EA039123B1 - Phosphoramidates for the treatment of hepatitis b virus - Google Patents

Abstract

Disclosed are phosphoramidate prodrug compounds of clevudine having the structural formulaor pharmaceutically acceptable salts thereof, pharmaceutical compositions comprising said compounds and methods of treating or preventing viral infections caused by DNA viruses using said compounds of clevudine, without myopathy problems.

Description

Disclosed are phosphoramidate prodrugs of clevudine having the structural formula

039123 Bl or their pharmaceutically acceptable salts, pharmaceutical compositions containing these compounds, and methods for the treatment or prevention of viral infections caused by DNA viruses, using these compounds of clevudine, without problems of myopathy.

State of the art

Hepatitis B virus (HBV) is an infectious disease that infects the liver, resulting in an acute infection with symptoms occurring after 45 to 160 days, or a chronic infection affecting 350 million people worldwide. An estimated 600,000 deaths occur each year as a result of HBV infection. HBV carries a 3.2 kb loose circular DNA (rcDNA) genome that is used to form covalently closed circular DNA (cccDNA) in the host cell. The cccDNA is then transcribed by RNA polymerase II, the host's DNA-dependent RNA polymerase, to form pregenomic RNA (pgRNA). The pgRNA is then used by the virus-encoded reverse transcriptase to form rcDNA. The goal of current treatments for chronic HBV infection is to reduce HBV replication and reduce the severity of liver damage.

Current treatments for chronic HBV infection include pegylated interferon-alpha and nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs). The NRTI is converted to the corresponding 5'-triphosphate, or diphosphate in the case of phosphonate-containing NRTIs, and reduces viral replication by inhibiting the HBV-encoded polymerase. Clevudine is an NRTI that is no longer considered for the treatment of chronic HBV due to drug-related skeletal myopathies that result from mitochondrial dysfunction in patients. Interestingly, clevudine triphosphate has been shown to be a competitive non-substrate inhibitor of HBV-encoded polymerase, and due to its long cellular half-life, is able to suppress HBV replication for an extended period of time after drug withdrawal.

Thus, novel compounds are needed for the treatment of HBV. The compositions and methods disclosed herein address this and other needs.

Brief description of the essence of the invention

In accordance with the objectives of the described materials and methods implemented and described in detail in the present invention, the disclosed subject matter of the invention in one aspect relates to compounds, compositions and methods for obtaining and using compounds and compositions. In specific aspects, the disclosed subject matter relates to clevudine derivatives and methods for making and using such derivatives. In addition, methods for treating HBV infections with the disclosed compounds are disclosed. Additional advantages will be set forth in part in the description which follows and in part will be apparent from the description or may be learned by practicing the aspects described below. The advantages described below will be realized and achieved with the elements and combinations specifically set forth in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory only, and are not limiting.

Brief description of graphic materials

The accompanying graphics, which are included in this document and form part of this document, illustrate several of the aspects described below.

Fig. 1 is a graph illustrating mean tissue levels of clevudine 5'-triphosphate in po treated EIDD-2173 rats;

fig. 2 is a graph illustrating the uptake and metabolism of EIDD-02173 (20 μM) by Huh-7 cells;

fig. 3 is a graph illustrating the tissue levels of clevudine nucleoside in rats PO treated with EIDD-2173 (5'-phosphoramidate).

Detailed Description of the Invention

The materials, compounds, compositions, and methods described herein will be better understood with reference to the following detailed description of specific aspects of the disclosed subject matter, figures, and examples included herein.

Before describing and disclosing materials, compounds, compositions, and methods, it should be emphasized that it is to be understood that the aspects described below are not limited to specific synthetic methods or specific reagents, as these may vary. In addition, it should be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

In addition, this document provides references to various publications. The descriptions of these publications are incorporated herein in their entirety by reference in order to more fully describe the state of the art in the field to which the disclosed substances relate. In addition, the disclosed references are also individually and specifically incorporated herein by reference to the material contained therein, which is discussed in the sentence in which the reference is made.

General definitions.

In the present description and the claims below, reference will be made to a number of terms, which are to be defined as having the following meaning.

In the description and claims, the word contain and other forms of this word, such as containing and contains, mean, including, but not limited to, and are not intended to be used

- 1 039123 include, for example, other additives, components, integers or steps.

In the description and the appended claims, the singular forms include references to the plural, unless the context clearly dictates otherwise. Thus, for example, a reference to a composition includes mixtures of two or more such compositions, a reference to an antibiotic includes mixtures of two or more such antibiotics, a reference to a compound includes mixtures of two or more of such compounds, and the like.

Optional or optional means that the event or circumstance described below may or may not occur, and that the description includes cases where the event or circumstance occurs and cases where it does not occur.

Although the numerical ranges and parameters establishing the broad scope of the invention are approximate, the numerical values given in the specific examples are as accurate as possible. Any numerical value, however, by its nature contains certain errors, necessarily resulting from the standard deviation found in the corresponding experimental data. In addition, where numerical ranges of varying scope are given herein, it is contemplated that any combination of these values, including the indicated values, may be used. In addition, ranges herein may be expressed as from about one particular value and/or to about another particular value. If such a range is specified, then the other aspect includes from one particular value and/or to another particular value. Similarly, if quantities are expressed as approximate by the word about before them, then it must be understood that the particular value constitutes another aspect. In addition, it should be understood that the endpoints of each range are significant both relative to the other endpoint and independently of the other endpoint. Unless otherwise indicated, the term about means within 5% (eg, within 2% or 1%) of the specific value as modified by the term about.

The term reduce, or other forms of the word, such as decrease or reduce, imply a decrease in the intensity of an event or characteristic (eg, a viral infection). It should be understood that this is usually evaluated relative to some standard or expected value, in other words, it is relative, but the standard or relative value need not always be mentioned. For example, a reduction in viral infection means a reduction in the amount of bacteria relative to a standard or control amount.

The term prevent, or other forms of the word, such as prevention or prevention, are intended to stop a particular event or characteristic, stabilize or slow down the development or progression of a particular event or characteristic, or minimize the likelihood that a particular event or characteristic will occur. Prevention does not require comparison with control, since it is usually more absolute than, for example, reduction. In this document, something can be reduced, but not eliminated, and something that is reduced can also be prevented. Likewise, something can be prevented but not reduced, and something that is prevented can also be reduced. It is to be understood that when the term reduce or prevent is used, the use of the other word is also explicitly disclosed unless specifically stated otherwise.

As used herein, the term treatment refers to obtaining beneficial or desirable clinical results. Beneficial or desired clinical results include, but are not limited to, any one or more of alleviation of one or more symptoms (e.g., infection), reduction in the extent of infection, stabilization (i.e., no worsening) of infection, prevention or slowing of the spread of infection, prevention or delaying the onset or recurrence of an infection; and delaying or slowing the progression of an infection.

The term patient preferably refers to a person in need of antibiotic treatment or treatment of any kind, and more preferably a person in need of such treatment to treat a viral infection. However, the term patient may additionally refer to non-human animals, preferably mammals such as dogs, cats, horses, cows, pigs, sheep, and non-human primates, among others, that are in need of antibiotic treatment.

It should be understood that herein the identifiers first and second are used only for ease of recognition of the various components and steps of the disclosed subject matter. Identifiers one and two do not imply any particular order, amount, preference, or importance of the components or steps modified by these terms.

Chemical definitions.

As used herein, the term composition refers to a product containing certain ingredients in certain amounts, as well as any product that is obtained, directly or indirectly, by combining certain ingredients in certain amounts.

References in the specification and claims to weight fractions of a particular element or component in a composition denote the weight ratio between the element or component and any other elements or components of the composition or article, whereby the proportion is expressed by weight.

Thus, in a mixture containing 2 wt.h.component X and 5 wt.h. component Y, X and Y are in a mass ratio of 2:5 and are present in this ratio regardless of the content of additional components in the mixture.

The mass percentage (wt.%) of the component, unless otherwise specifically indicated, is based on the total weight of the composition or composition in which the component is included.

As used herein, the term substituted is intended to include all acceptable substituents on organic compounds. Broadly accepted substituents include acyclic and cyclic, branched and straight, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Illustrative examples of substituents include, for example, those described below. Allowable substituents may be single or multiple, the same or different for the respective organic compounds. For the purposes of the present invention, heteroatoms, such as nitrogen, may contain hydrogen substituents and/or any valid substituents of the organic compounds described herein that correspond to the valency of the heteroatoms. The present description is by no means limited to the allowable substituents of organic compounds. In addition, the terms substitution or substituted include the implied condition that such a substitution is in accordance with the allowable valency of the substituted atom and the substituent, and that the substitution results in a stable compound, for example, a compound that does not undergo spontaneous transformations such as rearrangement, cyclization, elimination reactions. etc.

The term aliphatic as used herein refers to a non-aromatic hydrocarbon group and includes straight and branched, alkyl, alkenyl or alkynyl groups.

The term alkyl in this document means a branched or straight chain saturated hydrocarbon group containing from 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl , nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group may be substituted or unsubstituted. The alkyl group may be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, ketone, halogen, hydroxy , nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide or thiol as described below.

The symbols A ⁿ are used in this document only as a generic substitute in the following definitions.

The term alkoxy as used herein means an alkyl group attached via a single terminal ether bond; those. alkoxy may be defined as -OA ¹ where A ¹ is alkyl as defined above.

The term alkenyl in this document means a hydrocarbon group containing from 2 to 6 carbon atoms, with a structural formula containing at least one double carbon-carbon bond.

Asymmetric structures such as (A1A2)C=C(A ³ A ⁴ ) provide for the inclusion of both E and Z isomers. This may be implied in structural formulas herein where an asymmetric alkene is present, or may be explicitly indicated by the bond symbol C=C. The alkenyl group may be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, ketone, halogen, hydroxy , nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide or thiol as described below.

The term alkynyl in this document means a hydrocarbon group containing from 2 to 6 carbon atoms, with a structural formula containing at least one triple carbon-carbon bond. The alkynyl group may be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone , nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide or thiol as described below.

The term aryl as used herein means a group that contains any carbon-based aromatic group, including, but not limited to, benzene, naphthalene, phenyl, biphenyl, phenoxybenzene, and the like. The term heteroaryl means a group that contains an aromatic group containing at least one heteroatom in the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. The term non-heteroaryl, which is included in the term aryl, defines a group that contains an aromatic group that does not contain a heteroatom. The aryl and heteroaryl group may be substituted or unsubstituted. Aryl and heteroaryl groups may be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, ketone, halogen , hydroxy, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.

