KR20070015642A - 3-β-D-RIVOFURANOSYLTHIAZOLO[4,5-d]PYRIDIMINE NUCLEOSIDES AND USES THEREOF - Google Patents

Abstract

The present invention relates to pharmaceutical compositions containing 3-β-D-ribofuranosylthiazolo [4,5-d] pyridinine nucleosides and such compounds with immunomodulatory activity. The invention also relates to the therapeutic or prophylactic use of such compounds and compositions and to methods of treating the diseases and disorders described herein by administering an effective amount of such compounds.

Immunomodulatory, 3-β-D-ribofuranosylthiazolo [4,5-d] pyridinine nucleosides, pharmaceutical compositions

Description

３-β-D-ribofuranosylthiazolo[4,5-p]pyridine nucleoside and its use {3-β-D-RIVOFURANOSYLTHIAZOLO[4,5-d]PYRIDIMINE NUCLEOSIDES AND USES THEREOF}

1 is a graph showing plasma levels of isatoribine and interferon alpha in mice.

The present invention relates to a pharmaceutical composition containing 3-β-D-ribofuranosylthiazolo[4,5-d]pyridine nucleoside and such compounds having immunomodulatory activity. In addition, the present invention relates to the therapeutic or prophylactic use of such compounds and compositions and methods of treating diseases and disorders described herein by administering an effective amount of such compounds.

Over the past decades, great effort has been devoted to studying the potential for therapeutic use of D- and L-purine nucleoside analogs. A number of nucleoside analogs are recently marketed as antiviral agents including HIV reverse transcriptase inhibitors (AZT, ddI, ddC, d4T and 3TC).

In addition, various D- and L-purine nucleoside analogs have been explored in the study of immunomodulators. For example, guanosine analogs having substituents at the 7- and/or 8-position have been shown to stimulate the immune system [Reitz et al, J. Med. Chem., 37, 3561-78 (1994); Michael et al., J. Med. Chem., 36, 3431-36 (1993)]. In other studies, U.S. Patent No. 5,821,236 (Krenitsky et al.) discloses 6-alkoxy derivatives of arabinofuranosyl purine derivatives useful in the treatment of tumors. In addition, U.S. Patent No. 5,539,098 (Krenitsky et al.) discloses the 5-amino-6-methoxy-9-(β-D-arabinofuranosyl)-9H-purine 5′-O-propionyl and 5 Inhibitors of shingles virus including'-O-butyryl ester have been reported. 7-deazaguanosine and analogs have been shown to exhibit antiviral activity against several RNA viruses in mice, although these compounds lack antiviral properties in cell culture. In addition, 3-deazaguanine nucleosides and nucleotides have demonstrated a fairly broad range of antiviral activity against certain DNA and RNA viruses [Revankar et al., J. Med. Chem., 27, 1489-96 (1984)]. Certain 7- and 9-deazaguanine C-nucleosides show protective ability against the lethal attack of the Semliki Forest virus (SFV) [Girgis et al., J. Med. Chem., 33, 2750-55 (1990)]. Selected 6-sulfenamide and 6-sulfinamide purine nucleosides are described in US Pat. No. 4,328,336 (Robins et al.) to have significant antitumor activity.

Certain pyrimido[4,5-d]pyridine nucleosides are described in US Pat. No. 5,041,542 (Robins et al.) as effective in the treatment of L1210 in BDF1 mice. These specific nucleosides have been suggested to serve as immunomodulators [Bonnet et al., J. Med. Chem., 36, 635-53 (1993)]. In addition, Wang et al. International Patent Publication No. WO 98/16184 discloses that purine L-nucleoside compounds and analogs thereof are used to treat infections, parasitics, neoplasms, autoimmune diseases, or to regulate one aspect of the immune system. Are reporting. In addition, 3-β-D-ribofuranosylthiazolo[4,5-d]pyridimine, which has demonstrated significant immune activity, including in vivo activity against murine splenocyte proliferation and SFV, is disclosed in U.S. Patent Nos. 5,041,426 and 4,880,784 (Robins et al.).

