The present invention relates to salts of (S)-4-[(R)-6-(2-Chloro-4-fluoro-phenyl)-5- methoxycarbonyl-2-thiazol-2-yl-3,6-dihydro-pyrimidin-4-ylmet hyl]-morpholine-3-carboxylic acid and a salt former, the synthesis of the salts, and their use for the treatment of pathological disorders, particularly hepatitis B (HBV) infection.

Background of the Invention

HBV is a species of the hepadnaviridae family of viruses. HBV is a serious public health problem worldwide, with more than 400 million people especially in Asia-pacific regions chronically infected by this small enveloped DNA virus. Although most individuals seem to resolve the infection following acute symptoms, 15-40% of HBV patients will finally develop clinical diseases during their lifespan, most notably, hepatitis, liver cirrhosis, and hepatocellular carcinoma. Every year 500,000 to 1 million people die from the end stage of liver diseases caused by HBV infection.

HBV lifecycle begins with the binding of the "Dane" particle with an unidentified receptor on the surface of hepatocyte. Following entry, viral genome is delivered into nucleus where a covalently closed circular DNA (cccDNA) is formed through DNA repair of viral relaxed circular DNA. Unlike the mechanisms of most other DNA viruses, HBV cccDNA replicates through the retro transcription of a 1.1 -genome unit-length RNA copy (pregenomic RNA). Viral pregenomic RNA interacts with other two viral components, capsid protein and polymerase, as well as some host factors, to form capsid particles where viral DNA replication occurs. Most copies of the encapsidated genome then efficiently associate with the envelope proteins for virion assembly and secretion; a minority of these genomes is shunted to the nucleus, where they are converted to cccDNA. Currently, there are two types of anti-HBV agents on the market, nucleoside (tide) analogs targeting viral polymerase (lamivudine, adefovir, tenofovir, telbivudine and entecavir) and interferon modulating host immune functions. Mutations in the primary sequence of the polymerase that confer resistance to lamivudine and adefovir have been identified clinically and underlie a rebound of serum virus titers that 70% of treated patients experience within 3 years of the start of lamivudine therapy. Although resistance to telbivudine, adefovir, and entecavir occurs more rarely, it has been recorded. Interferon alpha is the other major therapy available for hepatitis B, but it is limited by a poor long-term response and debilitating side effects. Some viral genotypes do not show good responses to interferon therapy. Now, the standard of clinic cure of HBV infection is the loss and/or seroconversion of HBsAg. The majority (around or more than 90%) of treated patients fail to achieve this goal. This drawback is mainly due to the presence of a stable pool of viral cccDNA in nucleus that doesn't replicate itself, therefore, shows no accessibility to nucleoside (tide) analogs. HBV capsid protein plays essential roles in HBV replication. HBV has an icosahedral core comprising of 240 copies of the capsid (or core) protein. The predominant biological function of capsid protein is to act as a structural protein to encapsidate pre-genomic RNA and form immature capsid particles in the cytoplasm. This step is prerequisite for viral DNA replication. The HBV capsid spontaneously self-assembles from many copies of core dimers present in the cytoplasm. It has been shown that the formation of a trimeric nucleus and the subsequent elongation reactions occur by adding one dimeric subunit at a time until it is complete. Besides this function, capsid protein regulates viral DNA synthesis through different phosphorylation status of its C-terminal phosphorylation sites. When a near full-length relaxed circular DNA is formed through reverse-transcription of viral pregenomic RNA, an immature capsid becomes a mature capsid. On one hand, capsid protein might facilitate the nuclear translocation of viral relaxed circular genome by means of the nuclear localization signals located in the Arginine-rich domain of the C-terminal region of capsid protein. In nucleus, as a component of viral cccDNA minichromosome, capsid protein could play a structural and regulatory role in the functionality of cccDNA minichromosomes. Capsid protein also interacts with viral large envelope protein in endoplasmic reticulum and triggers the release of intact viral particles from hepatocytes.

There has been a couple of capsid related anti-HBV inhibitors reported. For example, phenylpropenamide derivatives, including compounds named AT-61 and AT-130 (Feld J. et al. Antiviral Research 2007, 168-177), and a class of thiazolidin-4-ones from Valeant R&D

(WO2006/033995), have been shown to inhibit pgRNA packaging. A recent study suggested that phenylpropenamides are, in fact, accelerators of HBV capsid assembly, and their actions result in the formation of empty capsids. These very interesting results illustrate the importance of the kinetic pathway in successful virus assembly.