- 3 039123

The term biaryl represents a specific type of aryl group and is included in the definition of aryl. Biaryl refers to two aryl groups that are connected to each other through a condensed ring structure, as in naphthalene, or which are connected through one or more carbon-carbon bonds, as in biphenyl.

The term cycloalkyl as used herein is a non-aromatic carbon ring of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The term heterocycloalkyl means a cycloalkyl group as defined above, wherein at least one of the carbon atoms of the ring is replaced by a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and the heterocycloalkyl group may be substituted or unsubstituted. The cycloalkyl group and the heterocycloalkyl group may be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, ketone, halogen, hydroxy , nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.

The term cycloalkenyl as used herein is a non-aromatic carbon ring of at least three carbon atoms and containing at least one double bond, i.e. C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like. The term heterocycloalkenyl refers to a type of cycloalkenyl group as defined above, wherein at least one of the carbon atoms of the ring is replaced by a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and the heterocycloalkenyl group may be substituted or unsubstituted. The cycloalkenyl group and the heterocycloalkenyl group may be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, ketone, halogen, hydroxy , nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.

As used herein, the term cyclic group is used to refer to both aryl groups and non-aryl groups (ie, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups), or both. Cyclic groups contain one or more ring systems, which may be substituted or unsubstituted. The cyclic group may contain one or more aryl groups, one or more non-aryl groups, or one or more aryl groups and one or more non-aryl groups.

The term aldehyde, as used herein, is represented by the formula -C(O)H. In this document, C(O) is an abbreviation for C=O.

As used herein, the terms amine or amino are represented by the formula NA'A ² A ³ where A ¹ , A ² and A ³ can independently be hydrogen, alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or a heterocycloalkenyl group as described above.

In this document, the term carboxylic acid is represented by the formula -C(O)OH. In this document, the term carboxylate is represented by the formula -C(O)O.

The term ester as used herein is represented by the formula -OC(O)A1 or -C(O)OA1, where A ¹ can be an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described higher.

The term ether as used herein is represented by the formula A1OA ² where A ¹ and A ² can independently be an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group as described above.

The term ketone herein is represented by the formula A1C(O)A ² where A ¹ and A ² independently of each other can be alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl group as described above .

The term halogen as used herein refers to the halogens fluorine, chlorine, bromine and iodine.

The term hydroxyl is represented herein by the formula -OH.

The term nitro is represented here by the formula -NO ₂ .

The term piano in this document is represented by the formula -CN.

The term azido is represented herein by the formula -N ₃ .

In this document, the term sulfonyl is used to refer to a sulfo-oxo group represented by the formula -S(O) ₂ A', where A ¹ can be hydrogen, alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl or a heterocycloalkenyl group as described above.

The term sulfonylamino or sulfonamide is represented herein by the formula

- 4 039123

-S(O)2NH2.

The term thiol in this document is represented by the formula -SH.

It is to be understood that the compounds provided herein may contain chiral centers. Such chiral centers may be in either the (R-) or (S-) configuration. The compounds provided herein may be enantiomerically pure or may be diastereomeric or enantiomeric mixtures. It is to be understood that the chiral centers of the compounds of the invention may undergo epimerization in vivo. Thus, one skilled in the art will appreciate that administering a compound in (R-) form is equivalent, for compounds that undergo epimerization in vivo, to administering a compound in (S-) form.

As used herein, the term substantially pure means sufficiently homogeneous to be assayed by standard methods of analysis such as thin layer chromatography (TLC), nuclear magnetic resonance (NMR), gel electrophoresis, high performance liquid chromatography (HPLC), and mass spectrometry ( MS), gas chromatography-mass spectrometry (GC-MS) and the like, used by those skilled in the art to evaluate such purity, appear free of easily detectable impurities. Or sufficiently pure such that further purification does not cause a detectable change in the physical and chemical properties, such as the enzymatic and biological activity of the specified substance. Both traditional and modern methods for purifying compounds to obtain substantially chemically pure compounds are known to those skilled in the art. However, a substantially chemically pure compound may be a mixture of stereoisomers.

Unless otherwise indicated, a formula with chemical bonds that are indicated only by solid lines, without wedges or dotted lines, covers each of the possible isomers, such as each enantiomer, diastereomer and meso compound, as well as mixtures of isomers, such as racemic or scalemic mixtures.

The term pharmaceutically acceptable ingredient means an ingredient that is suitable for use in humans and/or animals without undesirable side effects (such as toxicity, irritation and allergic reaction), commensurate with a reasonable benefit/risk ratio.

A pharmaceutically acceptable salt means a salt that is pharmaceutically acceptable and has the desired pharmacological properties. Such salts include salts which may be formed if the acidic protons present in the compound are capable of reacting with inorganic or organic bases. Suitable inorganic base salts include alkali metal salts such as sodium, potassium, magnesium, calcium and aluminum. Suitable salts with organic bases include salts formed with organic bases such as amine bases, for example ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids (eg hydrochloric and hydrobromic acid) and organic acids (eg acetic acid, citric acid, maleic acid, alkanyl arenesulfonic acids such as methanesulfonic acid and benzenesulfonic acid). If two acid groups are present, then the pharmaceutically acceptable salt may be a monoacid monosalt or a disol; similarly, if more than two acidic groups are present, some or all of such groups may be converted to salts.

Pharmaceutically acceptable excipient means an excipient which is normally used in the preparation of a pharmaceutical composition and which is generally safe, non-toxic and desirable and includes excipients which are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semi-solid or, in the case of an aerosol composition, gaseous.

The term pharmaceutically acceptable carrier means a carrier, such as a diluent, suspending agent, or vehicle for administering the described compounds to a patient. The carrier may be liquid or solid and is chosen according to the intended route of administration. Liposomes are also a pharmaceutical carrier. As used herein, the term carrier includes any and all solvents, dispersion media, bases, coatings, diluents, antibacterial and antifungal agents, isotonic and retarding absorption agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except where any conventional vehicle or agent is incompatible with the active ingredient, its use in therapeutic compositions is included.

The term "therapeutically effective amount" as used herein means that amount of an active compound or pharmaceutical agent that elicits a biological or medical response in a tissue, system, animal or human body expected by the researcher, veterinarian, physician, or other clinician.

Effective amounts of a compound or composition described herein for the treatment of a mammal may include from about 0.1 to about 1000 mg/kg of subject body weight per day, such as from about 1 to about 100 mg/kg/day, in particular from about 10 to about 100 mg/kg/day. Doses may be urgent or permanent. A wide range of dosages of the composition is considered to be both safe and effective.

Detailed references will be made below to specific aspects of the disclosed materials, compounds, compositions, articles of manufacture, and methods, examples of which are illustrated in the accompanying examples and figures.

Compositions.

To solve myopathic problems associated with clevudine, the synthesis of (S,S) and (S,R) diastereomers of clevudine phosphoramidate was carried out. The phosphoramidate fragment was used to deliver clevudine in the form of 5'-monophosphate to the liver in order to reduce: 1) systemic exposure to clevudine; and 2) the possibility of skeletal myopathy. Both phosphoramidates showed anti-HBV activity similar to that of clevudine, with slightly higher activity of the (S,S) diastereomer.

In a specific example, a compound of the formula is disclosed

or a pharmaceutically acceptable salt thereof, where each R ¹ is independently selected from one of the formulas

R ⁴ is hydrogen, benzyl, C _1-6 alkyl or cycloalkyl;

R ⁵ is hydrogen, deuterium, hydroxyl, cyano, azido, amino, substituted amino, aryl, heteroaryl, substituted aryl, C _1-6 αlkoxy, Cl _-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, or substituted heteroaryl; And

R ⁶ is Cl _-6 alkoxy, Cl _-6 alkyl, cycloalkyl, aryl, or substituted aryl, wherein when the group is substituted, it is substituted by one or more of the following: Cl _-6 alkyl, halogenated Cl _-6 alkyl, Cl _-6 alkoxy, C _2-6 alkenyl, C _2-6 alkynyl, aryl, heteroaryl, -C(O)H, amino, -C(O)OH, -OC(O)A ¹ or -C(O)OA1, A ¹ OA ² , halide, hydroxy, A ¹ C(O)A ² , nitro, sulfo-oxo, sulfonyl, sulfone, sulfoxide or thiol, where A ¹ and A ² are independently selected from C _1-6 alkyl, halogenated C _{1- 6} alkyl, C _2-6 alkenyl, C _2-6 α kynyl, aryl, heteroaryl or cycloalkyl, where aryl is naphthalene, phenyl, biphenyl or phenoxybenzene, cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, heteroaryl is an aromatic group, containing a heteroatom selected from nitrogen, oxygen, sulfur or phosphorus.

Compounds Revealed

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or a pharmaceutically acceptable salt thereof.

Application methods.

The compounds provided herein can be used to treat viral infectious diseases. Examples of viral infections include, but are not limited to, infections caused by RNA viruses (including negative helix RNA viruses, positive helix RNA viruses, double-stranded RNA viruses, and retroviruses) or DNA viruses. All strains, types and subtypes of RNA viruses and DNA viruses are included in the present invention.

Viruses are infectious agents that can usually replicate within the living cells of organisms. Viral particles (virions) usually consist of nucleic acids, a protein coat, and in some cases a lipid envelope that surrounds the protein coat. Virus shapes range from simple helical and icosahedral shapes to more complex structures. The virus-encoded protein subunits self-assemble to form a capsid, which usually requires the presence of the viral genome. Complex viruses can encode proteins that help the assembly of the capsid. The proteins associated with the nucleic acid are known as nucleoproteins, and the complex of the viral capsid proteins with the viral nucleic acid is called the nucleocapsid.

Viruses are transmitted in a variety of ways, including direct transmission or contact with body fluids such as blood, tears, semen, presperm, saliva, milk, vaginal secretions, lesions; droplet contact, fecal-oral contact, or as a result of an animal bite or birth. The virus contains DNA or RNA genes and is called DNA virus or RNA virus, respectively. The viral genome is single-stranded or double-stranded. Some viruses contain a genome that is partly double-stranded and partly single-stranded. For viruses with single-stranded DNA or RNA, the helices are called positive-sense (so-called plus helices) or negative-sense (so-called minus helices) depending on whether they are complementary to the viral messenger RNA (mRNA). The positive-sense RNA of the virus is identical to the mRNA of the virus and thus can be directly translated by the host cell. The negative-sense RNA of the virus is complementary to the mRNA and therefore must be converted to positive-sense RNA by RNA polymerase before translation. DNA nomenclature is similar to RNA nomenclature in that the coding strand for the viral mRNA is complementary to it (negative) and the non-coding strand is its copy (positive).