One of the possible purposes of immunomodulation involves stimulation or inhibition of Th1 and Th2 lymphokines. Type I (Th 1) cells produce interleukin 2 (IL-2), tumor necrosis factor (TNFα) and interferon gamma (IFNγ), which are primarily responsible for cell-mediated immunity such as delayed hypersensitivity and antiviral immunity. do. Type 2 (Th 2) cells produce interleukins, i.e. IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13, mainly as seen in response to allergens. It is involved in supporting the humoral immune response [see, eg, Mosmann, Annu. Rev. Immunol, 7, 145-73 (1989)]. D-guanosine analogs are described in vitro (Goodman, Int. J. Immunopharmacol, 10, 579-88 (1988); US Patent No. 4,746,651 to Goodman) and in vivo (Smee et al., Antiviral Res., 15, 229). (1991); Smee et al., Antimicrobial Agents and Chemotherapy, 33, 1487-92 (1989)) by inducing various effects on the lymphokines IL-1, IL-6, INFα and TNFα (directly). appear. However, the ability of D-guanosine analogs, such as 7-thio-8-oxoguanosine, to modulate direct type 1 or type 2 cytokines in T cells is not effective or has not been described.

In addition, it is known that oral administration of many purine nucleoside analogs has problems due to low absorption, low solubility, or degradation in the digestive tract under acidic or alkaline conditions or by the action of enzymes, and/or a combination of these phenomena. Therefore, there is a need for improved oral usability, tolerability, and administration of purine nucleoside analogs used to regulate one aspect of the immune system.

The present invention is useful as an immunomodulatory agent, 3-β-D-ribofuranosylthiazolo[4,5-d]pyridine nucleoside, a pharmaceutically acceptable prodrug thereof, pharmaceutically The above needs are addressed by the discovery of active metabolites, and pharmaceutically acceptable salts (such compounds, prodrugs, metabolites and salts are collectively referred to as "agonists").

In a general aspect, the invention relates to compounds of formula (I):

In the above formula,

R ¹ is independently H, -C(O)R ³ , or a racemic L- or D- amino acid group -C(O)CHNH ₂ R ⁴ (wherein R ³ is substituted or unsubstituted alkyl, and R ⁴ is H or substituted or unsubstituted alkyl);

R ² is H, OR ⁵ , or N(R ⁶ ) ₂ (wherein R ⁵ is independently H or alkyl, and R ⁶ is independently H, substituted or unsubstituted alkyl, cycloalkyl, or together with nitrogen To form a substituted or unsubstituted heterocycloalkyl ring);

When R ² is -OH, ^{at least one of the R 1} groups is a racemic L- or D- amino acid group -C(O)CHNH ₂ R ⁴ .

In a preferred embodiment, the invention is ^{that at least one of the R 1} groups is a racemic L- or D- amino acid group -C(O)CHNH ₂ R ⁴ (wherein R ⁴ is substituted or unsubstituted alkyl), The remaining R ¹ group is H, R ² is OR ⁵ or N(R ⁶ ) ₂ (wherein R ⁵ is independently selected from H or alkyl, and R ⁶ is H, independently substituted or unsubstituted alkyl, cyclo Alkyl, or together with nitrogen to form a substituted or unsubstituted heterocycloalkyl ring).

In another preferred embodiment, the invention is ^{that at least one of the R 1} groups is a racemic L-amino acid group -C(O)CHNH ₂ R ⁴ (wherein R ⁴ is substituted or unsubstituted alkyl), The remaining R ¹ group is H, R ² is OR ⁵ or N(R ⁶ ) ₂ (wherein R ⁵ is substituted alkyl and R ⁶ is independently H or substituted or unsubstituted alkyl), the formula ( It relates to the compound of I).

In another preferred embodiment, the present invention is ^{that at least one of the R 1} groups is a racemic L-amino acid group -C(O)CHNH ₂ R ⁴ (wherein R ⁴ is -CH(CH ₃ ) ₂ ), And the remaining R ¹ group is H and R ² is OH.

*In another aspect of the invention, the compounds of the invention are selected from:

In addition, the present invention relates to a compound of formula (I), a pharmaceutically acceptable prodrug of a prodrug or metabolite, a pharmaceutically active metabolite, and a pharmaceutically acceptable salt. In addition, an advantageous method of preparing the compound of formula (I) is described.

The compounds of formula (I) are useful as immune system enhancers and have specific immune system properties, including regulation, division, augmentation and/or enhancement of efficacy, or these compounds are intermediates to compounds having these properties. These compounds are predicted to affect at least natural killer cells, macrophage cells and lymphocyte cells of the host immune system. Because of these properties, the compounds are useful as antiviral and anti-tumor agents, or as intermediates for anti-viral and anti-tumor agents. These compounds can be used to treat diseased hosts by acting as an active ingredient in suitable pharmaceutical compositions.

