteria) in a subject as compared to a control. As defined herein, a therapeutically effective amount of a compound of the present invention may be readily determined by one of ordinary skill by routine methods known in the art.
In an embodiment, a therapeutically effective amount of a compound of the present invention ranges from about 0.1 to about 7 mg/kg body weight, suitably about 1 to about 5 mg/kg body weight, and more suitably, from about 2 to about 3 mg/kg body weight. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, or prevent a subject, from being afflicted with viral and/or bacterial infections and these factors include, but are not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject and other diseases present.
Moreover, a “treatment” or “prevention” regime of a subject with a therapeutically effective amount of the compound of the present invention may consist of a single administration, or alternatively comprise a series of applications. For example, the compound of the present invention may be administered at least once a week. However, in another embodiment, the compound may be administered to the patient from about one time per week to about once daily for a given treatment. The length of the treatment period depends on a variety of factors, such as the severity of the disease, the age of the patient, the concentration and the activity of the compounds of the present invention, or a combination thereof. It will also be appreciated that the effective dosage of the compound used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required. The compounds of the present invention may be administered before, during or after exposure to a viral and/or bacterial infection.
As used herein, “administered contemporaneously” means that two substances are administered to a subject such that they are both biologically active in the subject at the same time. The exact details of the administration will depend on the pharmacokinetics of the two substances in the presence of each other, and can include administering one substance within 24 hours of administration of the other, if the pharmacokinetics are suitable. Design of suitable dosing regimens are routine for one skilled in the art. In particular embodiments, two substances will be administered substantially simultaneously, i.e. within minutes of each other, or in a single composition that comprises both substances.
As used herein, and as well understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
“Palliating” a disease or disorder means that the extent and/or undesirable clinical manifestations of a disorder or a disease state are lessened and/or time course of the progression is slowed or lengthened, as compared to not treating the disorder.
The term “prevention” or “prophylaxis”, or synonym thereto, as used herein refers to a reduction in the risk or probability of a patient becoming afflicted with a viral and/or bacterial infection or manifesting a symptom associated with a viral and/or bacterial infection.
To “inhibit” or “suppress” or “reduce” a function or activity, such as ODCase activity, is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another conditions.
The term “subject” or “patient” or synonym thereto, as used herein includes all members of the animal kingdom, especially mammals, including human. The subject or patient is suitably a human.

II. Methods of the Invention

The present invention includes a method of treating or preventing viral infections and/or bacterial infections comprising administering to a subject in need thereof an anti-viral effective and/or an antibacterial effective amount compound selected from a compound of Formula I, tautomers thereof and pharmaceutically acceptable salts, solvates, and prodrugs thereof:
wherein,

wherein,

R⁴is selected from H, C₁-C₆alkyl and hydroxy-substituted-C₁-C₆alkyl;
One of R⁵and R⁶is hydrogen and the other is selected from H, OH and F and one of R^5′ and R^6′ is hydrogen and the other is selected from H, OH and F or R⁵and R⁶or R^5′ and R^6′ together may be ═O or ═CH₂;
R⁷is selected from H, F and OH;
R⁸is selected from H, C(O)C₁-C₆alkyl, P(O)(OH)₂, P(O)(OC₁-C₆alkyl)₂and P(O)(OC₁-C₆alkyl)OH;
R⁹is selected from H, N₃, CN, C₁-C₆alkyl; and
X—Y is selected from —CH₂—O—, —O—CH₂— and —S—CH₂—.