Antigenic variation or reassortment can produce new strains. Viruses undergo genetic modification through several mechanisms. Such mechanisms include a process called genetic drift, in which individual bases in DNA or RNA mutate to other bases. An antigenic shift occurs when there is a significant change in the genome of the virus. This may be the result of recombination or reassortment. RNA viruses often exist as quasi-species or clusters of viruses of the same species, but with slight differences in the nucleoside sequences of the genome.

In different types of viruses, the genetic material of the viruses and the way the material replicates vary. Replication of the genome of most DNA viruses occurs in the cell nucleus. If an appropriate receptor is present on the cell surface, then these viruses enter the cell by fusion with the cell membrane or by endocytosis. Most DNA viruses are completely dependent on the host's DNA and RNA synthesis apparatus and RNA processing apparatus. Replication usually occurs in the cytoplasm. RNA viruses typically use their own RNA replicase enzymes to make copies of their genome.

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The Baltimore classification of viruses is based on the mechanism by which mRNA is produced. In order to obtain proteins and self-replicate, viruses must generate mRNA from their genome, but different mechanisms are used to achieve this. Viral genomes can be single-stranded (ss) or double-stranded (ds), RNA or DNA, and may or may not use reverse transcriptase (RT). In addition, the ssRNAs of viruses can be in the sense (plus) or antisense (minus) sequence. In this classification, viruses are divided into seven groups: I, dsDNA viruses (eg, adenoviruses, herpes viruses, poxviruses); II, ssDNA viruses with (plus)sense DNA (eg, parvoviruses); III, dsRNA viruses (eg reoviruses); IV, (plus)ssRNA viruses with (plus)sense RNA (eg picornaviruses, togaviruses); V (minus)ssRNA viruses with (minus)sense RNA (eg, orthomyxoviruses, rhabdoviruses); VI, ssRNA-RT viruses with (plus)sense RNA and intermediate DNA in the life cycle (eg, retroviruses); and VII, dsDNA-RT viruses (eg, hepadnaviruses).

The hepatitis B virus is a hepadnavirus. The viral particle (virion) consists of an outer lipid shell and an icosahedral nucleocapsid core, which consists of a protein. The HBV genome consists of circular DNA, but the DNA is not fully double-stranded. One end of the chain is connected to the viral DNA polymerase. The virus replicates by reverse transcription through an intermediate form of RNA. Replication usually occurs in the liver, where it causes inflammation (hepatitis). The virus spreads into the blood, where specific proteins of the virus and antibodies to them are found in infected people. Blood tests to detect these proteins and antibodies are used to diagnose an infection.

The hepatitis B virus enters the cell by endocytosis. Because the virus replicates via RNA synthesized by the host enzyme, the viral genomic DNA must be transferred to the cell nucleus by the host's chaperones. The partially double-stranded viral DNA then becomes fully double-stranded and transformed into a covalently closed circular DNA (cccDNA), which serves as a transcriptional template for the viral mRNA. The virus includes four main serotypes (adr, adw, ayr, ayw) isolated on the basis of antigenic epitopes presented on envelope proteins, and eight genotypes (A-H) in accordance with the general variations in the nucleotide sequence of the genome.

Hepatitis B surface antigen (HBsAg) is typically used to screen for this infection. It is the first detectable viral antigen that appears during an infection. However, this antigen may be absent in the early stages of infection, and it may not be detected in the later stages of infection if the host removes it. The infectious virion contains an inner nuclear particle that encloses the viral genome. The icosahedral core consists of a core protein, alternatively known as hepatitis B core antigen or HBcAg. IgM antibodies to hepatitis B core antigen (anti-HBc IgM) can be used as a serological marker. Hepatitis B e antigen (HBeAg) may appear. The presence of HBeAg in host serum is associated with high rates of viral replication. Some variants of the hepatitis B virus do not produce the e antigen.

If the host is able to clear the infection, HBsAg levels usually fall below the detection threshold and IgG antibodies to hepatitis B surface antigen and core antigen (anti-HBs and anti-HBc IgG) develop. The time interval between the disappearance of HBsAg and the appearance of anti-HBs is called the seronegative window. An individual who is HBsAg seronegative and anti-HBs seropositive has cleared the infection or has been previously vaccinated. Individuals who remain HBsAg seropositive for at least six months are considered carriers of hepatitis B. Carriers of the virus may have chronic hepatitis B, manifested by elevated levels of serum alanine aminotransferase and inflammation of the liver, which can be detected by biopsy. Nucleic acid tests (PCR) have been developed to detect and measure the amount of HBV DNA in clinical specimens.

Acute infection with hepatitis B virus is accompanied by acute viral hepatitis. Acute viral hepatitis usually begins with symptoms of general malaise, loss of appetite, nausea, vomiting, body aches, mild fever, dark urine, and progresses to jaundice. Chronic infection with the hepatitis B virus may be asymptomatic or may be accompanied by chronic inflammation of the liver (chronic hepatitis), which can lead to cirrhosis. Chronic infection with hepatitis B increases the incidence of hapatocellular carcinoma (liver cancer).

During HBV infection, the host's immune response causes both damage to liver cells and clearance of the virus. The adaptive immune response, in particular virus-specific cytotoxic T lymphocytes (CTLs), contributes to most cases of liver injury associated with HBV infection. By destroying infected cells and producing antiviral cytokines capable of expelling HBV from viable hepatocytes, CTLs eliminate the virus. Although CTLs initiate and mediate liver injury, non-antigen-specific inflammatory cells can worsen CTL-induced immunopathology, and activated platelets at the site of infection can contribute to the accumulation of CTLs in the liver.

Therapeutic agents can stop viral replication, thus minimizing liver damage. In some embodiments of the invention, the proposed relates to those described in

- 8 039123 herein, methods of treating a subject diagnosed with HBV by administering a compound disclosed herein. In some embodiments of the invention, the subject is immunocompromised. In some embodiments, the compound is administered in combination with another antiviral agent such as lamivudine, adefovir, tenofovir, telbivudine, and entecavir, and/or the immune system modulators interferon a-2a and pegylated interferon a-2a (Pegasys). In some embodiments, the invention relates to the prevention of HBV infection in an immunocompromised subject at risk of infection by administering a pharmaceutical composition disclosed herein and optionally one or more antiviral agents. In some embodiments of the invention, the subject is at risk of infection due to the fact that his sexual partner is a subject diagnosed with HBV.

The compounds of the present invention may be administered in combination with a second antiviral agent such as abacavir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla, boceprevir, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz , emtricitabine, enfuvirtide, entecavir, famciclovir, fomivirsen, fosamprenavir, foscarnet, phosphonet, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, type III interferon, type II interferon, type I interferon, lamivudine, lopinavir, loviride, maraviroc , moroxidine, methysasone, nelfinavir, nevirapine, nexavir, oseltamivir, peginterferon a-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, ribavirin, rimantadine, ritonavir, pyramidin, saquinavir, sofosbovir, stavudine, telaprevir, telbivudine, tenofovir, tenofovir disoproxil , tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, salz itabine, zanamivir or zidovudine; and combinations thereof.

Compositions.

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

If the compounds of the invention contain both an acid group and a basic group, then the compounds of the present invention may additionally form internal salts, such compounds being included within the scope of the invention. If the compound of the present invention contains a hydrogen donor heteroatom (eg, NH), then the invention further includes salts and/or isomers formed by transferring a hydrogen atom to a basic group or atom in the molecule.

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

The compounds described herein can be administered in the form of a prodrug. The prodrug may include a covalently attached carrier that releases the active parent drug upon administration to a mammal. Prodrugs can be prepared by modifying the functional groups present on the compounds such that the modifying groups are cleaved off by routine manipulation or in vivo to form the parent compound. Prodrugs include, for example, compounds in which a hydroxyl group is bonded to any group that, when administered to a mammal, is cleaved off to form a free hydroxyl group. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol functional groups in compounds. Methods for constructing a compound as a prodrug are known, for example, from Testa and Mayer, Hydrolysis in Drug and Prodrag Metabolism, Wiley (2006). Typical prodrugs form the active metabolite by transformation of the prodrug by hydrolytic enzymes, for example by hydrolysis of amide, lactams, peptides, carboxylic acid esters, epoxides, or cleavage of inorganic acid esters. Ester prodrugs have been shown to be readily degraded in the body to release the corresponding alcohol. See, for example, Imai, Drug Metab Pharmacokinet. (2006) 21(3):

173-85, under Human carboxylesterase isozymes: catalytic properties and rational drag design.

Pharmaceutical compositions for use in the present invention generally contain an effective amount of a compound and a suitable pharmaceutically acceptable carrier. The formulations may be prepared in a manner known per se, which generally involves mixing at least one compound according to the invention with one or more pharmaceutically acceptable carriers, and, if desired, in combination with other pharmaceutically active compounds, if necessary under aseptic conditions. See U.S. Patent No. 6,369,086, U.S. Patent No. 6,372,778, U.S. Patent No. 6,369,087 and U.S. Patent No. 6,372,733 and other sources cited above, as well as standard references such as the latest edition of Remington's Pharmaceutical Sciences.

Typically, for pharmaceutical use, the compounds may be formulated into a pharmaceutical preparation containing at least one compound and at least one pharmaceutically acceptable carrier, diluent or excipient and, if necessary, one or more additional pharmaceutically active compounds.

The pharmaceutical preparations of the present invention are preferably in single dose form and may be suitably packaged in, for example, a box, blister, vial, bottle, sachet, ampoule, or any other suitable single-dose or multi-dose holder or container (which may be appropriately labeled), optionally with one or more package inserts containing information and/or instructions for use of the product. Typically such unit doses will contain 1 to 1000 mg, typically 5 to 500 mg, of at least one compound of the invention, such as about 10, 25, 50, 100, 200, 300, or 400 mg per unit dose.