In one aspect of the present invention, the compound of formula (I) is used to treat all kinds of viral diseases in mammals by administering a therapeutically effective amount of the compound to the mammal. Viral diseases considered to be treated with compounds of formula (I) include acute and chronic infections caused by viruses of both RNA and DNA. Without limiting the scope of viral infection in any way, the compounds of formula (I) include, in particular, adenovirus, cytomegalovirus, hepatitis A virus (HAV), hepatitis B virus (HBV), yellow fever virus. Flavivirus, and hepatitis C virus (HCV), herpes simplex type 1 and type 2, herpes zoster, human herpes virus 6, human immunodeficiency virus (HIV), human papilloma virus (HPV), type A Several causes of hemorrhagic fever, including influenza virus, influenza B virus, measles, parainfluenza virus, poliovirus, poxvirus (including smallpox and monkeypox virus), rhinovirus, respiratory syncytium virus (RSV), and arenavirus. Class of viruses (LCM, Junin virus, Machup virus, Guanarito virus and Lassa Fever), Bunyavirus (Hanta virus) and Rift Valley Fever), and pilovirus (Ebola and Marburg virus), West Nile virus, Lacrosse virus, California encephalitis virus, Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Western equine It is useful for the treatment of infections caused by tick-borne viruses such as encephalitis virus, Japanese encephalitis virus, Kysanur Forest virus, and Crimea-Congo hemorrhagic fever virus.

In another aspect, the compound of formula (I) is used to treat bacterial, fungal and protozoal infections in mammals by administering to the mammal a therapeutically effective amount of the compound. It is contemplated that all kinds of pathogenic microorganisms, including, but not limited to, organisms that are resistant to antistatic agents, are treatable by the compounds of the present invention. The ability of compounds of formula (I) to activate many components of the immune system does not go through the mechanisms of resistance commonly found to reduce susceptibility to antibiotics, and thus, by compounds of formula (I) There is a particular utility of the present invention for treating infections in mammals.

In another aspect of the invention, the compound of formula (I) is used to treat a tumor in a mammal by administering to the mammal a therapeutically effective amount of the compound. Tumors or cancers that are considered to be treated include those caused by viruses, whose effects are to inhibit the transformation of viral-infected cells into a neoplastic state, and to inhibit the spread of the virus from the transformed cells to other normal cells. And/or arresting the growth of viral transformed cells. Compounds of formula (I) are predicted to be useful against a wide variety of tumors, including, but not limited to, carcinoma, sarcoma and leukemia. These classes include breast cancer, colon cancer, bladder cancer, lung cancer, prostate cancer, gastric and pancreatic cancer, and lymphoid and myeloid leukemia.

In another aspect of the invention, a method of treating a mammal comprises administering a therapeutically and/or prophylactically effective amount of a medicament containing a compound of the invention. In this aspect, effects include interleukins, e.g., IL-1 to IL-12, and other cytokines such as TNF alpha, and interferons including interferon alpha, interferon theta and interferon gamma, and sub-effectors thereof. But not limited to the regulation of some of the mammalian immune systems, in particular the regulation of the cytokine activity of Th1 and Th2. When the modulation of Th1 and Th2 cytokines occurs, such regulation is the enhancement of both Th1 and Th2, inhibition of both Th1 and Th2, enhancement of either Th1 or Th2, and inhibition of the other, or Th1/Th2 levels. Either effect (such as generalized inhibition) on to occur at high concentrations, while other effects (such as enhancement of either Th1 or Th2 and the other suppression) may include bimodal regulation occurring at low concentrations.

In another aspect of the present invention, the pharmaceutical composition containing the compound of formula (I) is administered in a therapeutically effective dose to a mammal to which an anti-infective agent not included in the compound of formula (I) is administered. In a preferred aspect of the present invention, the pharmaceutical composition containing the compound of formula (I) is administered in a therapeutically effective dose together with an anti-infective agent that acts directly on an infectious agent that inhibits the growth of or kills the infectious agent.

In a preferred aspect of the present invention, a pharmaceutical composition comprising a therapeutically effective amount of a compound according to formula (I) provides improved oral usability and administration as an immunomodulatory agent. In another preferred aspect, a pharmaceutical composition comprising a therapeutically effective amount of a compound according to formula (I), the active structure is masked by the agent passing through the lymphatic tissue in the stomach to minimize activation of the lymphatic tissue, and improved oral tolerance Gives a surname.