In the method of the invention, R¹in the compounds of Formula I is selected from I, Br, C₁, N₃and NO₂. In embodiments of the invention, R¹in the compounds of Formula I is selected from I, Br or Cl. NO₂, N(C₁-C₄alkyl)₂, NHC(O)C₁-C₄alkyl and NHC(O)OC₁-C₄alkyl. In further embodiments of the invention, R¹in the compounds of Formula I is I.
In the method of the invention, R²in the compounds of Formula I is selected H, halo, C₁-C₆alkyl, C₁-C₆alkoxy, fluoro-substituted-C₁-C₆alkyl, fluoro-substituted-C₁-C₆alkoxy, N₃, NH₂and CN. In embodiments of the invention, R²in the compounds of Formula I is H. In further embodiments of the invention, R²in the compounds of Formula I is halo, suitably F, Br or I, more suitably F, when the method is for the treatment or prevention of bacterial infections.
In the method of the invention, R³in the compounds of Formula I is selected from OH, NH₂, H and NHC(O)C₁-C₆alkyl. In embodiments of the invention, R³in the compounds of Formula I is selected OH and NH₂. When R³in the compounds of Formula I is selected OH and NH₂, the compounds of formula I may exist as one of the following tautomers:
where W is O or NH. In embodiments of the invention W is O and the favoured tautomer is:
In the method of the invention, Z in the compounds of Formula I is selected from:
In an embodiment of the invention, Z is of the Formula II.
In the method of the invention, R⁴in the compounds of Formula I is selected from H, C₁-C₆alkyl and hydroxy-substituted-C₁-C₆alkyl. In an embodiment of the invention R⁴in the compounds of Formula I is H.
In the method of the invention, the compounds of Formula I include those in which one of R⁵and R⁶is hydrogen and the other is selected from H, OH and F and one of R^5′ and R^6′ is hydrogen and the other is selected from H, OH and F or R⁵and R⁶or R^5′ and R^6′ together may be ═O or ═CH₂. In an embodiment of the invention, R⁵and R^5′ are both OH and R⁶and R^6′ are both H. In a further embodiment of the invention, R⁵is H, R^5′ is OH and R⁶and R^6′ are both H.
In the method of the invention, R⁷in the compounds of Formula I is selected from H, F and OH, suitably H or OH.
In the method of the invention, R⁸in the compounds of Formula I is selected from H, C(O)C₁-C₆alkyl, P(O)(OH)₂, P(O)(OC₁-C₆alkyl)₂and P(O)(OC₁-C₆alkyl)OH. In embodiments of the invention, R⁸in the compounds of Formula I is selected from H, C(O)C₁-C₄alkyl, P(O)(OH)₂, P(O)(OC₁-C₄alkyl)₂and P(O)(OC₁-C₄alkyl)OH. In further embodiments of the invention, R⁸in the compounds of Formula I is selected from H, C(O)CH₃, P(O)(OH)₂, P(O)(OCH₃)₂and P(O)(OCH₃)OH. In still further embodiments of the invention, R⁸in the compounds of Formula I is selected from H, C(O)CH₃, and P(O)(OH)₂.
In the method of the invention, R⁹in the compounds of Formula I is selected from H, N₃, CN, C₁-C₆alkyl. Suitably R⁹is H.
In the method of the invention, X—Y in the compounds of Formula I is selected from —CH₂—O—, —O—CH₂— and —S—CH₂—. Suitably X—Y is —O—CH₂—.
It is an embodiment of the invention that R³is OH and Z is Formula II. In these compounds the keto tautomeric form is preferred. Accordingly, it is an embodiment of the invention that the compound of Formula I in the method of the invention has the following structure.
In specific embodiments of the invention, the compound of Formula I in the method of the invention for treating or preventing bacterial infections is selected from:

6-iodo uridine;
5-fluoro-6-iodo uridine;
6-iodo uridine-5′-O-monophosphate;
5-fluoro-6-iodo uridine-5′-O-monophosphate;
6-iodo uridine 5′-acetate;
5-fluoro-6-iodo uridine-5′-acetate;
6-iodo 2′-deoxyuridine;
5-fluoro-6-iodo 2′-deoxyuridine;
6-iodo 2′-deoxyuridine-5′-O-monophosphate;
5-fluoro-6-iodo 2′-deoxyuridine-5′-O-monophosphate, and
pharmaceutically acceptable salts, solvates, and prodrugs thereof.

In other embodiments of the invention, the compound of Formula I in the method of the invention for the treatment or prevention of viral infections is selected from:

6-iodo uridine;
6-iodo uridine-5′-O-monophosphate;
6-iodo uridine 5′-acetate;
6-iodo 2′-deoxyuridine;
6-iodo 2′-deoxyuridine-5′-O-monophosphate, and
pharmaceutically acceptable salts, solvates, and prodrugs thereof.

The present invention includes a method of treating or preventing viral infections comprising administering to a subject in need thereof an anti-viral effective amount compound selected from a compound of Formula I, tautomers thereof and pharmaceutically acceptable salts, solvates, and prodrugs thereof:
wherein,

R¹is selected from I, Br, C₁, N₃and NO₂;
R²is H;
R³is selected from OH, NH₂, H and NHC(O)C₁-C₆alkyl;
Z is selected from:

wherein,

The present invention includes a method of treating or preventing bacterial infections comprising administering to a subject in need thereof an antibacterial effective amount compound selected from a compound of Formula I, tautomers thereof and pharmaceutically acceptable salts, solvates, and prodrugs thereof:
wherein,