The compounds can be administered by various routes, including oral, ophthalmic, rectal, transdermal, subcutaneous, intravenous, intramuscular, or nasal, depending primarily on the particular formulation. The compound is usually administered in an effective amount, which means any amount of the compound, after appropriate administration, is sufficient to achieve the desired therapeutic or prophylactic effect in the subject to which it is administered. Usually, depending on the condition to be prevented or treated and the route of administration, such an effective amount will be from 0.01 to 1000 mg per 1 kg of body weight of the patient per day, more often from 0.1 to 500 mg, for example from 1 to 250 mg, for example about 5, 10, 20, 50, 100, 150, 200, or 250 mg per kg of patient body weight per day, which may be administered as a single daily dose divided into one or more doses per day. The amount(s) to be administered, routes of administration, as well as the treatment regimen, may be determined by the attending physician depending on such factors as the age, sex and general condition of the patient, the nature and severity of the disease/symptoms to be treated. See U.S. Patent No. 6,372,778, U.S. Patent No. 6,369,087, U.S. Patent No. 6,369,086 and U.S. Patent No. 6,372,733 and other sources cited above, as well as standard references such as the latest edition of Remington's Pharmaceutical Sciences.

In the case of an oral administration form, the compound may be mixed with suitable additives such as excipients, stabilizers or inert diluents, and may be formulated into a suitable administration form such as tablets, coated tablets, hard capsules, by conventional means, aqueous, alcoholic or oily solutions. Examples of suitable inert carriers are gum arabic, magnesia, magnesium carbonate, potassium phosphate, lactose, glucose or starch, in particular corn starch. In this case, the composition can be processed both in the form of dry and wet granules. Suitable oil excipients or solvents are vegetable or animal oils such as sunflower oil or fish oil. Suitable solvents for aqueous or alcoholic solutions are water, ethanol, sugar solutions or mixtures thereof. Polyethylene glycols and polypropylene glycols are also suitable as additional excipients for other forms of administration. In the case of immediate release tablets, said compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, fillers, disintegrants, diluents and lubricants known in the art.

When administered by nasal spray or inhalation, the compositions may be prepared according to techniques well known in the pharmaceutical art and may be prepared as solutions in saline, using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability. , fluorocarbons and/or other solubilizing or dispersing agents known in the art. Suitable pharmaceutical compositions for administration as aerosols or sprays are, for example, solutions, suspensions or emulsions of the compounds of the present invention or their physiologically tolerable salts in a pharmaceutically acceptable solvent such as ethanol or water, or mixtures of such solvents. If necessary, the preparation may additionally contain other pharmaceutical excipients such as surfactants,

- 10 039123 emulsifiers and stabilizers, as well as propellants.

In the case of subcutaneous or intravenous administration, the compounds, if necessary, together with conventional substances such as solubilizers, emulsifiers or other excipients, are introduced into a solution, suspension or emulsion. In addition, the compounds can be lyophilized and the resulting lyophilizates used, for example, for the preparation of injections or infusion preparations. Suitable solvents are, for example, water, saline sodium chloride solution or alcohols, for example ethanol, propanol, glycerol, sugar solutions such as glucose or mannitol solutions, or mixtures of the various solvents mentioned. Injectable solutions or suspensions may be formulated according to the known art, using suitable non-toxic, suitable parenteral diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or moisturizing agents. and suspending agents such as sterile, light, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.

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

In some embodiments of the invention, it is contemplated that these compositions may be sustained release formulations. Typical sustained release formulations use an enteric coating. Typically, a barrier is applied to the oral drug to control the site of absorption of the drug in the digestive system.

Enteric coatings prevent the drug from being released before it reaches the small intestine. Enteric coatings may contain polysaccharide polymers such as maltodextrin, xanthan, scleroglucan, dextran, starch, alginates, pullulan, hyaluronic acid, chitin, chitosan, and the like; other natural polymers such as proteins (albumin, gelatin, etc.), poly-L-lysine; sodium polyacrylate; poly(hydroxyalkyl methacrylates) (e.g., poly(hydroxyethyl methacrylate); carboxypolymethylene (e.g., Carbopol™); carbomer; polyvinylpyrrolidone; gums such as guar gum, gum arabic, karaya gum, gatti gum, locust bean gum, tamarind gum, gellan gum , gum tragacanth, agar, pectin, gluten, etc., polyvinyl alcohol, ethylene-vinyl alcohol copolymer, polyethylene glycol (PEG), and cellulose ethers such as hydroxymethyl cellulose (HMC), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC) , methylcellulose (MC), ethylcellulose (EC), carboxyethylcellulose (CEC), ethylhydroxyethylcellulose (EHEC), carboxymethylhydroxyethylcellulose (CMHEC), hydroxypropylmethylcellulose (HPMC), hydroxypropylethylcellulose (HPEC) and sodium carboxymethylcellulose (Na-CMC); and copolymers and/or (simple) blends of any of the above polymers Some of the above polymers can be cross-linked using standard techniques .

The choice of polymer will be determined by the nature of the active ingredient/drug used in the composition of the invention, as well as the desired release rate. In particular, as will be appreciated by one of skill in the art, for example, in the case of HPMC, a higher molecular weight will generally result in a slower release rate of the drug from the composition. In addition, in the case of HPMC, different degrees of substitution of methoxy groups and hydroxypropoxy groups will cause a change in the release rate of the drug from the composition. In this respect, as noted above, it may be desirable to provide coating compositions of the invention in which the polymeric carrier is made using a mixture of two or more polymers, for example of different molecular weight, to obtain a particular desired or desired release profile.

Microspheres of polylactide, polyglycolide and their poly(lactide-co-glycolide) copolymers can be used to prepare sustained release protein delivery systems. Proteins can be enclosed in poly(lactide-co-glycolide) depot microspheres in several ways, including the formation of a water-in-oil emulsion with a protein in an aqueous phase and a polymer in an organic solvent phase (emulsion method), the formation of a suspension of a solid in oil, in wherein the solid protein is dispersed in a solvent-based polymer solution (suspension method), or by dissolving the protein in a solvent-based polymer solution (dissolution method). Poly(tylene glycol) (PEGylation) can be attached to proteins to increase the in vivo half-life of circulating therapeutic proteins and reduce the likelihood of an immune response.

Additionally, liposomal suspensions (including liposomes targeted to viral antigens) can be prepared by conventional methods for preparing pharmaceutically acceptable carriers. This may be appropriate in the delivery of free nucleosides, acyl nucleosides, or phosphate ester prodrugs of the nucleoside compounds of the present invention.

- 11 039123

It is to be understood that the nucleosides of the present invention contain multiple chiral centers and may exist and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention includes any racemic, optically active, diastereomeric, polymorphic, or stereoisomeric form of a compound of the invention, or mixtures thereof, that have the useful properties described herein. It is well known in the art how to prepare optically active forms (eg, by resolution of the racemic form using recrystallization methods, by synthesis from optically active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase).

The carbon atoms in the nucleoside are chiral, their non-hydrogen substituents (base and CHOR groups, respectively) can be in the cis (on one side) or trans (on opposite sides) position with respect to the sugar ring system. Therefore, the four optical isomers are represented by the following configurations (when the sugar fragment is oriented in the horizontal plane so that the oxygen atom is behind): cis (both groups at the top, which corresponds to the configuration of natural β-O-nucleosides), cis (both groups at the bottom, which corresponds to the artificial e-L configuration), trans (C2' substituent above and C4' substituent below), and trans (C2' substituent below and C4' substituent above). D-nucleosides are cis-nucleosides in the natural configuration, and L-nucleosides are cis-nucleosides in the artificial configuration.

Likewise, most amino acids are chiral (denoted as L- or D-, with the L-enantiomer being the natural configuration) and can exist as separate enantiomers.

Examples of methods for producing optically active materials are known in the art and include at least the following: i) physical separation of crystals - a technique in which macroscopic crystals of individual enantiomers are manually separated. This method can be applied if crystals of individual enantiomers exist, ie. the material is a conglomerate, and the crystals are visually different; ii) simultaneous crystallization - a technique in which individual enantiomers are individually crystallized from a solution of a racemate, this can only be carried out if the latter is a conglomerate in the solid state; iii) enzymatic separation - a technique for partial or complete separation of a racemate based on the different rates of reaction of enantiomers with an enzyme; iv) asymmetric enzymatic synthesis - a synthetic procedure in which at least one synthetic step uses an enzymatic reaction to produce an enantiomerically pure or enriched synthetic precursor of the desired enantiomer; v) chemical asymmetric synthesis - a synthetic procedure in which the target enantiomer is synthesized from an achiral precursor under conditions that give asymmetry (i.e. chirality) in the product, which can be achieved using chiral catalysts or chiral excipients; vi) separation of diastereomers - a technique in which a racemic compound is reacted with an enantiomerically pure reagent (chiral excipient) that converts individual enantiomers to diastereomers. The resulting diastereomers are separated by chromatography or crystallization based on more pronounced structural differences, and then the chiral auxiliary reagent is removed to obtain the target enantiomer; vii) asymmetric first and second order conversion - a technique whereby diastereomers from a racemate are balanced so that the diastereomer from the target enantiomer predominates in solution, or the diastereomer from the target enantiomer preferentially crystallizes, which disrupts the equilibrium in such a way that, ultimately, practically all material is converted to the crystalline diastereomer from the desired enantiomer. The target enantiomer is then released from the diastereomer; viii) kinetic resolution - this technique refers to achieving partial or complete resolution of a racemate (or further resolution of a partially resolved compound) based on unequal reaction rates of enantiomers with a chiral non-racemic reagent or catalyst under kinetic conditions; ix) enantioselective synthesis from non-racemic precursors - a synthetic method by which the target enantiomer is obtained from non-chiral starting materials and in which the stereochemical integrity is not or only minimally disturbed during the synthesis; x) chiral liquid chromatography - a technique in which the enantiomers of a racemate are separated in a liquid mobile phase based on differences in their interaction with the stationary phase. The stationary phase may be made of a chiral material, or the mobile phase may contain additional chiral material to induce interaction differences; xi) chiral gas chromatography, a technique whereby a racemate is evaporated and enantiomers are separated based on differences in their interaction in a gaseous mobile phase with a column containing a stationary phase of a non-racemic chiral adsorbent; xii) extraction with chiral solvents - a technique in which enantiomers are separated based on the selective dissolution of one of the enantiomers in a particular chiral solvent; xiii) trance

- 12 039123 port through chiral membranes - a technique in which the racemate is brought into contact with a thin barrier membrane. The barrier typically separates two miscible liquids, one of which contains a racemate, whereby a driving force, such as a difference in concentration or pressure, causes preferential transport across the barrier membrane. The separation occurs as a result of the non-racemic chiral nature of the membrane, which allows only one enantiomer from the racemate to pass through. In one embodiment of the invention, chiral chromatography is used, including fluid bed chromatography. A wide range of chiral stationary phases is commercially available.