Heteroaryldihydropyrimidines or HAP, including compounds named Bay 41-4109, Bay 38-7690 and Bay 39-5493, were discovered in a tissue culture -based screening (Deres K. et al. Science 2003, 893). These HAP analogs act as synthetic allosteric activators and are able to induce aberrant capsid formation that leads to degradation of the core protein. HAP analogs also reorganized core protein from preassembled capsids into noncapsid polymers, presumably by interaction of HAP with dimers freed during capsid 'breathing', the transitory breaking of individual intersubunit bonds. Bay 41-4109 was administered to HBV infected transgenic mouse or humanized mouse models and demonstrated in vivo efficacy with HBV DNA reduction (Deres K. et al. Science 2003, 893; Brezillon N. et al. PLoS ONE 2011, e25096). It was also shown that bis-ANS, a small molecule that acts as a molecular 'wedge' and interferes with normal capsid- protein geometry and capsid formation (Zlotnick A. et al. J. Virol. 2002, 4848-4854).

Summary of the invention

The present invention provides organic acid salts as therapeutic agents for treating diseases and disorders, such as viral infections caused by hepatitis B virus.

In one embodiment, the invention provides a salt comprising at least two components: (A) the compound according to Formula I or a pharmaceutically acceptable stereoisomer, or tautomer thereof; and

(B) a salt former. The salt formers are acids. Illustrative of acids that can be used as salt formers are HC1, tartaric acid, fumaric acid, succinic acid, malonic acid, glutaric acid, adipic acid, benzoic acid, acetic acid, a-keto glutaric acid and anthranilic acid.

According to one embodiment the salt former is tartaric acid, while for some other embodiments the salt former can be an acid selected from HC1, benzoic acid, acetic acid or succinic acid.

Salts according to the present invention can comprise the compound of Formula I and tartrate, the compound of Formula I and HC1, the compound of Formula I and zinc benzoate, the compound of Formula I and zinc acetate, or the compound of Formula I and succinate.

The inventive salts of the compound of Formula I and an acid are crystalline and have characteristic X-ray powder diffraction peaks at a diffraction angle of 2Θ as further illustrated in Table 2. The inventive salts were further characterized by IR absorption and Raman

spectroscopy.

In one embodiment, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and a salt comprising at least two components: (A) the compound according to Formula I or a pharmaceutically acceptable stereoisomer or tautomer thereof and (B) and a salt former. According to an embodiment, the salt of the inventive pharmaceutical composition comprises the compound of Formula I and tartaric acid as the salt former. According to another embodiment, the invention provides a method for treating a disease or condition by administering to a subject in need thereof a therapeutically effective amount of the acid salt according to claim 1. Illustrative of diseases or conditions treated using the inventive salt of the compound of Formula I and an acid are viral infections, in particular hepatitis B infection.

According to an embodiment of this method, the salt of the compound of Formula I and an acid is administered in combination with a therapeutically effective amount of a second therapeutic agent selected from the group consisting of antiviral agents, immunomodulatory agents, a- interferon, β-interferon, pegylated a-interferon, pegylated β-interferon, ribavirin, polymerase inhibitors, and toll receptor-like modulators. Specifically, the acid salts of the present invention can be used together with the pegylated a-interferons PEGASYS® (Roche) or Peglntron® (MSD), lamivudine, adefovir dipivoxil, entecavir, telbivudine, and tenofovir disoproxil for the treatment or prophylaxis of HBV infections.

Acid derived salts in accordance with the invention are synthesized by combining a solution or a suspension of the salt former with a solution or a suspension of the compound of Formula I or a pharmaceutically acceptable stereoisomer or tautomer thereof to obtain a combined solution, followed by supersaturating said solution to initiate formation of salts.

Solvents used to solubilize the salt former and the compound of Formula I include organic solvents, water, or a mixture of water and an organic solvent. Illustrative of the class organic solvents suitable for solubilizing the salt former include without limitation methanol, ethanol, propanol, isopropanol, isopropyl acetate, hexane, heptane, toluene, acetone, acetonitrile, dioxane, tetrahydrofuran (THF), ethyl acetate, or combinations thereof.

Brief description of the figures

Figure 1 - XRPD (X-ray powder diffraction) pattern of Form A of the tartaric acid salt of (S)-4- [(R)-6-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2-thiazo l-2-yl-3,6-dihydro-pyrimidin-4- ylmethyl]-morpholine-3-carboxylic acid (compound of Formula I).