wherein,

All of the compounds of Formula I have more than one asymmetric centre. Where the compounds according to the invention possess more than one asymmetric centre, they may exist as diastereomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present invention. In suitable embodiments of the invention, the stereochemistry is that found in the natural form of uridine as depicted above. It is to be understood that while, the relative stereochemistry of the compounds of Formula I is suitably as shown above, such compounds of Formula I may also contain certain amounts (e.g. less than 20%, preferably less than 10%, more preferably less than 5%) of compounds of Formula I having alternate stereochemistry.
In further embodiments, the present invention includes a use of a compound selected from a compound of Formula I as defined above, and pharmaceutically acceptable salts, solvates, and prodrugs thereof, for the prevention or treatment of viral and/or bacterial infections as well as a use of a compound selected from a compound of Formula I as defined above, and pharmaceutically acceptable salts, solvates, and prodrugs thereof, for the preparation of a medicament for the prevention or treatment of viral and/or bacterial infections.
The present invention further includes a method of preventing or treating an infection of one or more bacteria and/or one or more virues in a subject comprising administering to the subject an anti-viral effective amount and/or an antibacterial effective amount of a compound selected from a compound of Formula I as defined above, and pharmaceutically acceptable salts, solvates, and prodrugs thereof.
The present invention also includes a use of a compound of Formula I as defined above, and pharmaceutically acceptable salts, solvates, and prodrugs thereof, to prevent or treat an infection of a virus and/or bacterium in a subject as well as a use of a compound of Formula I as defined above, and pharmaceutically acceptable salts, solvates, and prodrugs thereof, to prepare a medicament to prevent or treat an infection of a virus and/or bacterium in a subject
According to another aspect of the present invention, there is included a pharmaceutical composition for the treatment or prevention of viral and/or bacterial infections comprising an anti-viral effective amount and/or an antibacterial effective amount of a compound selected from a compound of Formula I as defined above, and pharmaceutically acceptable salts, solvates, and prodrugs thereof, and a pharmaceutically acceptable carrier or diluent.
The compounds of the invention are suitably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo.
The compositions containing the compounds of the invention can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (2003—20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999. On this basis, the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
The compounds of Formula I may be used pharmaceutically in the form of the free base, in the form of salts, solvates and as hydrates. All forms are within the scope of the invention. Acid and basic addition salts may be formed with the compounds of the invention for use as sources of the free base form even if the particular salt per se is desired only as an intermediate product as, for example, when the salt is formed only for the purposes of purification and identification. All salts that can be formed with the compounds of the invention are therefore within the scope of the present invention.
In accordance with the methods of the invention, the described compounds of the invention, may be administered to a patient in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. The compounds of the invention may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump or transdermal administration and the pharmaceutical compositions formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.
A compound of the invention may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the compound of the invention may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
A compound of the invention may also be administered parenterally. Solutions of a compound of the invention can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. A person skilled in the art would know how to prepare suitable formulations. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003—20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersion and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists.
Compositions for nasal administration may conveniently be formulated as aerosols, drops, gels and powders. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomizing device. Alternatively, the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use. Where the dosage form comprises an aerosol dispenser, it will contain a propellant which can be a compressed gas such as compressed air or an organic propellant such as fluorochlorohydrocarbon. The aerosol dosage forms can also take the form of a pump-atomizer.
Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, wherein the active ingredient is formulated with a carrier such as sugar, acacia, tragacanth, or gelatin and glycerine. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter.
The compounds of the invention, may be administered to an animal alone or in combination with pharmaceutically acceptable carriers, as noted above, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard pharmaceutical practice.