Some of the compounds described herein contain olefinic double bonds and, unless otherwise indicated, both E and Z geometric isomers are included.

In addition, some of the nucleosides described herein may exist as tautomers, such as keto-enol tautomers. The compounds of the present invention include single tautomers as well as mixtures thereof.

Examples

The following examples are presented to illustrate methods, compositions, and results in accordance with the disclosed subject matter. These examples are not intended to cover all aspects of the subject matter disclosed herein, but rather illustrate typical methods, compositions, and results. These examples are not intended to exclude equivalents and variations of the present invention, which are obvious to a person skilled in the art.

Efforts have been made to ensure accuracy in terms of numbers (eg quantities, temperatures, etc.), but some errors and deviations must be taken into account. Unless otherwise indicated, parts are parts by weight, temperatures are in °C or room temperature, and pressures are at or near atmospheric. There are numerous variations and combinations of reaction conditions, such as temperature, pressure, component concentrations, and other ranges and reaction conditions, that can be used to optimize the purity and yield of the product resulting from the described process. To optimize such process conditions, only reasonable and routine experimentation is required.

Example 1 HBV assay.

HepG2.2.15 cells (100 µl) in RPMI 1640 medium containing 10% fetal bovine serum are placed in all wells of a 96-well plate at a density of 1 x 10 ⁴ cells per well, and the plate is incubated at 37°C in medium containing 5% CO ₂ for 24 hours. After incubation, six 10-fold serial dilutions of test compound in RPMI1640 medium containing 10% fetal bovine serum are added to separate wells of the plate in three parallel experiments. As a control, medium containing only the virus is added to six wells on the plate. The tablet is incubated for 6 days at 37°C in a medium containing 5% CO2. Culture medium on day 3 is replaced with medium containing the indicated concentrations of each compound. One hundred microliters of supernatant was removed from each well for viral DNA analysis by qPCR, and cytotoxicity was assessed by XTT staining of the cell culture monolayer on the sixth day.

Ten microliters of the cell culture supernatant was taken on the sixth day, diluted with qPCR dilution buffer (40 µg/ml fragmented salmon sperm DNA) and boiled for 15 minutes. Real time quantitative PCR was performed in 386-well plates using an Applied Biosystems 7900HT sequencing system and SDS 2.4 supporting software. Five microliters (5.0 µl) of boiled DNA for each sample and serial 10-fold dilutions of the quantitative DNA standard are processed by real-time PCR using the Q-Platinum SuperMix-UDG quantitative PCR mixture (Invitrogen) and DNA specific oligonucleotide primers (IDT, Coralville, Iowa) HBV-AD38-qF1 (5'-CCG TCT GTG CCT TCT CAT CTG-3') (SEQ ID NO: 1), HBV-AD38-qR1 (5'-AGT CCA AGA GTY CTC TTA TRY AAG ACC TT-3') (SEQ ID NO: 2) and HBV-AD38-qP1 (5'-FAM CCG TGT GCA /ZEN/CTT CGC TTC ACC TCT GC-3'-BHQ1) (SEQ ID NO: 3) at the final concentrations of 0.2 μM for each primer in a total volume of the reaction mixture of 15 μl. The copy number of HBV DNA in each sample is interpolated from standard curve data using SDS.24 software and the data is imported into Excel for analysis.

The 50% cytotoxic concentration for test materials is obtained by measuring the reduction of XTT tetrazolium dye on processed tissue culture plates. XTT is metabolized by the mitochondrial enzyme NADPH oxidase to a soluble formazan product in metabolically active cells. XTT solution is prepared daily as a stock solution of 1 mg/ml in PBS. The stock solution of phenazine methosulfate (MSF) is prepared as 0.15 mg/ml in PBS and stored in the dark at -20°C. The XTT/MSF solution is prepared immediately before use by adding 40 µl of MSF to 1 ml of XTT solution. Fifty microliters of XTT/MSF are added to each well of the plate and the plate is incubated for 2-4 hours at 37°C. An incubation period of 2-4 hours was empirically determined to be in the linear range for XTT dye recovery with the indicated number of cells for each assay. Instead of covers, devices for gluing the tablet are used. The sealed plate is inverted several times to mix the soluble formazan product and the plate is read at 450 nm (reference wavelength 650 nm) using a Molecular Devices SpectraMax Plus 384 spectrophotometer. Data are recorded using Softmax 4.6 software and imported into Excel. for analysis. The data are presented in table. 1.

____________________________________________________________________________Table 1

Structure and number Efficacy analysis against HBV Cytotoxicity (SS ₅₀ µM) ec ₅₀ (µM) HepG2 CC50 (µM) Huh7 HepG2 WhRSZ CEM A204 IEC6 H9s A549 Vero WITH HN |]^ O^N _ f—OH And he EIDD-02020 Clevudin 0.46 >100 >400 >400 >400 >400 >400 >400 >400 >400 >400 i bn Q EIDD-02173 1.71 >100 >400 >400 >400 140 330 307 344 >400 >400 About ^F ό EIDD-Q2174 4.29 >100 290 >400 >400 >400 374 338 311 >400 >400 9 Η 9 1 ⁰ О 1 OO=F-0Fh ο γ 2.9 >100 ° 6 1 OQ=F’«OPh 6100-02309 AJ 2.39 85.1 0 S^NT, Ι-ΟΗ he EIDD-02333-1 0 ^H 1 V ⁰ Η ⁰ I xA Υ II Η II S Νρ |—0-ΡΝ.Χ Αγ ' »ό EIDD-02334-1 0 NGT 1 0 U 0 1 ^s JtrAR EIDD-02335-1 nh ₂ JO (ΤΉ X f-OH X 3TS (lamivudine) >100 >100 >100 0.14 >100 >100 >100 >10 >400 >400 >400

- 14 039123

Example 2 Preparation of (S')-2-[-(S)-2-(2,3,4,5,6-ntafluorophenoxy)phenoxyphosphorylamino]pronionic acid isopropyl ester

In a flask containing (S)-isopropyl-2-aminopropanoate hydrochloride (72.0 g, 430 mmol) are placed phenyl ester of phosphoric acid dichloride (64.2 ml, 430 mmol) and dichloromethane (DCM, 1200 ml). The mixture was cooled from -70 to -78° C. in a dry ice/acetone bath and then treated with triethylamine (120 mL, 859 mmol) dropwise over 30 minutes. The mixture was stirred at -70 to -78°C for 30 minutes, then allowed to warm to room temperature and stirred for 1 hour.

Next, the reaction mixture is cooled to 0-5°C in an ice bath and added over 30 min to a solution of 2,3,4,5,6-pentafluorophenol (79 g, 430 mmol) and triethylamine (59.9 ml, 430 mmol) in 100 ml dichloromethane. The resulting mixture was stirred at -70 to -78°C for 30 minutes, then warmed to ambient temperature and stirred for 2 hours.

The solids are filtered off and the solid is washed with 200 ml of ethyl acetate. The filtrate and washings are concentrated by vacuum distillation to a semi-solid residue. The semi-solid residue is dissolved in 500 ml of ethyl acetate and washed with water and sodium chloride solution. The aqueous layers are re-extracted with 50 ml of ethyl acetate. The combined organic layer was dried over anhydrous MgSO ₄ and concentrated to give 210 g of crude racemic product (100% yield). The racemic product appears to be a 1:1 mixture of two diastereomers according to NMR examination.

The kinetic separation of the racemic product to obtain the target SS-diastereomer is carried out based on the following protocol.

1) The crude racemic mixture is suspended in 500 ml of 20% ethyl acetate/hexane and added to a solution of 5 g of pentafluorophenol, 10 ml of triethylamine and 100 mg of dimethylaminopyridine in 20 ml of 20% ethyl acetate/hexane. The reaction mixture is heated at 45-50°C for 30 min and the suspension is stirred overnight. A white solid is separated by filtration and washed with 200 ml of 20% ethyl acetate/hexane and 100 ml of hexane. The product is dried at 40° C. under vacuum to give a white solid (wt: 98 g). According to the NMR study, the product appears to be essentially an SS diastereomer.

2) The filtrate and washings from the above reaction are combined and concentrated to give a semi-solid which is essentially the SS diastereomer as shown by NMR along with other impurities. The resulting residue is dissolved in 150 ml of ethyl acetate and washed with 50 ml of 1N. HCl, water and 5% K ₂ CO ₃ solution. The organic layer is dried and concentrated. The white residue is suspended in 100 ml of 20% ethyl acetate/hexane and the solid is separated by filtration. The precipitate is then washed with 20% ethyl acetate/hexane, hexane and dried. The weight of the white solid obtained is 22 g. According to 1H- and ³¹ P-NMR studies, the product appears to be mainly the SS diastereomer. Total weight of product after separation: 120 g (61.6% yield).

Example 3 Synthesis of 2-chloro-nitrophenylphosphoramidate (5)

A solution of phenyl dichlorophosphate (60 g, 42.5 ml, 284 mmol) in dichloromethane (300 ml) was cooled to 0° C. and then treated with (S)-isopropyl-2-aminopropanoate hydrochloride (47.7 g, 284 mmol). The mixture is further cooled to -78°C and treated dropwise with a solution of triethylamine (57.6 g, 79 ml, 569 mmol) in methylene chloride (300 ml) over 1 hour. The reaction mixture is heated at 0°C for 30 min and then treated with a premixed mixture of 2-chloro-4-nitrophenol (46.9 g, 270 mmol) and triethylamine (28.8 g, 39.6 ml, 284 mmol) in dichloromethane (120 ml) for 20 min. After 2 hours at 0° C., the mixture is filtered through a fritted funnel and the collected filtrate is concentrated to dryness. The crude resin was dissolved in MTBE (500 ml) and washed successively with 0.2M K ₂ CO ₃ solution (2x100 ml) followed by 10% sodium chloride solution (3x75 ml). The organic layer is dried

- 15 039123 over sodium sulfate, filtered and concentrated to dryness on a rotary evaporator to obtain a mixture of diastereomers (100 g, 93%) as a pale yellow oil.