Figure 2 - FT-Raman spectra of Form A of the tartaric acid salt of (5 ^, )-4-[(R)-6-(2-Chloro-4- fluoro-phenyl)-5-methoxycarbonyl-2-thiazol-2-yl-3,6-dihydro- pyrimidin-4-ylmethyl]- morpholine-3-carboxylic acid (compound of Formula I).

Detailed description of the invention

The present invention provides salts that comprise the compound of Formula I and an acid as the salt former. The inventive acid derived salts and their pharmaceutical compositions are useful in treating or preventing viral infections, in particular viral infections caused by hepatitis B virus.

It should be noted that the term "comprising" (and its grammatical variations) is used in the inclusive sense of "having" or "including" and not in the exclusive sense of "consisting only of '. The terms "a," "an" and "the" as used herein are understood to encompass the plural as well as the singular.

The phrases "active pharmaceutical ingredient" or "API" refer to a substance, for example a compound or a biologic in a pharmaceutical composition that elicits a biological activity.

Within the context of the invention, the term "immunomodulator" refers to natural or synthetic products capable of modifying the normal or aberrant immune system through stimulation or suppression.

The term "preventing" refers to the ability of a compound or composition of the invention to prevent a disease identified herein in patients diagnosed as having the disease or who are at risk of developing such disease. The term also encompasses preventing further progression of the disease in patients who are already suffering from or have symptoms of such disease.

The term "patient" or "subject" means an animal (e.g., cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit, guinea pig, etc.) or a mammal, preferably a human, including chimeric and transgenic animals and mammals.

The term a "therapeutically effective amount" refers to an amount of the compound of the invention sufficient to provide a benefit in the treatment or prevention of disease, to delay or minimize symptoms associated with disease, or to cure or ameliorate the disease or infection or causes thereof. In particular, a therapeutically effective amount means an amount sufficient to provide a therapeutic benefit in vivo. Used in connection with an amount of a compound of the invention, the term preferably encompasses a non-toxic amount that improves overall therapy, reduces or avoids symptoms or causes of disease, or enhances the therapeutic efficacy of or synergies with another therapeutic agent.

The terms "treat", "treating" and "treatment" refer to the amelioration or eradication of a disease or symptoms associated with a disease. In certain embodiments, such terms refer to minimizing the spread or worsening of the disease resulting from the administration of one or more prophylactic or therapeutic agents to a patient with such a disease. Some compounds described herein can have asymmetric centers and therefore can exist as enantiomers and diastereomers. A compound of the invention can be in the form of a single enantiomer (optical isomer), a diastereomer, or as a mixture of enantiomers, including a racemic mixture. Optical isomers of the compounds of the invention can be obtained by known techniques such as asymmetric synthesis, chiral chromatography, or via chemical separation of stereoisomers through the employment of optically active resolving agents.

Unless otherwise indicated, "stereoisomer" means one stereoisomer of a compound that is substantially free of other stereoisomers of that compound. Thus, a stereomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound. A typical stereomerically pure compound comprises greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, for example greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, or greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound.

If there is a discrepancy between a depicted structure and a name given to that structure, then the depicted structure controls. Additionally, if the stereochemistry of a structure or a portion of a structure is not indicated with, for example, bold or dashed lines, the structure or portion of the structure is to be interpreted as encompassing all stereoisomers of it. Those skilled in the art of organic synthesis will know if the compounds are prepared as single enantiomers from the methods used to prepare them.

Acid derived salts of the compound of Formula I

( _l S')-4-[(R)-6-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbo nyl-2-thiazol-2-yl-3,6-dihydro- pyrimidin-4-ylmethyl]-morpholine-3-carboxylic acid illustrated below as Formula I exists as an amorphous powder in the free base form.

The free base, however, has a strong hygroscopic behavior leading to an undesired uptake of water. Accordingly, there's no suitable manufacturing process to consistently synthesize pharmaceutical grade material and thus, the free base is unsuitable as a pharmaceutical agent. These disadvantages are circumvented by the tartaric acid salt which is a non-hygroscopic and which allows that material of suitable quality for the further galenical processing can be produced consistently. The inventive salts, synthesized using protocols described below, address several drawbacks and challenges associated with the synthesis and pharmaceutical development of the free base form of the compound of Formula I as a therapeutic for treating HBV infections. As further described below, the inventive salts are synthesized by contacting (S)-4-[(R)-6-(2-Chloro-4-fluoro-phenyl)- 5-methoxycarbonyl-2-thiazol-2-yl-3,6-dihydro-pyrimidin-4-ylm ethyl]-morpholine-3-carboxylic acid (compound of Formula I) and at least one salt former.