The compounds of the invention, can be used alone or contemporaneously with other agents that inhibit ODCase activity or in combination with other types of treatment (which may or may not modulate ODCase) for treating viral and/or bacterial infections.

III. Methods of Preparing Compounds of the Invention

In accordance with another aspect of the present invention, the compounds of the invention can be prepared by processes analogous to those established in the art. In particular, reactions for functionalizing the 5 and/or 6 position of a uracil, cytosine or thymine ring are well known. For example, treatment of uracil, cytosine or thymine with a strong base, such as an alkyl lithium or lithium diisopropyl amide, at reduced temperatures, such at about −60° C. to about −90° C., followed by reaction with a reagent of the Formula R¹-LG, where R¹is as defined in Formula I and LG is a suitable leaving group, such as halo, provides a compound substituted at the 6-position of the pyrimidine ring with R¹. Compounds substituted with a suitable leaving group, such as I or Br, at the 5-position of the pyrimidine ring of uracil or cytosine are commercially available or are known in the art. These compounds may be converted to their corresponding anions at reduced temperatures, such at about −60° C. to about −90° C., and reacted with a reagent of the Formula R²-LG, wherein R²is as defined in Formula I and LG is a suitable leaving group, such as halo to provide a compound substituted at the 5-position of the pyrimidine ring with R². Conversion of various R¹groups into other R¹groups can be done using standard chemistries known to those skilled in the art.
Pyrimidine compounds may be reacted with a reagent of the formula Z-LG, wherein Z is as defined in Formula I and LG is a suitable leaving group, under standard conditions to provide nucleosides of Formula I or precursors to Formula I. Such reactions would be well known to those skilled in the art. Substitution of the appropriate R¹, R²and/or R³groups on the pyrimidine ring may be done before or after the coupling of the pyrimidine ring with Z.
Pyrimidine compounds and reagents of the Formula Z-LG are commercially available or may be prepared using methods known in the art. Acylation or addition of the phosphate group on to the 5′ position of the nucleoside may be performed using known reactions.
In some cases the chemistries outlined above may have to be modified, for instance by use of protective groups, to prevent side reactions due to reactive groups, such as reactive groups attached as substituents. This may be achieved by means of conventional protecting groups, for example as described in “Protective Groups in Organic Chemistry” McOmie, J. F. W. Ed., Plenum Press, 1973 and in Greene, T. W. and Wuts, P. G. M., “Protective Groups in Organic Synthesis”, John Wiley & Sons, 3^rdEdition, 1999.
The formation of a desired compound salt is achieved using standard techniques. For example, the neutral compound is treated with an acid or base in a suitable solvent and the formed salt is isolated by filtration, extraction or any other suitable method.
The formation of solvates of the compounds of the invention will vary depending on the compound and the solvate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions.
Prodrugs of the compounds of Formula I may be, for example, conventional esters formed with available hydroxy, thiol, amino or carboxyl group. For example, available hydroxy or amino groups may be acylated using an activated acid in the presence of a base, and optionally, in inert solvent (e.g. an acid chloride in pyridine). Some common esters which have been utilized as prodrugs are phenyl esters, aliphatic (C₈-C₂₄) esters, acyloxymethyl esters, carbamates and amino acid esters.
The present invention includes radiolabeled forms of the compounds of the invention, for example, compounds of the invention labeled by incorporation within the structure ³H, ¹¹C or ¹⁴C or a radioactive halogen such as ¹²⁵I and ¹⁸F. A radiolabeled compound of the invention may be prepared using standard methods known in the art. For example, tritium may be incorporated into a compound of the invention using standard techniques, for example by hydrogenation of a suitable precursor to a compound of the invention using tritium gas and a catalyst. Alternatively, a compound of the invention containing radioactive iodo may be prepared from the corresponding trialkyltin (suitably trimethyltin) derivative using standard iodination conditions, such as [¹²⁵I] sodium iodide in the presence of chloramine-T in a suitable solvent, such as dimethylformamide. The trialkyltin compound may be prepared from the corresponding non-radioactive halo, suitably iodo, compound using standard palladium-catalyzed stannylation conditions, for example hexamethylditin in the presence of tetrakis(triphenylphosphine) palladium (0) in an inert solvent, such as dioxane, and at elevated temperatures, suitably 50-100° C. Further, a compound of the invention containing a radioactive fluorine may be prepared, for example, by reaction of K[¹⁸F]/K222 with a suitable precursor compound, such as a compound of Formula I comprising a suitable leaving group, for example a tosyl group, that may be displaced with the ¹⁸F anion.
The following non-limiting examples are illustrative of the present invention:

VI. Examples

Example 1

Synthesis of 6-iodo uridine (Ia) and 6-iodo-uridine-5′-O-monophosphate (Ib)

Compounds Ia and Ib were synthesized from uridine. Introduction of the iodo moiety at the C-6 position of protected uridine was achieved using lithium diisopropylamide followed by treatment with iodine.^xxxivDeprotection with TFA followed gave compound Ia, and the subsequent phosphorylation with phosphorus oxychloride afforded the mononucleotide Ijb^{xxxv,xxxvi,xxxvii}Then, the compound Ib was transformed into its ammonium salt by neutralization with 0.5 M NH₄OH solution at 0° C. and freeze-dried to get the ammonium salt as a powder.
(a) 5′-O-(t-Butyldimethylsilyl)-2′,3′-O-isopropylidene uridine. A stirred suspension of uridine (1 g, 4.1 mmol) in anhydrous acetone (50 mL) was treated with H₂SO₄(0.5 mL) drop wise at room temperature and the resulting mixture was stirred for an additional hour. The reaction was then neutralized with Et₃N and was concentrated. The crude mixture was purified by column chromatography (5-8% MeOH:CHCl₃) to afford 2′,3′-O-isopropylidene uridine (1.15 g, quant.) as a white solid. ¹H NMR (CDCl₃) d ppm 1.36 (s, 3H, —CH₃), 1.57 (s, 3H, —CH₃), 3.80 (dd, 1H, H-5′), 3.91 (dd, 1H, H-5″), 4.26-4.30 (m, 1H, H-4′), 4.95 (dd, 1H, H-3′), 5.02 (dd, 1H, H-1-2′) 5.56 (d, 1H, H-1′), 5.72 (d, 1H, H-5), 7.36 (d, 1H, H-6).
A stirred solution of 2′,3′-O-isopropylidene uridine (0.2 g, 0.7 mmol) in anhydrous CH₂Cl₂(3 mL) was treated with imidazole (0.095 g, 1.4 mmol) and TBDMSiCl (0.105 g, 0.7 mmol) at 0° C. The reaction mixture was brought to room temperature and stirred for an additional hour. The solvent was evaporated under vacuum and the crude was dissolved in ethyl acetate (30 mL), washed with water (15 mL), brine (15 mL) and dried (Na₂SO₄). Evaporation of the solvent and purification of the crude by column chromatography (5% MeOH in CHCl₃) yielded 5′-O-(t-butyldimethylsilyl)-2′,3′-O-isopropylidene uridine (0.27 mg, 96% yield) as a foam: ¹H NMR (CDCl₃) d ppm 0.10 (s, 6H, CH₃), 0.90 (s, 9H, CH₃), 1.36 (s, 3H, CH₃) 1.59 (s, 3H, CH₃), 3.79 (dd, 1H, H5′), 3.92 (dd, 1H, H-5″), 4.30-4.33 (m, 1H, H-4′), 4.67 (dd, 1H, H-3′), 4.75 (dd, 1H, H-2′), 5.66 (d, 1H, H-5), 5.96 (dd, 1H, H-1′), 7.68 (d, 1H, H-6), 8.47 (brs, H-1, —NH).
(b) 5′-O-(t-Butyldimethylsilyl)-6-iodo-2′,3′-O-isopropylidene uridine. A stirred solution of LDA (0.62 mL, 1.3 mmol, 2.0 M solution in THF) in anhydrous THF (2 mL) was treated with 5′-O-(t-butyldimethylsilyl)-2′,3′-O-isopropylidene uridine (0.25 g, 0.6 mmol) dissolved in 1.5 mL anhydrous THF, at −78° C. After stirring for 1 h, iodine (0.16 g, 0.6 mmol) in anhydrous THF (2 mL) was added and the mixture was stirred for an additional 5 h at the same temperature. The reaction was quenched with AcOH (0.3 mL), then brought to room temperature and dissolved in ethyl acetate (25 mL). The organic layer was washed with saturated NaHCO₃solution (10 mL), 5% Na₂S₂O₃solution (10 mL), brine (10 mL) and dried (Na₂SO₄). Evaporation of the solvent and purification of the crude by column chromatography (hexanes-ethyl acetate, 70:30) gave 5′-O-(t-butyldimethylsilyl)-6-iodo-2′,3′-O-isopropylidene uridine (0.224 g, 68%) as a yellow foam: ¹H NMR (CDCl₃) d ppm 0.06 (s, 6H, CH₃), 0.89 (s, 9H, 3CH₃), 1.35 (s, 3H, CH₃) 1.56 (s, 3H, CH₃), 3.76-3.86 (m, 2H, H5′, H-5″), 4.15-4.20 (m, 1H, H-4′), 4.81 (dd, 1H, J=4.2, 6.3 Hz, H-3′), 5.18 (dd, 1H, J=2.0, 6.3 Hz, H-2′), 6.09 (s, 1H, H-5), 6.45 (dd, 1H, J=2.0 Hz, H-1′), 8.78 (brs, 1H, NH).
(c) 6-Iodo-uridine (Ia). A stirred solution of 5′-O-(t-butyldimethylsilyl)-6-iodo-2′,3′-O-isopropylidene uridine (0.300 g, 0.572 mmol) was treated with 50% aqueous TFA (3 mL) at 0° C., brought to room temperature and stirred for 2 h in the dark. Evaporation of the solvent and purification of the crude by column chromatography (10-15% EtOH in CHCl₃) afforded 6-iodo uridine Ia (0.182 g, 0.49 mmol, 86%) as a light brown solid. UV (H₂O): λ_max=268 nm (e=8975); ¹H NMR (D₂O) δ ppm 3.77 (dd, 1H, H-5′), 3.91 (dd, 1H, H-5″), 3.978-4.032 (m, 1H, H-4′), 4.43 (t, 1H, H-3′), 4.84 (dd, 1H, H-2′), 6.06 (d, 1H, H-1′), 6.67 (s, 1H, H-5). HRMS (ESI) calculated for C₉H₁₁N₂O₆NaI (M+Na⁺) 392.9554, found 392.9565.
(d) 6-Iodo uridine-5′-O-monophosphate (Ib). A stirred solution of H₂O (0.034 g, 1.89 mmol) and POCl₃(0.28 mL, 2.97 mmol) in anhydrous acetonitrile (3 mL) was treated with pyridine (0.261 mL, 3.24 mmol) at 0° C. and stirred for 10 min. 6-Iodo uridine (0.250 g, 0.67 mmol) was added and the mixture was stirred for an additional 5 h at 0° C. The reaction mixture was then quenched with 25 mL of cold water and continued stirring for an additional hour. The evaporation of the solvent and purification of the crude by column chromatography (Dowex ion-exchange basic resin, 0.1 M formic acid) afforded 6-iodo uridine-5′-O-monophosphate (Ib) (0.207 g, 68%) as a syrup. UV (H₂O): λ_max=267 nm (e=2890); ¹H NMR (D₂O) δ ppm 3.78 (dd, 1H, H-5′), 3.91 (dd, 1H, H-5″), 3.98-4.03 (m, 1H, H-4′), 4.43 (t, H-3′), 4.84 (dd, 1H, H-2′), 6.05 (d, 1H, H-1′), 6.67 (s, 1H, H-5). ³¹P NMR (D₂O) δ ppm 2.214. HRMS (ESI, negative) calculated for C₉H₁₁N₂O₉PI (M⁻) 448.9252, found 448.9263.