When the following terms are used herein, these terms are used as defined below:

The term “comprising” is used in an open, non-limiting sense.

The term “nucleotide” consists of any pentose or modified pentose attached to a specific position in the heterocycle, or at the same position in the original position or analog of a purine (9-position) or pyrimidine (1-position). Represents a compound.

The term “purine” refers to a nitrogen containing bicyclic heterocycle.

The term “pyrimidine” refers to a nitrogen containing monocyclic heterocycle.

The term “D-nucleoside” refers to a nucleoside compound (eg, adenosine) having a D-ribose sugar moiety.

The term “L-nucleoside” refers to a nucleoside compound having an L-ribose sugar moiety.

The term "alkyl" as used herein denotes a straight or branched chain alkyl group having 1 to 12 carbon atoms. Examples of alkyl groups include methyl (Me, which may be structurally represented by "/"), ethyl (Et), n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl (tBu) , Pentyl, isopentyl, tertiary-pentyl, hexyl, isohexyl, and the like.

The term "alkoxy" denotes -O-alkyl. Illustrative examples include methoxy, ethoxy, propoxy, and the like.

The term "halogen" denotes chlorine, fluorine, bromine or iodine. The term “halo” denotes chloro, fluoro, bromo or iodo.

The term “cycloalkyl” denotes a saturated or partially saturated monocyclic or fused polycyclic or spiro polycyclic carbocycle having 3 to 12 ring atoms per ring. Illustrative examples of cycloalkyl groups include the following moieties:

“Heterocycloalkyl” is a saturated or partially saturated monocyclic, or fused polycyclic or spiro polycyclic ring structure having 3 to 12 ring atoms selected from C atoms, N, O and S heteroatoms per ring. Represents. Illustrative examples of heterocycloalkyl groups include:

The term “aryl” (Ar) denotes a monocyclic, or fused polycyclic or spiro polycyclic aromatic carbocyclic (ring structure in which all ring atoms are carbon) having 3 to 12 ring atoms per ring. Illustrative examples of aryl groups include the following moieties:

The term “substituted” means that a particular group or moiety carries one or more substituents. The term “unsubstituted” means that a particular group does not carry a substituent.

Substituted alkyl, cycloalkyl or heterocycloalkyl is halogen (F, Cl, Br or I), lower alkyl (C _{1 -6} ), -OH, -NO ₂ , -CN, -CO ₂ H, -O-lower Alkyl, -aryl, -aryl-lower alkyl, -CO ₂ CH ₃ , -CONH ₂ , -OCH ₂ CONH ₂ , -NH ₂ , -SO ₂ NH ₂ , haloalkyl (e.g. -CF ₃ ,- CH ₂ CF ₃ ), -O-haloalkyl (eg, -OCF ₃ , -OCHF ₂ ) and the like.

The term “immunomodulator” refers to a natural or synthetic product capable of modifying a normal or abnormal immune system through enhancement or inhibition.

The term “preventing” is the ability of a compound or composition of the present invention to refer to the ability to prevent an identified disease in a patient diagnosed with or at risk of developing the disease. The term also includes preventing further progression of the disease in a patient who already has or has symptoms of such disease.

The term "treating" means

(i) preventing a disease, disease, and/or condition from occurring in an animal that may have a disease, condition and/or condition, but has not yet been diagnosed;

(ii) inhibiting a disease, disease or condition, ie, stopping its progression; And

(iii) to alleviate a disease, disease or condition, ie to cause regression of the disease, disease and/or condition.

The terms “α” and “β” refer to the specific stereochemical form of a substituent on an asymmetric carbon in the derived chemical structure. All of the compounds described herein exist in the form of D-furanosyl.

The compounds of the present invention may exhibit a tautomerization phenomenon. While formula (I) cannot clearly represent all possible tautomeric forms, formula (I) is intended to represent any tautomeric form of the represented compound, and only in the specific compound form represented by the formula shown. It should be understood as not limiting. For example, for formula (I), whether the substituents are shown as enol or keto, it should be understood that they represent the same compound (as shown by way of example below).

Some of the compounds of the present invention may exist as single stereoisomers (ie, virtually no other stereoisomers), racemates, and/or mixtures of enantiomers and/or diastereomers. Such single stereoisomers, racemates and mixtures thereof are intended to be within the scope of the present invention. Preferably, the optically active compounds of the invention are used in optically pure form.