Several acids have been evaluated as salt formers. Illustrative salt formers include without limitation oxalic acid, HC1, L-aspartic acid, maleic acid, saccharin, 2-aminobenzoic acid, L- glutamic acid, L-threonine, 4-aminobenzoic acid, a-ketoglutaric acid, l-hydroxy-2-naphthoic acid, malonic acid, gentisic acid, salicylic acid, tartaric acid, fumaric acid, galactaric acid, citric acid, D-glucuronic acid, β-cyclodextrin, hydroxypropyl-P-cyclodextrin, 4- amino salicylic acid, nicotinamide, L(-)-malic acid, hippuric acid, glycolic acid, L(-)pyroglutamic acid, γ- cyclodextrin, benzoic acid, succinic acid, glutaric acid, adipic acid, 4-hydroxybenzoic acid, (+)camphoric acid, sorbic acid, nicotinic acid, orotic acid, urea, methylparabene, propylparabene, L(-)-lactamide, L-ascorbic acid, cinnamic acid, D,L-mandelic acid, acetic acid, vanillic acid and methyl-4-hydroxybenzoate. Table 1 illustrates physical characteristics of exemplary salts comprising the compound of Formula I and an acid salt former in accordance with the present invention. As illustrated in Table 1 and further described below, many of the exemplified salts have superior aqueous solubility permitting their designation as Class I drugs based on the Biopharmaceutics

Regulatory Systems (BCS) classification system.

Table 1

The inventive salts of the compound of Formula I and an acid were further characterized using X-ray powder diffraction (XRPD), infrared (IR) and Raman spectroscopy. The 2Θ values from X-ray powder diffraction studies for several salts in accordance with the invention are listed in Table 2.

Table 2

Pharmaceutical compositions and dosages

The present invention also is directed to pharmaceutical formulations of the inventive salts of the compound of Formula I and an acid and the use of such pharmaceutical formulations for the treatment of disease conditions such as viral infections, in particular viral infections caused by hepatitis B virus. In one aspect, therefore, the present invention relates to a pharmaceutical formulation of a salt comprising the compound of Formula I or a pharmaceutically acceptable stereoisomer, tautomer, or solvate and at least one salt former as well as pharmaceutically acceptable excipients.

The inventive compositions may further contain in accordance with accepted practices of pharmaceutical compounding, one or more additional therapeutic agents, pharmaceutically acceptable diluents, adjuvants, stabilizers, emulsifiers, preservatives, colorants, buffers, or flavor imparting agents. Suitable excipients are well known to those skilled in the art of pharmacy, and non-limiting examples of suitable excipients are provided herein. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the way in which the dosage form will be administered to a patient. For example, oral dosage forms such as tablets may contain excipients not suited for use in parenteral dosage forms. The suitability of a particular excipient may also depend on the specific active ingredients in the dosage form. Pharmaceutical compositions suitable for single unit dosages that comprise a salt of the invention and a pharmaceutically acceptable carrier are also encompassed within the scope of the present disclosure. Pharmaceutical formulations as well as single unit dosage forms according to the present invention may be suitable for oral, mucosal (including sublingual, buccal, rectal, nasal, or vaginal), parenteral (including subcutaneous, intramuscular, bolus injection, intra-arterial, or intravenous), transdermal, or topical administration. Sterile formulations of the inventive salt are also contemplated.

Pharmaceutical formulations as well as single unit dosage forms can contain various amounts of the salt. For instance, the amount of salt in the pharmaceutical formulations as well as single unit dosage forms may range from about 0.1 mg to about 1000 mg. For some formulations and single dosage forms the amount of salt is from about 50 mg to about 750 mg, from about 50 mg to about 500 mg, from about 100 mg to about 250 mg, from about 10 mg to about 100 mg, or about 1 mg to about 10 mg. Anhydrous pharmaceutical compositions comprising a salt of the invention also are encompassed within the scope of this disclosure. Since moisture and/or humidity are routinely encountered during the manufacture, storage, handling, packaging, shipment and use of pharmaceutical formulations, methods for preparing anhydrous pharmaceutical compositions and dosage forms of the disclosure using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions known to those skilled in the formulations art are within the scope of this disclosure. To maintain the anhydrous nature of a pharmaceutical composition of the salts, the inventive pharmaceutical formulations should be packaged and stored under conditions that maintain its anhydrous nature. Accordingly, anhydrous compositions are preferably packaged using materials known to prevent exposure to water. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

Salts in accordance with the present invention are therapeutics for the treatment of diseases or disorders associated with improper cell division, improper immune function, or to treat viral infections. Treatment may be effected by administering one or more therapeutically effective doses of the inventive salts to a patient or subject in need of treatment.