Example 2

Synthesis of compounds Ic and Id

Introduction of the iodo moiety at the C-6 position of fully protected uridine was achieved through LDA and iodine, and further substitution of the iodo by the azido group produced the 6-azido derivative shown in the above scheme.^xxxviiiDeprotection of the isopropylidene and t-butyldimethylsilyl groups using trifluoroacetic acid yielded 6-azido-uridine Ic. Monophosphorylation of Id with phosphorus oxychloride to afford its mononucleotide followed by the reduction of the azido group with Pd/C gave the compound 6-amino-uridine-5′-O-monophosphate Ic in good yield.^xxxix,xl,xli
(a) 6-Azido-5′-O-(t-butyldimethylsilyl)-2′,3′-O-isopropylidene uridine. 5′-O-(t-Butyldimethylsilyl)-2′,3′-O-isopropylidene-6-iodo uridine (0.25 g, 0.48 mmol) was dissolved in dry DMF (3 mL) and NaN₃(0.034 g, 0.53 mmol) was added. The reaction mixture was stirred at room temperature for 1 hr in the dark. Organic solvent was evaporated under vacuum and the crude was dissolved in ethyl acetate (15 mL), washed with brine and dried (Na₂SO₄). Organic layers were evaporated and the crude was purified by silica gel column chromatography (1% EtOH:CHCl₃). Purification of the compound and solvent evaporation were performed in the dark to yield the title compound 6-azido-5′-O-(t-butyldimethylsilyl)-2′,3′-O-isopropylidene uridine (0.19 g, 0.44 mmol) in 92% yield as a light brown solid. ¹H NMR (CDCl₃) d 0.06 (s, 6H), 0.89 (s, 9H), 1.34 (s, 3H) 1.54 (s, 3H), 3.74-3.85 (m, 2H), 4.08-4.15 (m, 1H), 4.80 (dd, 1H, J=4.8, 6.3 Hz), 5.14 (dd, 1H, J=1.5, 6.3 Hz), 5.50 (s, 1H), 6.09 (dd, 1H, J=1.5 Hz), 9.12 (brs, 1H).
(b) 6-Azido uridine (Ic). A stirred solution of 6-azido-5′-O-(t-butyldimethylsilyl)-2′,3′-O-isopropylidene uridine (0.300 g, 0.683 mmol) was treated with 50% aqueous trifluoroacetic acid (3 mL) at 0° C. The reaction mixture was then brought to r.t. and was stirred for an additional hour. Evaporation of the solvent and purification of the crude by column chromatography (10-15% EtOH in CHCl₃) gave 6-azido uridine Ic (0.17 g, 0.61 mmol) in 89% yield as a light brown solid. UV (H₂O): λ_max=285 nm; ¹H NMR (D₂O) δ 3.77 (dd, 1H, J=5.4, 12.0 Hz), 3.89-4.00 (m, 2H), 4.43 (t, J=6.9 Hz 1H), 4.77 (dd, 1H, J=3.6, 6.9 Hz), 5.76 (s, 1H), 6.07 (d, 1H, J=3.6 Hz). HRMS (ESI) Calculated for C₉H₁₁N₅O₆Na (M+Na⁺) 308.0601, found 308.0597.
(c) 6-Azido uridine-5′-O-monophosphate (Id). A stirred solution of water (0.03 g, 1.89 mmol) and POCl₃(0.28 mL, 2.97 mmol) in anhydrous acetonitrile (3 mL) was treated with pyridine (0.26 mL, 3.24 mmol) at 0° C. and stirred for 10 min. 6-Azido uridine Ic was added (0.25 g, 0.68 mmol) and the mixture was stirred for an additional 5 hr at 0° C. The reaction mixture was quenched with 25 mL of cold water and the stirring was continued for another hour. Evaporation of the solvent and purification of the crude by column chromatography (Dowex ion-exchange basic resin, 0.1 M formic acid) gave the mononucleotide Id (0.23 g, 0.63 mmol) in 60% yield as syrup. UV (H₂O) λ_max=283 nm; ¹H NMR (D₂O) δ 3.78-3.85 (m, 1H), 3.89-4.00 (m, 2H), 4.34 (t, J=6.9 Hz 1H), 4.80 (m, 1H), 5.73 (s, 1H), 6.04 (brs, 1H). ³¹P NMR (D₂O) δ ppm 2.47. HRMS (ESL negative) Calculated for C₉H₁₁N₅O₉P (M⁻) 364.0299, found 364.0307.

Example 3

Anti-Viral Activity

Molecules containing the core structure, formula I, with specific substitutions at C-6 position (R₁) of the pyrimidine moiety are either noncovalent or covalent inhibitors of orotidine monophosphate decarboxylase (ODCase). The molecular structures listed above also include, but are not limited to, all chemically-reasonable tautomeric forms of the above structures as well as the prodrugs forms that release the above mentioned compounds and their tautomers. These molecules, described above, exhibit antiviral activities and protect the cells from viral infections. Such molecules can be used in the treatment of viral infections either alone or in combination with other methods of treatment.
Selected compounds of Formula I were incubated with MDCK cells (for influenza virus A/WSN/33) and L2 cells (for MHV-1 infection—a mouse SARS like corona virus representing a model for human SARS-like corona virus) for 5 hrs with 100 μM compounds. These compound-treated cells were then infected with the appropriate virus with a dose equivalent to kill 100% cells within 24 h. Results for compounds Ia and Ib are depicted in FIGS. 4 and 5. Selected compounds of Formula I were evaluated for their effect on the human primary blood cells for any indications of toxicity. Compound Ia did not inhibit primary cells significantly.