Example 4 Separation of diastereomers of compound 5

The diastereomeric mixture 5 (28 g, 63.2 mmol) was dissolved in a 2:3 mixture of ethyl acetate-texane (100 ml) and cooled to -20°C. After 16 hours, the resulting white solid was isolated by filtration and dried under high vacuum to give a mixture of diastereomers S _P :R _P 16:1 (5.5 g, 19.6%). The mother liquor is concentrated and the resulting residue is dissolved in ethyl acetatetetexane 2:3 (50 ml). After 16 hours at -10° C., the resulting white solid was collected and dried under high vacuum to give a mixture of diastereomers S _P :R _P 1:6 (4 g, 14%). A mixture of diastereomers S _P :R _P 16:1 (5.5 g, 12.4 mmol) is suspended in hot hexane (50 ml) and slowly treated with ethyl acetate (approximately 10 ml) until complete dissolution. After cooling to 0°C, the resulting white solid is separated by filtration, washed with hexane and dried under high vacuum to obtain the diastereomer S _p of compound 6 (4.2 g, 76%) as a single isomer.

A mixture of diastereomers S _P :R _P 1:6 (4 g, 12.4 mmol) is suspended in hot hexane (50 ml) and slowly treated with ethyl acetate (approximately 5 ml) until complete dissolution. After cooling to 0°C, the resulting white solid is separated by filtration, washed with hexane and dried under high vacuum to obtain the diastereomer _RP of compound 7 (3.2 g, 80%) as a single isomer.

Example 5 Synthesis of Compound 8

1-((2S,3S,4S,5S-3-fluoro-4-hydroxy-5(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidin-2,4(1H,3H) -dione (300 mg, 1.153 mmol) and THF (15 ml) The suspension is cooled in an ice bath under nitrogen atmosphere tert-butyl magnesium chloride (2.260 ml, 2.260 mmol) is added using a syringe to form a clear solution The mixture is stirred at ambient temperature medium for 30 min and cooled again to 0° C. A solution of compound 4 in THF (20 ml) was added via syringe over 10 min at 0° C. The resulting yellowish solution was stirred at room temperature overnight.

The reaction mixture is cooled to 0°C and quenched with 5 ml of 2N. HCl. The reaction mixture was allowed to warm to room temperature and stirred for 30 minutes. Then 30 ml of toluene are added and the resulting layers are separated. The organic layer is washed with 1N. HCl (1x20 ml), water (20 ml), 5% aq. K2CO3 solution (2x30 ml) and sodium chloride solution (30 ml). All aqueous layers are re-extracted with toluene (30 ml) and washed with 5% K2CO3 (1x30 ml) and sodium chloride solution (30 ml). The combined organic layers are dried over anhydrous MgSO ₄ and concentrated to give an oily residue. The product is purified on 15 g silica gel eluting with 1% and then 2.5% MeOH/DCM. The product is obtained in 2.5% MeOH/DCM as a single spot of product.

Example 6 Preparation of Compound 9

The compound is isolated from the reaction mixture for the synthesis of compound 8.

- 16 039123

Example 7 Synthesis of Compound 10

ABOUT

1-((2S,3S,4S,5S)-3-fluoro-4-hydroxy-5(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidin-2,4(1H,3H )-dione (400 mg, 1.537 mmol) and THF (20 ml), the suspension is cooled in an ice bath under nitrogen atmosphere and tert-butyl magnesium chloride (1.691 ml, 1.691 mmol) is added dropwise over 10 min. The resulting mixture was stirred at 0° C. for 30 minutes, then allowed to warm to room temperature and stirred for 30 minutes. A solution of (2S)-isopropyl-2-(((2-chloro-4-nitrophenoxy)(phenoxy)phosphoryl)amino)propαnoate (817 mg, 1.845 mmol) in THF (150 mL) was added dropwise at room temperature over 10 -50 min. The resulting solution is allowed to stir at ambient temperature overnight.

The reaction mixture is cooled to 0°C and quenched with 5 ml of 2N. HCl. The reaction mixture is then allowed to warm to room temperature and stirred for 30 minutes. Then add 30 ml of toluene and the layers are separated. The organic layer is washed with 1N. HCl (1x20 ml), water (20 ml), 5% aq. K ₂ CO ₃ solution (2x30 ml) and sodium chloride solution (30 ml). All aqueous layers are re-extracted with toluene (30 ml), washed with 5% K ₂ CO ₃ solution (1x30 ml) and sodium chloride solution (30 ml). The combined organic layers are dried over anhydrous MgSO ₄ and concentrated to give an oily residue.

The product is purified on 15 g silica gel eluting with 1% and then 2.5% MeOH/DCM. The product is obtained in 2.5% MeOH/DCM.

Example 8 Preparation of compound 11 o A Α ϊ ι ό

I oo \u003d Aorp 11

Λγ ^Η

The compound is isolated from the reaction mixture for the synthesis of compound 10.

Example 9 Synthesis of Compound 13

A stirrer is installed in a pressurized pressure tube, 12 (0.132 g, 0.25 mmol), triethylamine (12.5 ml) and water (12.5 ml) are placed. The tube is sealed and heated at 35° C. overnight with stirring. After 16 hours the reaction mixture was cooled to room temperature, the tube was opened and the contents were transferred to a round bottom flask. The mixture is concentrated on a rotary evaporator to give 200 mg of crude product, which is partitioned between water and dichloromethane (30 ml each). The organic layer was discarded and the aqueous layer was concentrated on a rotary evaporator to give ~150 mg of crude product, which was dissolved in MeOH and immobilized on celite. Automated flash chromatography on a Combiflash column (12 g, gradient iPrOH to iPrOH:conc. NH ₄ OH:water 7:2:1) gave the product as wet aluminum salt. The solid was dissolved in water, frozen in a dry ice bath, and lyophilized to give 13 (0.089 g, 81%) as a flaky white solid which was determined to be ~94% pure by ¹ H NMR analysis.

1H NMR (400 MHz, MeOH-d ₄ ) δ 7.66 (t, J=1.4 Hz, 1H), 6.18 (dd, J=15.4 Hz, 4.3 Hz, 1H), 5 .00 (ddd, J=52.6Hz, 4.2Hz, 3.3Hz, 1H), 4.42 (ddd, J=19.8Hz, 4.7Hz, 3.4Hz, 1H) , 4.05-3.95 (m, 3H), 3.76 (dc, J=8.9 Hz, 7.0 Hz, 1H), 1.92 (d, J=1.2 Hz, 3H) , 1.35 (d, J=7.0 Hz, 1H);

1H NMR (400 MHz, D2O) δ 7.65 (t, J=1.3 Hz, 1H), 6.25 (dd, J=15.2 Hz, 4.4 Hz, 1H), 5.16 ( ddd, J=51.9 Hz,

- 17 039123

4.3Hz, 3.5Hz, 1H), 4.48(ddd, J=19.9Hz, 5.4Hz, 3.5Hz, 1H), 4.10-3.93(m, 3H ), 3.62 (dc, J=8.7 Hz, 7.0 Hz,

1H), 1.88 (d, J=1.2 Hz, 3H), 1.28 (d, J=7.1 Hz, 3H);

1H NMR (400 MHz, DMSO-d ₆ ) δ 7.64 (t, J=1.4 Hz, 1H), 6.13 (dd, J=13.2 Hz, 4.9 Hz, 1H), 5 .07 (dt,

J=53.3Hz, 4.6Hz, 1H), 4.26(dt, J=20.4Hz, 4.7Hz, 1H), 3.92-3.75(m, 3H), 3 .45-3.35 (m, 1H, overlapping with broad water peak), 1.80 (d, J=1.2 Hz, 3H), 1.14 (d, J=7.2 Hz, 3H);

¹³ C NMR (100 MHz, DMSO-d ₆ ) δ 177.1, 163.8, 150.3, 136.5, 109.0, 95.4 (d, J=190.4 Hz), 82.1 (d, J=16.6 Hz), 81.9 (t, J=7.1 Hz), 72.9 (d, J=23.3 Hz), 62.6, 50.8, 19.8 (d, J=6.5 Hz), 12.1;

³¹ P NMR (162 MHz, DMSO-d6) δ 6.04 (s); ESI-MS: m/z 412.0 ([M+H]+).

Example 10 Synthesis of Compound 15

¹⁴ 15

Clevudine (14) (0.143 g, 0.55 mmol) was placed in a round bottom flask and dried overnight at 50° C. in a vacuum oven. The flask is removed and cooled to room temperature under nitrogen atmosphere, the solid residue is dissolved in trimethyl phosphate (1.375 ml) with stirring. The solution is cooled to 0° C. and phosphoryl trichloride (0.126 g, 0.825 mmol) is added dropwise via syringe. The reaction mixture was stirred at 0° C. for 3 hours, after which TLC analysis showed little progress. A second aliquot of phosphoryl trichloride (0.422 g, 2.75 mmol) is added dropwise via syringe and the mixture is stored at -5° C. in a freezer overnight. After 20 h at the same temperature, the mixture was poured into water (20 ml) and the aqueous layer was washed with chloroform (2x20 ml). Then the aqueous layer was neutralized by adding concentrated aqueous ammonia to pH 7, washed again with chloroform (1x20 ml) and concentrated on a rotary evaporator (bath temperature 25°C). The resulting crude semi-solid is suspended in MeOH and immobilized on celite. Automated flash column chromatography (12 g Combiflash, iPrOH to iPrOH:conc. NH4OH:water 7:2:1 gradient) gave 200 mg of a wet white solid. The solid was dissolved in water, frozen in a dry ice bath, and lyophilized to give 15 (0.055 g, 29%) as a flaky white solid, determined to be ~95% pure by 1 H NMR analysis.

1H NMR (400 MHz, D2O) δ 7.69 (t, J=1.5 Hz, 1H), 6.28 (dd, J=15.6 Hz, 4.4 Hz, 1H), 5.18 ( ddd, J=51.8Hz, 4.4Hz, 3.3Hz, 1H), 4.51 (ddd, J=19.6Hz, 5.2Hz, 3.2Hz, 1H), 4, 16-4.01 (m, 3H), 1.89 (d, 1.2 Hz, 3H);

1H NMR (400 MHz, DMSO-J6) δ 7.60 (s, 1H), 6.11 (dd, J=13.8 Hz, 4.6 Hz, 1H), 5.03 (dt, J=53 .2 Hz, 4.4 Hz, 1H), 4.29 (dt, J=20.2 Hz, 3.6 Hz, 1H), 3.90 (wm, 3H), 1.79 (d, 1 .2 Hz, 3H);

¹³ C NMR (100 MHz, DMSO-J6) δ 163.8, 150.3, 136.6, 109.0, 95.3 (d, J=190.0 Hz), 82.1 (d, J= 16.5 Hz), 81.9 (t, J=6.4 Hz), 72.6 (d, J=23.5 Hz), 62.6, 12.2;

³¹ P NMR (162 MHz, DMSO-d6) δ 0.01 (s);

ESI-MS: m/z 339.0 ([M+H]+).