As generally understood by those of skill in the art, an optically pure compound having one chiral center (i.e., one asymmetric carbon) is a compound consisting of substantially one of two possible enantiomers (i.e., enantiotropically pure). , Optically pure compounds with more than one chiral center are simultaneously diastereomerically pure and enantiomerically pure compounds. Preferably, the compounds of the invention are in an optically pure form of at least 90%, ie at least 90% of a single isomer (80% enantiomeric selectivity ("ee") or diastereomeric selectivity ("de")), More preferably 95% or more (90% ee or de), more preferably about 97.5% or more (95% ee or de), very preferably 99% or more (98% ee or de). Is used.

Additionally, formula (I) is intended to include solvated as well as unsolvated forms of the identified structures. For example, formula (I) includes both hydrated and unhydrated forms of the compounds of the structure shown. Other examples of solvates include structures in combination with isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, or ethanolamine.

In addition to the compounds of formula (I), the present invention includes pharmaceutically acceptable prodrugs, pharmaceutically active metabolites, and pharmaceutically acceptable salts of such compounds and metabolites.

A “pharmaceutically acceptable prodrug” is a compound that can be converted to a specified compound, or to a pharmaceutically acceptable salt of such a compound, before exhibiting a pharmacological effect under physiological conditions or by solvent decomposition. In general, prodrugs have improved chemical stability, improved patient tolerance and suitability, improved bioavailability, extended duration of action, improved organ selectivity, improved formulation (e.g., increased water solubility), and /Or formulated with substances intended for reduced side effects (eg toxicity). As described in the following documents, the prodrugs can be readily prepared from compounds of formula (I) using known methods in the art [see: Burger's Medicinal Chemistry and Drug Chemistry, 1, 172-178, 949- 982 (1995); Bertolini et al., J. Med. Chem,, 40, 2011-2016 (1997); Shan, et al., J. Pharm. Sci., 86 (7), 765-767; Bagshawe, Drug Dev, Res., 34, 220-230 (1995); Bodor, Advances in Drug Res., 13, 224-331 (1984); Bundgaard, Design of Prodrugs (Elsevier Press 1985); Larsen, Design and Application of Prodrugs, Drug Design and Development (Krogsgaard-Larsen et al., eds., Harwood Academic Publishers, 1991); Dear et al., J. Chromatogr. B, 748, 281-293 (2000); Spraul et al., J. Pharmaceutical & Biomedical Analysis, 10, 601-605 (1992); and Prox et al., Xenobiol., 3, 103-112 (1902)].

“Pharmaceutical active metabolite” is intended to mean a pharmacologically active product of a specified compound or salt thereof produced through metabolism in the body. After introduction into the body, most drugs are chemically capable of altering their physical properties and biological effects. These metabolic shifts, which generally affect the polarity of compounds of formula (I), alter the way drugs are distributed and secreted from the body, but in some cases, drug metabolism is therapeutic. Required for effectiveness, for example, anticancer agents of the antimetabolite class must be converted to their active form after delivery to cancer cells.

Since most drugs undergo metabolic transformation to some extent, the biochemical reactions that act on drug metabolism are numerous and varied. The main site of drug metabolism is the liver, but other organs may also be involved.

A characteristic feature of many of these modifications is that although sometimes polar drugs produce less polar products, the metabolites or "metabolites" are more polar than the parent drug. Substances with a high lipid/water partition coefficient, which pass easily through the membrane, also readily back-diffusion from tubular urine through renal tubular cells into plasma. Thus, these substances tend to have low renal clearance and long-term persistence in the body. If drugs are metabolized to more polar compounds, which are compounds with a lower partition coefficient, their tubular reuptake will be greatly reduced. Moreover, in the proximal renal tubule, and in the parenchymal liver cells, specific secretion mechanisms for anions and cations act on highly polar substances.

As a specific example, phenacetin (acetofenetidine) and acetanilide are both mild analgesics and antipyretics, but are now widely used in the body as p-hydroxyacetanilide (acetanilide), a more polar and more effective metabolite. Transformed. When a certain amount of acetanilide is administered to a person, subsequent metabolites peak and decay in the plasma sequentially. During the first hour, acetanilide becomes the main plasma component. In the second hour, the metabolite acetaminophen concentration peaks as the levels of acetanilide decrease. Finally, after a few hours, the main plasma component is inactive and is an additional metabolite that can be secreted from the body. Thus, the plasma concentration of the drug itself, as well as one or more metabolites, can be pharmacologically important.