Example 4

Antibacterial Activity

ODCase gene, pyrF, was obtained from the complete genomes of the respective bacteria through ATCC. The pyrF gene was amplified from the genome of S. aureus M252 using two synthetic primers: Dir 5′-CCGGAATTCATGATGAAAGATTTACCAATTATTGCATTAG [SEQ ID NO: 1] and Rev 5′-CGGAAGCTTTTATACTAACCAACTTTCTTTAATTTTATGATAACTTTCG [SEQ ID NO: 2].
The pET expression system was used to express ODCase from pyrF. The cloning strategy can use any generic strategies, herein a double digestion of pET24a(+) expression vector and pyrF separately using EcoRI and HindIII restriction enzyme was used to produce digestion products with sticky ends. The sticky digestion products were then ligated together producing pyrF::pET24a(+) cloned DNA. pET24a(+) vector was isolated from pET24a(+)/NovaBlue stock cells which were grown overnight at 37° C. in LB growth medium containing 30 mg/mL kanamyocin. pET24a(+) vector was isolated from the NovaBlue cells via QIAprep spin Miniprep kit using microcentrifuge Protocol. The isolated vector was double digested in 50 uL solution containing 1× EcoRI buffer, 1 uL EcoRI (20 000 U/mL) and 0.5 uL HindIII (20 000 U/mL) producing sticky ends. The digested vector was separated from the restriction enzyme by gel purification by QIAquick Gel Extraction Kit Protocol using microcentrifuge and quantified on 1% agarose gel with 2-log ladder as standard. In addition to quantifying DNA, 1% agarose gel was run with undigested vector to confirm that the vector was digested.
pyrF from the PCR mixture was also double digested in 50 uL solution using the same conditions described above. The separation of the pyrF from restriction enzymes was carried out by QIAquick Gel Extraction Kit Protocol using Microcentrifuge, and the collected gene was quantified on 1% agarose gel. The digested/purified pyrF was then ligated to the digested/purified pET24a(+) using T4 Ligase. The ligation reaction was carried out in 10 uL solution containing 70 ng of vector and mass of gene that was 5× that of the vector. The reaction was incubated at 37° C. for 3 hours. 5 uL of the ligation mixture was then used to transform the ligation product (pyrF::pET24a(+)) to NovaBlue cells and was plated onto Agar/LB/Kanamycin plates. The pyrF::pET24a(+) vector was isolated from NovaBlue cells and used to transform BL21(DE3) cells. To induce ODCase expression, pyrF::pET24a(+)/BL21(DE3) was grown in 1 L TB growth medium containing 30 mg/mL Kanamyocin. The cells were grown at 37° C. until OD₆₀₀=0.6. Once OD₆₀₀=0.6, the cells were induced by the addition of IPTG to a final concentration of 0.3 mM. The induced cells were incubated for 16 hours at 25° C. The cells were harvested by centrifugation and the collected pellet was stored at −80° C. until use. Harvested cells were dissolved in 30 mL of 20 mM Tris (pH 7.8) buffer and sonicated to release the proteins within the cell followed by centrifugation to collect insoluble components, including membranes and proteins as a pellet. The pellet was discarded and the supernatant was directly loaded to a 100 mL DEAE anion exchange column. Bound protein was eluted by an increasing sodium chloride gradient (0.1 to 0.4 M sodium chloride). Samples from the elution fraction were run on 15% SDS gel and stained with Coomassie Blue staining solution to determine protein content and purity of the fractions. The proteins were then loaded to a 320 mL Sephacryl S-200 HiPrep26-60 size exclusion column. The proteins were eluted using 1×PBS buffer at pH 7.2. The fractions containing the enzyme were concentrated and desalted by buffer exchange with 20 mM Tris (pH 7.8).
ODCase from H. pylori and other bacterial strains can be obtained using the above general procedure. This enzyme is then used to evaluate ODCase inhibitory activities of nucleotide derivatives.
Enzyme inhibition assays were performed using standard protocols (see for example Poduch, E.; Bello, A. M.; Tang, S.; Fujihashi, M.; Pai, E. F.; Kotra, L. P. J. Med. Chem. 2006, 49, 4937-4935).
Antimicrobial activities are carried out using standard protocols with various dilutions of the inhibitors such as the broth dilution method, and MIC₅₀and other relevant parameters are computed.
The K₁for compound Ib against Staphylococcus aureus ODCase was less than 50 nM indicating that this compound is a potent antibacterial agent for this microorganism.
While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present application is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.

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Claims

1. A method of treating or preventing viral and/or bacterial infections comprising administering to a subject in need thereof a anti-viral effective amount and/or an antibacterial effective amount of compound selected from a compound of Formula I, tautomers thereof and pharmaceutically acceptable salts, solvates, and prodrugs thereof:

wherein,

R¹is selected from I, Cl, Br, N₃and NO₂;

R²is selected from H, halo, C₁-C₆alkyl, C₁-C₆alkoxy, fluoro-substituted-C₁-C₆alkyl, fluoro-substituted-C₁-C₆alkoxy, N₃, NH₂and CN;

R³is selected from OH, NH₂, H and NHC(O)C₁-C₆alkyl;

Z is selected from:

wherein,

R⁴is selected from H, C₁-C₆alkyl and hydroxy-substituted-C₁-C₆alkyl;

One of R⁵and R⁶is hydrogen and the other is selected from H, OH and F and one of R^5′ and R^6′ is hydrogen and the other is selected from H, OH and F or R⁵and R⁶or R^5′ and R^6′ together may be ═O or ═CH₂;

R⁷is selected from H, F and OH;

R⁸is selected from H, C(O)C₁-C₆alkyl, P(O)(OH)₂, P(O)(OC₁-C₆alkyl)₂and P(O)(OC₁-C₆alkyl)OH,

R⁹is selected from H, N₃, CN, C₁-C₆alkyl; and

X—Y is selected from —CH₂—O—, —O—CH₂— and —S—CH₂—.

2. The method according to claim 1, wherein R¹in the compounds of Formula I is selected from I, Br and Cl.

3. The method according to claim 2, wherein R¹in the compounds of Formula I is I.

4. The method according to claim 1, wherein R²in the compounds of Formula I is H.

5. The method according to claim 1, wherein R²in the compounds of Formula I is F.

6. The method according to claim 1, wherein R³in the compounds of Formula I is selected from OH and NH₂.

7. The method according to claim 6, wherein R³in the compounds of Formula I is OH and the compound of Formula I has the following tautomeric structure:

8. The method according to claim 1, wherein in the compounds of Formula I, Z is Formula II.

9. The method according to claim 1, wherein R⁴in the compounds of Formula I is H.

10. The method according to claim 1, wherein, R⁵and R^5′ are both OH and R⁶and R^6′ are both H.

11. The method according to claim 1, wherein R⁵is H, R^5′ is OH and R⁶and R^6′ are both H.

12. The method according to claim 1, wherein R⁷in the compounds of Formula I is H or OH.

13. The method according to claim 1, wherein R⁸in the compounds of Formula I is selected from H, C(O)C₁-C₄alkyl, P(O)(OH)₂, P(O)(OC₁-C₄alkyl)₂and P(O)(OC₁-C₄alkyl)OH.