Example 11 General procedure for the preparation of 5'-triphosphates.

The nucleoside analog is dried under high vacuum at 50°C for 18 h and then dissolved in anhydrous trimethyl phosphate (0.3M). After the addition of PROTON-SPONGE™ (1.5 mol. equiv.), the mixture was cooled to 0°C and treated dropwise with phosphoryl chloride (1.3 mol. equiv.) using a syringe over 15 minutes. The mixture was stirred at 0° C. for 4-6 h monitored by TLC (isopropanol: conc. NH4OH: water 7:2:1). After reaching more than 85% conversion to monophosphate, the reaction mixture is treated with a mixture of bis(tri-n-butylammonium pyrophosphate) (3 mol.eq.) and tributylamine (6 mol.eq.) in anhydrous DMF (1 ml). After 20 min at 0°C with TLC control (NH4OH:isopropanol:water 11:7:2), the mixture is treated with 20 ml of 100 mM triethylammonium bicarbonate (TEAB), stirred for 1 h at room temperature and then extracted with ether ( 3x15 ml). The aqueous layer was then purified by anion exchange chromatography on DEAE SEPHADEX™ A-25 resin (11 x 200 mm) using a buffer gradient of 50 mM (400 ml) to 600 mM (400 ml) TEAB. Fractions of 10 ml were analyzed by TLC (NH4OH:isopropanol:water, 11:7:2). Fractions containing triphosphate (eluted at 500 mM TEAB) are combined and concentrated using a rotary evaporator (bath <25°C). The resulting solid was diluted with demineralized water (10 ml) and concentrated by lyophilization.

Example 12 Synthesis of clevudin-5'-triphosphate (16)

- 18 039123 about

ηνΆ^ ί J ⁰⁰⁰ (ΑΆ o-Ao-Ao-^-oh ZhG 0H 0H 0Η ^F Ch η he

Compound 16 was synthesized using the general procedure for the synthesis of 5'-triphosphate.

Example 13 Synthesis of Compound 17

To a suspension of 14 (0.980 g, 3.77 mmol) in anhydrous CH ₂ Cl ₂ (37.7 ml), imidazole (0.769 g, 11.30 mmol), DMAP (0.046 g, 0.377 mmol) and TBS triflate (2.162 ml, 9.42 mmol) at 0°C under argon. The resulting reaction mixture was stirred at 0°C for 1 h. Then the reaction mixture was warmed to room temperature and stirred for 24 h. The reaction mixture was washed with water, sodium chloride solution and dried over Na ₂ SO ₄ . After removing the solvent, the resulting colorless residue was loaded onto an ISCO column (40 g silica gel). Fractions containing product were collected and condensed on a rotary evaporator to give a colorless residue which turned to a white foam under high vacuum to give 17 (1.8303 g, 99% yield).

Example 14 Synthesis of Compound 18

DMAP ( _0.915 g, 7.49 mmol) and triethylamine ( _1.096 ml, 7 .86 mmol). After cooling to 0° C., 2,4,6-triisopropylbenzene-1sulfonyl chloride (2.268 g, 7.49 mmol) is added in one portion. The resulting yellow reaction mixture was warmed to room temperature and stirred for 22 h. TLC showed unreacted starting material, so the reaction mixture was heated to 40°C and stirred for another 24 h. The reaction mixture was cooled to 0°C, add solution 2, 6-dimethylphenol (1.372 g, 11.23 mmol), DABCO (0.082 ml, 0.749 mmol) and triethylamine (1.566 ml, 1.23 mmol) in CH ₂ Cl ₂ (7.49 ml). Upon completion of the addition, the orange reaction mixture was heated to rt and stirred for 2 days. TLC shows that the original nucleoside is completely consumed, so the reaction mixture is diluted with CH ₂ Cl ₂ , washed with NaHCO ₃ solution, sodium chloride solution and dried over Na ₂ SO ₄ . The organic layer was condensed on a rotary evaporator and the resulting orange residue was loaded onto an ISCO column (120 g silica gel, 16x150 mm). All fractions containing the desired product are collected and condensed on a rotary evaporator to give a yellow residue (2.07 g, 93% yield), which turns to a yellow foam under high vacuum.

Example 15 General Conditions for Nucleotide Coupling.

The target nucleic base (5 eq.) is transferred to a dry flask under argon and suspended in HMDS (2 ml/mmol nucleic base). A catalytic amount of ammonium sulfate (1-3 mg) is added to the reaction vessel and the suspension is refluxed for 1-8 hours. During the reaction, the white suspension becomes transparent. The reaction vessel is left to cool to room temperature, and excess HMDS is removed under reduced pressure. The resulting residue is dissolved in dry DCE (5 ml/mmol of carbohydrate), followed by the addition of the desired carbohydrate at room temperature. Finally, undiluted TMSOTf (5.5 eq.) is added to the solution with stirring. The reaction is quenched with saturated sodium bicarbonate solution. The organic layer is separated, dried over MgSO ₄ , filtered and concentrated under reduced pressure. The desired protected nucleoside is purified on silica gel eluting with DCM/MeOH 9:1.

- 19 039123

Example 16 General Conditions for Nucleotide Coupling.

The target nucleic base (5 eq.) is transferred to a dry flask under argon and suspended in HMDS (2 ml/mmol nucleic base). A catalytic amount of ammonium sulfate (1-3 mg) is added to the reaction vessel and the suspension is refluxed for 1-8 hours. During the reaction, the white suspension becomes transparent. The reaction vessel is left to cool to room temperature, and excess HMDS is removed under reduced pressure. The resulting residue is dissolved in dry DCE (5 ml/mmol of carbohydrate), followed by the addition of the desired carbohydrate at room temperature. Finally, undiluted TMSOTf (5.5 eq.) is added to the solution with stirring. The reaction is quenched with saturated sodium bicarbonate solution. The organic layer is separated, dried over MgSO ₄ , filtered and concentrated under reduced pressure. The desired protected nucleoside is purified on silica gel eluting with DCM/MeOH 9:1.

Example 17 General Desilylation Reaction Conditions

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

Example 18 General conditions for debenzoylation.

To a solution of the protected nucleoside prepared under the basic conditions of the condensation reaction (0.126 g, 0.315 mmol) was added NH3 in MeOH (7M, 1.573 mL, 11.01 mmol). The reaction _mixture was allowed to stir at room _temperature or with gentle heating in a sealed tube for 4.5 h. obtaining the target nucleoside.

Example 19 Synthesis of 5'-deuterated nucleoside.

The suitably protected nucleoside is suspended in methylene chloride (40 ml, partially soluble). After stirring at room temperature for 30 minutes, the mixture is treated successively with PDC, acetic anhydride and then t-butanol. The mixture is continued to stir at room temperature. After 4 hours, TLC data (5% methanol in DCM) and LC-MS show only a small amount of starting material remaining. The mixture is filtered through a bed of silica gel loaded into a 150 ml fritted funnel. The material on silica was eluted with ethyl acetate. The collected filtrate is concentrated under reduced pressure. The crude dark oil was purified by silica gel chromatography (25 mm x 175 mm) with a gradient of hexane:ethyl acetate 2:1 to ethyl acetate. The pure fractions are collected and concentrated to give a white gum. The material is placed under high vacuum for 2 days and used in the next step without further purification.

The 5'-protected nucleoside is dissolved in ethanol 200 and then treated with solid sodium boron deuteride. The mixture becomes homogeneous, it is then heated to 80°C. After 12 hours a white/pale yellow precipitate is formed. The mixture is left to cool to room temperature. TLC (5% methanol in methylene chloride) showed complete conversion of starting material. The mixture is cooled to 0°C in an ice bath and then slowly quenched with acetic acid (approximately 1 ml). The clear solution is warmed to room temperature and then partitioned between ethyl acetate (30 ml) and sodium chloride solution (3 ml). The organic phase is concentrated and then purified by silica gel chromatography (19 mm x 180 mm) using 5% methanol in methylene chloride as mobile phase.

Example 20 Synthesis of bis-POM-5'-monophosphate prodrugs.

Place ((hydroxyphosphoryl)bis(oxy))bis(methylene)bis-(2,2dimethylpropanoate) (0.229 g, 0.703 mmol) into a 50 ml flask, add dry THF (4 ml) to obtain a colorless solution. The flask is evacuated and filled with argon. Triethylamine (0.108 ml, 0.773 mmol) is then added dropwise. After stirring for 30 minutes at room temperature, the desired nucleoside analog is added. The reaction mixture is cooled to 0°C and then N-ethyl-Nisopropylpropan-2-amine (0.245 ml, 1.406 mmol), bis-(2-oxooxazolidin-3-yl)phosphine chloride (0.224 g, 0.879 mmol) and 3-nitro -1H-1,2,4-triazole (0.100 g, 0.879 mmol). The reaction mixture is allowed to stir overnight, gradually warming to room temperature. Then the reaction mixture was diluted with ethyl acetate and quenched with saturated NaHCO ₃ . The organic layer is separated, dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The crude material was purified by column chromatography (12 g ISCO column) eluting with a gradient of 100% DCM to 5% MeOH in DCM to give the desired product.

Example 21 Synthesis of 2-substituted-b-Ata-nucleoside analogs.

Place 1,3,5-tri-O-benzoyl-L-α-ribose into a dry flask under an argon atmosphere. The carbohydrate is dissolved in dry DCM and the reaction flask is cooled to 0°C. Position 2 is activated by adding trifluoromethanesulfonic anhydride, mesyl chloride or SO ₂ Cl ₂ followed by imidazole. Next, the appropriate nucleophile is added to the reaction flask at 0°C. After completion of the reaction according to TLC, the reaction mixture was quenched with water. The reaction mixture with water is placed in a separating funnel. The organic layer is separated and washed with sodium chloride solution. The organic layer is dried over magnesium sulfate, filtered and concentrated under reduced pressure. The product is loaded onto an ISCO column and purified. The product is then processed under basic condensation reaction conditions and general debenzoylation conditions to give the desired nucleoside analogs.