Oral dosage forms

Inventive compositions suitable for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions. See generally, Remington 's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990). Such oral dosage forms contain predetermined amounts of active ingredients in an intimate admixture with at least one excipient. Liquid formulations can further contain sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations of a salt of the invention.

Suitable oral compositions in accordance with the invention include without limitation tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, syrups or elixirs. Because of their ease of administration, storage and packaging, tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid excipients are employed. If desired, tablets can be coated by standard aqueous or nonaqueous techniques. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients, namely the salt of the compound of Formula I and an acid, with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary.

For tablet compositions the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients is used for the manufacture of tablets. Examples of such excipients include without limitation inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin, gums such as acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The binder or filler used for a pharmaceutical composition of the invention is typically present in from about 50 to about 99 weight percent of the pharmaceutical composition or dosage form.

Tablets can be prepared by compression or molding and may be uncoated or coated to delay disintegration and absorption in the gastrointestinal tract and thereby to provide a sustained therapeutic action over a desired time period. Exemplary time delay materials include without limitation glyceryl monostearate or glyceryl distearate.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

In addition to tablets and capsules, a salt of the invention may be formulated as syrups, suspensions, dispersible powders and granules that are suitable for oral administration. When formulated as an oral suspension, the pharmaceutical composition can contain dispersing or wetting agents, such as naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with a fatty acid, a long chain aliphatic alcohol, or partial esters of fatty acids. Illustrative of such products are polyoxyethylene stearate,

heptadecaethyleneoxycetanol, a hexitol such as polyoxyethylene sorbitol monooleate, or polyethylene sorbitan monooleate. Oral suspensions may also contain one or more

preservatives, for example ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin. Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, and flavoring and coloring agents.

Delayed release dosage forms

The inventive salts can be administered by controlled release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Patent Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; and 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5, 120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,566, each of which is incorporated herein by reference. Such dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example,

hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled-release formulations are known to those of ordinary skill in the formulary art. The disclosure thus encompasses single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gel caps, and caplets that are adapted for controlled-release.

All controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled counterparts. Ideally, the use of a controlled-release preparation in medical treatment is characterized by administration of a known quantity of the active agent over a pre-determined time interval, such as a 2 days, 3, days, 4 days, 5 days, 6 days, 1 week, 2weeks, 3 weeks, 4 weeks, or from 2- 12 months, 3-12 months, 4- 12 months, 5- 12 months, 6- 12 months, 7-12 months, 8- 12 months, 9- 12 months, 10-12 months, 11- 12 months, or 1 year. Advantages of controlled-release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance. In addition, controlled-release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the drug, and can thus affect the occurrence of side (e.g., adverse) effects.

Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release the same or other amounts of drug to maintain the same levels of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, temperature, enzymes, water, or other physiological conditions or compounds. Parenteral dosage forms

Parenteral dosage forms can be administered to patients by various routes including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intra- arterial injections. Because their administration typically bypasses patients' natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry and/or lyophylized products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection (reconstitutable powders), suspensions ready for injection, and emulsions. Suitable vehicles that can be used to provide parenteral dosage forms of the disclosure are well known to those skilled in the art. Examples include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Parenteral formulations can include compounds that increase the solubility of one or more of the active pharmaceutical ingredients or an inventive salt of the compound of Formula I and an acid in accordance with the invention.

Kits

The disclosure provides a pharmaceutical pack or kit comprising one or more containers comprising a salt in accordance with the invention and can optionally include one or more containers containing a pharmaceutically acceptable carrier. In other embodiments, the disclosure provides a pharmaceutical pack or kit comprising one or more containers comprising a salt in accordance with the invention, one or more containers comprising an additional therapeutic agent and one or more containers containing a pharmaceutically acceptable carrier. A kit or a pack in accordance with the invention may comprise one or more containers or packets comprising one or more of the pharmaceutical ingredients necessary for formulating the inventive salt into a pharmaceutical composition suitable for administration as well as devices such as one or more syringes for administering a pharmaceutical composition of the inventive salt.