14. The method according to claim 13, wherein R⁸in the compounds of Formula I is selected from H, C(O)CH₃, P(O)(OH)₂, P(O)(OCH₃)₂and P(O)(OCH₃)OH.

15. The method according to claim 14, wherein R⁸in the compounds of Formula I is selected from H, C(O)CH₃, and P(O)(OH)₂.

16. The method according to claim 1, wherein R⁹in the compounds of Formula I is H.

17. The method according to claim 1, wherein X—Y is O—CH₂—.

18. The method according to claim 1, wherein the compound of Formula I has the following structure:

19. The method according to claim 1, wherein the viral infection is an RNA viral infection.

20. The method according to claim 19, wherein the RNA viral infection is from a viral family selected from Flaviviridae, Bunyaviridae and Togaviridae, or is from a virus selected from hepatitis C virus (HCV), hepatitis B virus (HBV), herpes viruses, influenza virus, human immuno virus (HIV), polio virus, Coxsackie A and B viruses, Rhino virus, small pox virus, Ebola virus, West Nile virus and coronavirus.

21. The method according to claim 1, wherein the bacterial infection is from a bacterium selected from H. pylori, S. aureus, B. anthracis, Mycobacterial species (such as M. tuberculosis, M. leprae and M. avium), P. aueruginosa, Streptococcal species and Pneumocystis carinii.

22. A method of treating or preventing viral infections comprising administering to a subject in need thereof an anti-viral effective amount of a compound selected from a compound of Formula I, tautomers thereof and pharmaceutically acceptable salts, solvates, and prodrugs thereof:

wherein,

R¹is selected from I, Br, C₁, N₃and NO₂;

R²is H;

R³is selected from OH, NH₂, H and NHC(O)C₁-C₆alkyl;

Z is selected from:

wherein,

R⁴is selected from H, C₁-C₆alkyl and hydroxy-substituted-C₁-C₆alkyl;

R⁷is selected from H, F and OH;

R⁸is selected from H, C(O)C₁-C₆alkyl, P(O)(OH)₂, P(O)(OC₁-C₆alkyl)₂and P(O)(OC₁-C₆alkyl)OH;

R⁹is selected from H, N₃, CN, C₁-C₆alkyl; and

X—Y is selected from —CH₂—O—, —O—CH₂— and —S—CH₂—.

23. The method according to claim 22, wherein the viral infection is an RNA viral infection.

24. The method according to claim 23, wherein the RNA viral infection is from a viral family selected from Flaviviridae, Bunyaviridae and Togaviridae, or is from a virus selected from hepatitis C virus (HCV), hepatitis B virus (HBV), herpes viruses, influenza virus, human immuno virus (HIV), polio virus, Coxsackie A and B viruses, Rhino virus, small pox virus, Ebola virus, West Nile virus and coronavirus.

25. The method according to claim 22, wherein the compound of Formula I is selected from:

6-iodo uridine;

6-iodo uridine-5′-O-monophosphate;

6-iodo uridine 5′-acetate;

6-iodo 2′-deoxyuridine 5′-acetate, and

pharmaceutically acceptable salts, solvates, and prodrugs thereof.

26. A method of treating or preventing bacterial infections comprising administering to a subject in need thereof an antibacterial effective amount compound selected from a compound of Formula I, tautomers thereof and pharmaceutically acceptable salts, solvates, and prodrugs thereof:

wherein,

R¹is selected from I, Br, C₁, N₃and NO₂;

R³is selected from OH, NH₂, H and NHC(O)C₁-C₆alkyl;

Z is selected from:

wherein,

R⁴is selected from H, C₁-C₆alkyl and hydroxy-substituted-C₁-C₆alkyl;

R⁷is selected from H, F and OH;

R⁹is selected from H, N₃, CN, C₁-C₆alkyl; and

X—Y is selected from —CH₂—O—, —O—CH₂— and —S—CH₂—.

27. The method according claim 26, wherein the bacterial infection is from a bacterium selected from H. pylori, S. aureus, B. anthracis, Mycobacterial species (such as M. tuberculosis, M. leprae and M. avium), P. aueruginosa, Streptococcal species and Pneumocystis carinii.

28. The method according to claim 26, wherein the compound of Formula I is selected from:

6-iodo uridine;

5-fluoro-6-iodo uridine;

6-iodo uridine-5′-O-monophosphate;

5-fluoro-6-iodo uridine-5′-O-monophosphate;

6-iodo uridine 5′-acetate;

5-fluoro-6-iodo uridine-5′-acetate;

6-iodo 2′-deoxyuridine;

5-fluoro-6-iodo 2′-deoxyuridine;

6-iodo 2′-deoxyuridine-5′-O-monophosphate;

5-fluoro-6-iodo 2′-deoxyuridine-5′-O-monophosphate, and

pharmaceutically acceptable salts, solvates, and prodrugs thereof.