"Pharmaceutically acceptable salt" is intended to mean a salt that has the biological efficacy of the free acids and bases of the specified compound, and is biologically or otherwise preferred. The compounds of the present invention may be sufficiently acidic or sufficiently basic, or may have both functional groups, and thus may react with a number of inorganic or organic bases, and inorganic and organic acids to form pharmaceutically acceptable salts. Illustrative pharmaceutically acceptable salts include salts prepared by reaction of a compound of the present invention with an inorganic acid or an organic acid, or an inorganic base. For example, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decano Eate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butin-1 ,4-dioate, hexine-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, Phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, γ-hydroxybutyrate, glycolate, tartrate, methane-sulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfo Salts including nates and mandelates.

When the compound of the present invention is a base, the preferred pharmaceutically acceptable salt is any suitable method available therein, e.g., the free base is converted to an inorganic acid (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.), Or organic acids (e.g., acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid), pyranosidylic acid (e.g., glucuronic acid, or galacturonic acid), alpha-hydroxy Manufactured by treatment with acids (e.g., citric acid or tartaric acid), amino acids (e.g., aspartic acid or glutamic acid), aromatic acids (e.g., benzoic acid or cinnamic acid), sulfonic acid (e.g., p-toluenesulfonic acid or ethanesulfonic acid), etc. Can be.

When the compound of the present invention is an acid, the preferred pharmaceutically acceptable salts are prepared by any suitable method, e.g., free acids, inorganic or organic bases such as amines (primary, secondary or tertiary amines), alkalis. It can be produced by treatment with a metal hydroxide or alkaline earth metal hydroxide. Illustrative examples of suitable salts include organic salts derived from amino acids (e.g. glycine and arginine), ammonia, primary, secondary and tertiary amines, and cyclic amines (e.g. piperidine, morpholine and piperazine), And inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

When the agent is a solid, those skilled in the art should understand that the compounds and salts of the present invention may exist in different crystalline or polymorphic forms, all of which are included within the scope of the present invention and within the formulas specified.

A further aspect of the present invention comprises a pharmaceutically acceptable carrier or diluent and a therapeutically effective amount of a compound of formula (I), a pharmaceutically acceptable salt, hydrate, ester, solvate, prodrug, metabolite, or stereoisomer. It relates to a pharmaceutical composition comprising.

The compounds of formula (I) are useful for the preparation of pharmaceutical formulations which are included in therapeutically effective amounts as mixtures or with excipients or carriers suitable for enteral or parenteral application. As such, the formulation of the present invention suitable for oral administration is in the form of individual units, such as capsules, sachets, tablets, troches or lozenges, each containing a predetermined amount of the active ingredient, in the form of a powder or granules, or In the form of a suspension or solution in an aqueous liquid or non-aqueous liquid; Or in the form of an oil-in-water emulsion or a water-in-oil emulsion. The active ingredient may also be present in the form of bolus, soft or paste.

The compositions will generally be formulated in unit dosage form, such as tablets, capsules, aqueous suspensions or solutions. Such formulations generally include solid, semi-solid or liquid carriers. Exemplary carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, mineral oil, cocoa butter, theobroma oil, alginate, tragacanth, gelatin, syrup, methyl cellulose, poly Oxyethylene sorbitan monolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and the like.

Particularly preferred formulations include (a) diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, dried corn starch and glycine; And/or (b) a lubricant such as silica, talc, stearic acid, magnesium or calcium salts thereof, and polyethylene glycol, as well as tablets and gelatin capsules comprising the active ingredient.

Further, the tablet may include a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidone; Carriers such as lactose and corn starch; Disintegrants such as starch, agar, alginic acid or its sodium salt, and effervescent mixtures; And/or absorbents, colorants, flavors and sweeteners. The compositions of the present invention may be sterilized and/or contain adjuvants such as preservatives, stabilizers, swelling or emulsifying agents, solution promoters, salts for regulating osmotic pressure, and/or buffers. Additionally, the compositions of the present invention may also contain therapeutically valuable substances. Aqueous suspensions may contain emulsifying and suspending agents in combination with the active ingredient. All oral dosage forms may additionally contain sweetening and/or flavoring and/or coloring agents.