Kits or packs may include buffers, compounds to adjust the tonicity of the pharmaceutical carrier and compounds to alter the hydrophilic-lipophilic balance (HLB) so as to improve delivery and bio-distribution. Optionally associated with the container(s) present in a kit or a pack can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

Combination therapy

In one aspect, a therapeutically effective dose of a salt of the invention may be administered along with a therapeutically effective dose of the combination drug (additional therapeutic agent) to a patient or subject in need of treatment. The person of skill in the art will recognize that a dose of the salt of the compound of Formula I and an acid may be administered separately, say within seconds, minutes or hours before or after the administration of the combination drug or the two doses may be administered together. The additional therapeutic agents include, but are not limited to antibiotics, antiemetic agents, antidepressants, and antifungal agents, antiinflammatory agents, antiviral agents, anticancer agents, immunomodulatory agents, a- interferons, β-interferons, pegylated a-interferon, pegylated β-interferon, ribavirin, alkylating agents, hormones, cytokines, or toll receptor-like modulators.

In addition to the above therapeutic agents, the present invention encompasses combination therapy regimens that include a salt of the compound of Formula I and an acid in combination with a therapeutically effective amount of a polymerase inhibitor, for example, lamivudine, adefovir, entecavir and telbivudine. Therapeutic regimens comprising the combination of three drugs are also encompassed within the scope of the invention. For instance, such a regimen can encompass the administration of therapeutically effective doses of the salt of the compound of Formula I and an acid, a polymerase inhibitor and an interferon or a pegylated interferon. The percentages of each compound or a pharmaceutically acceptable salt or tautomer thereof, present in a composition can also vary. For example, in some embodiments, the composition can include an amount of the salt of the compound of Formula I and an acid or a pharmaceutically acceptable salt or tautomer in the range of about 1% to about 98% (weight/weight). The composition can contain the inventive salt of the compound of Formula I and an acid or a pharmaceutically acceptable salt or tautomer thereof, in the range of about 5% to about 80%, about 10% to about 70%, about 15% to about 60%, about 20% to about 50% and about 30% to about 40% (weight/weight). The inventive salt can be administered or formulated in combination with other antiviral agents too. Useful antiviral agents include, but are not limited to, protease inhibitors, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and nucleoside analogs. The antiviral agents include but are not limited to zidovudine, didanosine, stavudine, combivir, abacavir, adefovir, dipivoxil, cidofovir, ribavirin and its analogs, levovirin, viramidine, isatoribine, pirfenidone, or its analogs, acyclovir, gangcyclovir, vidarabine, idoxuridine, trifluridine, and as well as foscarnet, amantadine, rimantadine, saquinavir, indinavir, amprenavir, lopinavir, ritonavir, the a-interferons; β-interferons; clevadine, entecavir, pleconaril.

When administered in combination with an additional therapeutic agent, the salt and the other therapeutic agent can act additively or, more preferably, synergistically. In one embodiment, a composition comprising a salt of the present invention is administered concurrently with the administration of another therapeutic agent, which can be part of the same composition or in a different composition from that comprising the compounds of the disclosure. In another embodiment, the salt may be administered prior to or subse quent to administration of another therapeutic agent. In a separate embodiment, a salt of the present invention is administered to a patient who has not previously undergone or is not currently undergoing treatment with another therapeutic agent, particularly an antiviral agent. Examples

The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention.

General experimental conditions

Intermediates and final compounds were purified by flash chromatography using one of the following instruments: i) Biotage SP1 system and the Quad 12/25 Cartridge module, ii) ISCO combi-flash chromatography instrument. Silica gel brand and pore size: i) KP-SIL 60 A, particle size: 40-60 μΜ; ii) CAS registry NO: Silica Gel: 63231-67-4, particle size: 47-60 micron silica gel; iii) ZCX from Qingdao Haiyang Chemical Co., Ltd, pore: 200-300 or 300-400.

Intermediates and final compounds were purified by preparative HPLC on reversed phase column using XBridge™ Prep-C18 (5 μιη, OBDTM 30 x 100 mm) column or SunFire™ Prep- C18 (5 μιη, OBDTM 30 x 100 mm) column. Waters AutoP purification System (Column:

XBridgeTM Prep-C18, 30 x 100 mm, Sample Manager 2767, Pump 2525, Detector: Micromass ZQ and UV 2487, solvent system: acetonitrile and 0.1% ammonium hydroxide in water). For SFC chiral separation, intermediates were separated by chiral column (Daicel chiralpak IC, 5 μιη, 30 x 250 mm) column using Mettler Toledo SFC-Multigram III system, solvent system: 95% C0 ₂ and 5% IPA (0.5% TEA in IPA), back pressure lOObar, detection UV@ 254nm.