Example 22 Synthesis of 2-fluoro-L-Ara-nucleoside analogues

Place 1,3,5-tri-O-benzoyl-L-α-ribose into a dry flask under an argon atmosphere. The carbohydrate is dissolved in dry DCM and the reaction flask is cooled to 0°C. Position 2 is activated by adding trifluoromethanesulfonic anhydride. Then tetrabutylammonium fluoride in MeCN is added to the reaction flask at 0°C. After completion of the reaction according to TLC, the reaction mixture was quenched with water. The reaction mixture with water is placed in a separating funnel. The organic layer is separated and washed with sodium chloride solution. The organic layer is dried over magnesium sulfate, filtered and concentrated under reduced pressure. The product is loaded onto an ISCO column and purified. The product is then processed under basic condensation reaction conditions and general debenzoylation conditions to give the desired nucleoside analogs.

Example 23 Synthesis of 2-chloro- or bromo-L-Ara-nucleoside analogues.

Place 1,3,5-tri-O-benzoyl-L-α-ribose into a dry flask under an argon atmosphere. The carbohydrate is dissolved in dry DCM and the reaction flask is cooled to 0°C. Then triphenylphosphine with carbon tetrachloride or carbon tetrabromide is added to the reaction flask at 0°C. After completion of the reaction according to TLC, the reaction mixture was quenched with water. The reaction mixture with water is placed in a separating funnel. The organic layer is collected and washed with sodium chloride solution. The organic layer is dried over magnesium sulfate, filtered and concentrated under reduced pressure. The product is loaded onto an ISCO column and purified. The product is then processed under basic condensation reaction conditions and general debenzoylation conditions to give the desired nucleoside analogs.

Example 24 Synthesis of L-Ara-nucleoside analogs with substituents at the carbon atom in position 2.

Place 1,3,5-tri-O-benzoyl-L-α-ribose into a dry flask under an argon atmosphere. The carbohydrate is dissolved in dry DCM and the reaction flask is cooled to -78°C. DMSO and oxyl chloride are then added to the reaction flask. After stirring for 1 hour, a solution of trimethylamine in DCM is added to the reaction flask. After consumption of the starting material, the reaction is quenched with water. The organic layer is separated, washed with sodium chloride solution, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The product is loaded onto an ISCO column and purified.

The resulting 2-keto intermediate was placed in a dry flask under argon and dissolved in dry THF. Then the reaction flask is cooled to -78°C. The appropriate organometallic reagent is then added. After consumption of the starting material, the reaction mixture is quenched with water. The organic layer is separated, washed with sodium chloride solution, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The product is loaded onto an ISCO column and purified.

Under stirring, methyl 2-chloro-2-oxoacetate is added dropwise to a solution of the protected carbohydrate and 4-DMAP in acetonitrile at room temperature under nitrogen using a syringe. The mixture was stirred at room temperature for 2 h and then diluted with EtOAc.

The resulting organic solution is successively washed with sat. aq. NAHCO3 solution, water and sodium chloride solution (1x120 ml each), dried over MgSO ₄ , filtered and concentrated on a rotary evaporator. The obtained crude product is dried under high vacuum overnight to obtain the desired product. The crude product mixture was used in its entirety in the next step without further purification.

Solid AIBN was added in one portion to a solution of the above product and tributyltin hydride in toluene while stirring under nitrogen at reflux. The mixture is refluxed for 2 hours and then cooled to room temperature. The volatiles are removed by rotary evaporation and the crude residue is dissolved in a small amount of PhMe. Flash chromatography on Combiflash yields the desired product, which is further processed under basic condensation reaction conditions and general debenzoylation conditions.

Example 25 Cell Incubation and Analysis Procedure

Huh-7 cells are seeded at a density of 0.5 x 10 -6 cells per well in 1 ml of complete medium in 12-well tissue culture plates. The cells are left overnight at 37°C/5% CO ₂ . The initial 40 μm solution of the test product is prepared in 100% DMSO. From the 40 µM stock solution, prepare a 20 µM solution of the test product in 25 ml of complete DMEM medium. For compound treatment, medium is removed from the wells and 1 ml of a 20 μM solution in complete DMEM medium is added to the appropriate wells. Additionally prepare a separate tablet with cells, but without adding compounds. The plates are incubated at 37° C./5% CO ₂ for the following time points: 1, 3, 6 and 24 hours. After incubation at the indicated time points, the cells are washed twice with 1 ml of DPBS. Cells are extracted by adding to each well treated with test product 500 µl of 70% methanol/30% water, supplemented with an internal standard. The raw plate blank is extracted with 500 μl of 70% methanol/30% water per well. Samples are centrifuged at 16,000 rpm for 10 min at 4°C. Samples are analyzed by LC-MS/MS using an ABSCIEX 5500 QTRAP LC-MS/MS system and a Hypercarb (PGC) column.

Example 26 Methodology for a pharmacokinetic experiment in rats.

Rats are allowed to acclimate for 2 or more days after arrival. Rats are weighed on the day before dosing to calculate the dose volume. Rats are administered the drug p/o at a dose of 50 mg/kg, 10 mg/kg and 5 ml/kg. Rats were sampled at 6 time points: 1, 2, 3, 4, 6 and 24 hours (3 rats per time point for study drug). Rats are sacrificed and organs removed (see below). For blood collection, rats are sacrificed with CO ₂ at the appropriate time point indicated above. Blood is collected by cardiac puncture (0.3 ml) at each time point. After blood collection, the organs of the rats are removed (see below). The blood is processed by gently inverting the Li-heparin blood tubes 2 or 3 times to mix thoroughly. The tubes are then placed in a rack in ice water until they are suitable for centrifugation (<1 h). Once they have become suitable, the blood is centrifuged at ~2000 x g for 10 min in a refrigerated centrifuge to obtain plasma. Then, using a pipette, 200 µl of plasma is transferred into a labeled 1.5 ml Eppendorf tube in ice water. Plasma is frozen on dry ice or in a refrigerator. Samples are stored at -80°C until analysis. Organs of slaughtered rats are removed. Organs (lungs, liver, kidneys, spleen and heart) are removed, placed in a test tube and immediately frozen in liquid nitrogen. The tubes are then transferred to dry ice. Samples are stored in cryogenic tissue vials. Samples are analyzed by LC-MS/MS using an ABSCIEX 5500 QTRAP LC-MS/MS system and a Hypercarb (PGC) column.

Claims (17)

1. A compound having the following formula: or a pharmaceutically acceptable salt thereof, where each R ¹ is independently selected from one of the formulas R ⁴ is hydrogen, benzyl, O _1-6 alkyl or cycloalkyl; R ⁵ is hydrogen, deuterium, hydroxyl, cyano, azido, amino, substituted amino, aryl, heteroaryl, substituted aryl, O _1-6 alkoxy, O _1-6 alkyl, O _2-6 alkenyl, O _2-6 alkynyl, or substituted heteroaryl; And R ⁶ is O _1-6 alkoxy, O _1-6 alkyl, cycloalkyl, aryl or substituted aryl, wherein when the group is substituted it is substituted with one or more of the following: 0 _1-6 alkyl, halogenated O _1-6 alkyl , O _1-6 alkoxy, O _2-6 alkenyl, O _2-6 alkynyl, aryl, heteroaryl, -C(O)H, amino, -C(O)OH, -OC(O)A1 or -C(O )OA ¹ , A1OA ² , halide, hydroxy, A1C(O)A ² , nitro, sulfo-oxo, sulfonyl, sulfone, sulfoxide or thiol, where A ¹ and A ² are independently selected from C _1-6 alkyl, - 22 039123 halogenated C _1-6 alkyl, C _{2 -} balkenyl, C ₂ . ₆ alkynyl, aryl, heteroaryl or cycloalkyl, where aryl is naphthalene, phenyl, biphenyl or phenoxybenzene, cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, heteroaryl is an aromatic group containing a heteroatom selected from nitrogen, oxygen, sulfur or phosphorus . 2. A compound according to claim 1, selected from the following: 3. The compound according to claim 2, having the following formula: 4. A compound according to claim 1, wherein the aryl is phenyl, 1-naphthyl, 2-naphthyl, or 4-substituted phenyl. 5. A compound according to claim 1, wherein the aryl is 4-chlorophenyl or 4-bromophenyl. 6. A compound according to claim 1 wherein R ³ is fluoromethyl, difluoromethyl, trifluoromethyl or hydroxymethyl. 7. A compound according to claim 1 wherein R ⁴ is methyl, ethyl, isopropyl, cyclopentyl, cyclohexyl, neopentyl or benzyl. 8. A compound according to claim 1 wherein R ⁴ is methyl, ethyl, isopropyl, cyclopentyl, cyclohexyl or neopentyl. 9. A compound according to claim 1 wherein R ⁶ is methyl, ethyl or t-butyl. 10. A pharmaceutical composition for the treatment or prevention of a viral infection, containing a therapeutically effective amount of a compound according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. 11. Pharmaceutical composition according to claim 10, characterized in that the viral infection is caused by an infectious agent containing a DNA virus. 12. Pharmaceutical composition according to claim 10, characterized in that the DNA virus contains hepadnaviruses. 13. Liposomal composition for the treatment or prevention of a viral infection, containing a therapeutically effective amount of a compound according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. 14. A method of treating or preventing infections caused by DNA viruses, which comprises administering to a subject in need thereof an effective amount of a compound of claim 1, or a pharmaceutically acceptable salt thereof. 15. The method according to claim 14, characterized in that the DNA virus contains hepadnaviruses. 16. A method of treating or preventing infections caused by DNA viruses, comprising administering to a subject in need thereof an effective amount of a compound having the structure as defined in claim 2 - 23 039123 or a pharmaceutically acceptable salt thereof and additional administration of an antiviral agent selected from abacavir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla, boceprevir, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine , enfuvirtide, entecavir, famciclovir, fomivirsen, fosamprenavir, foscarnet, phosphonet, ganciclovir, ibacitabin, imunovir, idoxuridine, imiquimod, indinavir, inosine, type III interferon, type II interferon, type I interferon, lamivudine, lopinavir, loviride, maraviroc, moroxidine , metisasone, nelfinavir, nevirapine, nexavir, oseltamivir, peginterferon a-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, ribavirin, rimantadine, ritonavir, pyramidin, saquinavir, sofosbovir, stavudine, telaprevir, telbivudine, dipzoranil, tenofovir, tenofovir , trifluridine, trizivir, tromantadine, trivada, valacic lovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, zidovudine, and combinations thereof. 17. A method of treating or preventing infections caused by DNA viruses, comprising administering to a subject in need thereof an effective amount of a compound of claim 2, or a pharmaceutically acceptable salt thereof.