LC/MS spectra of compounds were obtained using a LC/MS (Waters™ Alliance 2795- Micromass ZQ), LC/MS conditions were as follows (running time 6 min): Acidic condition: A: 0.1% formic acid in H ₂ 0; B: 0.1% formic acid in acetonitrile;

Basic condition: A: 0.01% NH ₃ H ₂ 0 in H ₂ 0; B: acetonitrile;

Neutral condition: A: H ₂ 0; B: acetonitrile.

Mass spectra (MS): generally only ions which indicate the parent mass are reported, and unless otherwise stated the mass ion quoted is the positive mass ion (MH) ⁺ .

NMR Spectra were obtained using Bruker Avance 400 MHz. The microwave assisted reactions were carried out in a Biotage Initiator Sixty microwave synthesizer.

All reactions involving air-sensitive reagents were performed under an argon atmosphere.

Reagents were used as received from commercial suppliers without further purification unless otherwise noted.

The following examples were prepared by the general methods outlined in the schemes above. They are intended to illustrate the meaning of the present invention but should by no means represent a limitation within the meaning of the present invention:

Preparation of ( _t S')-4-r(R)-6-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbo nyl-2-thiazol-2-yl-3,6- dihydro-pyrimidin-4-ylmethyll -morpholine-3-carboxylic acid To a stirred solution of (R)-6-bromomethyl-4-(2-chloro-4-fluoro-phenyl)-2-thiazol-2-y l-l,4- dihydro-pyrimidine-5-carboxylic acid methyl ester (0.049 g, 0.11 mmol) and (S)-morpholine-3- carboxylic acid (Aldrich, CAS: 106825-79-0) (0.044 g, 0.17 mmol) in 1,2-dichloroethane (5 mL) was added dropwise DIPEA (0.078 mL, 0.45 mmol). The reaction mixture was stirred at room temperature until the disappearance of starting material which was checked by LC/MS. The mixture was diluted with EtOAc (50 mL) and washed successively with saturated aqueous

NH ₄ CI solution and brine. The organic layer was separated and dried over Na ₂ S0 ₄ . The solvent was concentrated in vacuo and the crude product was purified by prep-HPLC to give (5)-4-[(Λ)- 6-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2-thiazol-2-y l-3,6-dihydro-pyrimidin-4- ylmethyl]-morpholine-3-carboxylic acid (compound of Formula (I)) as described in

WO2014/037480.

Preparation of ( _t S ^, )-4-r(R)-6-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbony l-2-thiazol-2-yl-3,6- dihvdro-pyrimidin-4-ylmethyll-morpholine-3-carboxylic acid-tartaric acid salt

200.0 g ( _l S')-4-[(R)-6-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbo nyl-2-thiazol-2-yl-3,6-dihydro- pyrimidin-4-ylmethyl]-morpholine-3-carboxylic acid (RO6889678-000) were slurried in 1500 g of isopropyl acetate. The suspension was stirred and heated to 50°C. A clear yellow solution was obtained. 60.6 g L(+) tartaric acid were suspended in 200 g of methanol. The suspension was stirred and heated to 50 °C to dissolve the solid completely. The clear methanolic tartaric acid solution was added within 1 h to the isopropyl acetate solution of RO6889678-000. After complete addition of the methanol the obtained suspension was aged for min 15 at 50 °C and subsequently cooled down to 20 °C in 6 - 12 h. The suspension was stirred for 2 hour at 20 °C. The solid was isolated by filtration and rinsed by 500 g of isopropyl acetate.

The product was dried at 40 °C / 0-5 mbar until weight constant, approx 12- 16 h.

Yield: 234.9 g (90%) of (5)-4-[(R)-6-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbonyl-2- thiazol- 2-yl-3,6-dihydro-pyrimidin-4-ylmethyl]-morpholine-3-carboxyl ic acid-tartaric acid salt.

X-Ray Powder Diffraction (Stoe Stadi P)

XRPD patters were recorded at ambient conditions with a STOE STADI P diffractometer (CuKal radiation, primary Ge-monochromator, strip detector (Mythen IK), 3° to 42° 2-theta angular range, 0.5° 2-theta detector step width, 20s per step measurement time). The samples were analyzed without further processing (e.g. grinding or sieving) of the material.

XRPD-data

Form A of ( _l S')-4-[(R)-6-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbo nyl-2-thiazol-2-yl-3,6- dihydro-pyrimidin-4-ylmethyl]-morpholine-3-carboxylic acid (RO6889678-000) can be characterized by its XRPD pattern obtained with a Cu Ka radiation having characteristic peaks expressed in degrees 2Theta at approximately: 7.3, 7.7, 9.4, 17.3, 17.8, 17.9, 20.9 and 22.7.

Figure 1 shows the XRPD pattern of a typical lot of Form A of (5 ^, )-4-[(R)-6-(2-Chloro-4-fluoro- phenyl)-5-methoxycarbonyl-2-thiazol-2-yl-3,6-dihydro-pyrimid in-4-ylmethyl]-morpholine-3- carboxylic acid-(2R,3R)-2,3-dihydroxy-tartaric acid salt.

IR spectroscopy

The ATR FTIR spectra were recorded without any sample preparation using a ThermoNicolet iS5 FTIR spectrometer with an iD5 ATR accessory and DTGS detector. The spectral range is between 4000 cm- 1 and 650 cm-1, resolution 2 cm-1 and at least 50 co-added scans were collected. Happ-Genzel apodization was applied. Using ATR FTIR will cause the relative intensities of infrared bands to differ from those seen in a transmission FTIR spectrum using KBr disc or nujol mull sample preparations. Due to the nature of ATR FTIR, the bands at lower wavenumber are more intense than those at higher wavenumber.

IR analysis:

Form A of ( _l S')-4-[(R)-6-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbo nyl-2-thiazol-2-yl-3,6- dihydro-pyrimidin-4-ylmethyl]-morpholine-3-carboxylic acid-(2R,3R)-2,3-dihydroxy-tartaric acid salt can be characterized by IR spectroscopy having characteristic bands approximately at wavenumbers positions (cm-1): 659, 671, 756, 818, 832, 879, 907, 968, 1075, 1105, 1165, 1224, 1303, 1379, 1438, 1476, 1494, 1515, 1540, 1603, 1676, 1747, 2937, 3098, 3278, 3343 and 3460.

Characteristic Bands (cm- 1) in the IR Spectra for the Form A of RO6889678-004 E150115.302 HBVR08301 (Error is + 1 cm-1). Wavenumbers in red are unique for this form and could be used in claims.

Raman Spectroscopy

FT-Raman analysis

The Raman spectrum was collected in the spectral range of 4000-50 cm ^"1 with a Bruker

MultiRam FT-Raman spectrometer, equipped with a NdYAG 1064 nm laser and a liquid nitrogen cooled Germanium detector. The laser power at the sample was about 200 mW, 2 cm ^"1 resolution was used and 512 scans were co-added. The apodization used was Blackman-Harris 4- term.

Characteristic Bands (cm ¹ ) in the FT-Raman Spectra for the Form A of (5 ^, )-4-[(R)-6-(2-Chloro- 4-fluoro-phenyl)-5-methoxycarbonyl-2-thiazol-2-yl-3,6-dihydr o-pyrimidin-4-ylmethyl]- morpholine-3-carboxylic acid-tartaric acid salt, E150115.901 HBVR08301 (Error is + 1cm ^"1 )): Form A of ( _l S')-4-[(R)-6-(2-Chloro-4-fluoro-phenyl)-5-methoxycarbo nyl-2-thiazol-2-yl-3,6- dihydro-pyrimidin-4-ylmethyl]-morpholine-3-carboxylic acid-tartaric acid salt can be

characterized by Raman spectroscopy having characteristic bands approximately at wavenumbers positions (cm- 1): 53, 73, 146, 235, 304, 428, 688, 767, 837, 907, 1063, 1166, 1230, 1269, 1290, 1358, 1402, 1439, 1478, 1517, 1542, 1607, 1680, 2953 and 3112.

Figure 2 shows the FT-Raman spectra of a typical lot of (S)-4-[(R)-6-(2-Chloro-4-fluoro- phenyl)-5-methoxycarbonyl-2-thiazol-2-yl-3,6-dihydro-pyrimid in-4-ylmethyl]-morpholine-3- carboxylic acid-tartaric acid salt.