Each of these compositions is prepared by conventional mixing, granulating or coating methods and contains about 0.1 to 75% active ingredient, preferably about 1 to 50% active ingredient. Tableting may be made by compressing or molding the active ingredient, optionally with one or more accessory ingredients. Compressed tablets can be made by compressing the active ingredient into a free flowing form such as a powder or granules, which is mixed with any binder, lubricant, inert diluent, surfactant or dispersant in a suitable machine. Molded tablets can be made by molding in a suitable machine a mixture of the powdered active ingredient and a suitable carrier moistened with an inert liquid diluent.

When administered parenterally, the compositions may be presented in unit dose sterile injectable form (isotropic aqueous solution, suspension or emulsion) with a pharmaceutically acceptable carrier. Such carriers are preferably non-toxic, parenterally acceptable, and are non-therapeutic diluents or solvents. Examples of such a carrier include water; Aqueous solutions such as brine (isotonic sodium chloride solution), Ringer's solution, dextrose solution, and Hanks' solution; Water-insoluble carriers such as 1,3-butanediol, fixed oils (eg, corn oil, cottonseed oil, peanut oil, sesame oil, and synthetic mono- or diglycerides), ethyl oleate, and isopropyl myristate.

Oily suspensions can be formulated according to the art known in the art using suitable dispersing or wetting agents and suspending agents. Among these acceptable solvents or suspending media are sterilized fixed oils. For this purpose, any bland fixed oil can be used. Fatty acids such as oleic acid and glyceride derivatives thereof, especially polyoxyethylated forms thereof, including olive oil and perm oil, are also useful in the preparation of injectable solutions. Such oil solutions or suspensions may also contain long chain alcohol diluents or dispersants.

Sterilized saline is the preferred carrier, and such compounds are often sufficiently water soluble to be possible as a solution for all foreseeable needs. The carrier may contain minor amounts of additives, such as substances that enhance solubility, isotonicity and chemical stability (eg, antioxidants, buffers and preservatives).

When administered rectally, the compositions will generally be formulated in unit dosage form such as suppositories or sachets. Such compositions can be prepared by mixing the compound with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature so that it dissolves in the rectum to release the compound. Typical excipients include cocoa butter, beeswax and polyethylene glycol or other fatty emulsions or suspensions.

Formulations suitable for nasal or oral administration (self-propelled powder dispersion formulations) may comprise from about 0.1 to about 5% w/w of active ingredient or, for example, about 1% w/w of active ingredient. Additionally, some formulations can be formulated as sublingual troches or lozenges.

In addition, the compounds of the present invention may be administered topically, particularly if the condition being treated for treatment relates to an easily accessible area or organ by topical application, including the eye, skin or lower intestine.

For topical application in ophthalmic or ophthalmic use, the present compounds are isotonic, as micronized suspensions in pH-adjusted sterile saline, or, preferably, with or without preservatives such as benzylalkonium chloride. It can be formulated as a solution in pH-adjusted sterile saline. Alternatively, the compound can be formulated as an ointment such as petrolatum.

For topical application to the skin, the compounds may be used in combination with one or more components of, for example, mineral oil, liquid petrolatum, white petrolatum, flophyllene glycol, polyoxyethylene compound, polyoxypropylene compound, emulsifying wax and water. It may be formulated as a suitable ointment that is suspended or dissolved in a mixture and contained. Alternatively, the present compounds may contain one or more components of, for example, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. It may be formulated as a suitable lotion or cream that is contained by being suspended or dissolved in a mixture of with.

Topical application to the lower gastrointestinal tract can be implemented as a rectal suppository formulation (see above) or a suitable enema formulation.

The formulation may be provided in a unit dosage form, and may be prepared by any method well known in the pharmacy field. All methods include the step of bringing into association the active ingredient with a carrier that constitutes one or more accessory ingredients. In general, the formulation is formulated by uniformly and intimately binding the active ingredient with a liquid carrier or a particulate solid carrier or both, and then, if necessary, shaping the product into the desired formulation.

The pharmaceutical composition of the present invention is used in a therapeutically effective amount, which amount may depend on the desired release profile, the concentration of the pharmaceutical composition required for the sensitizing effect, and the time at which the pharmaceutical composition must be released for treatment.

The compounds of formula (I) of the present invention are preferably administered as capsules or tablets containing single or divided doses of the compound, or as sterile solutions, suspensions, or emulsions for parenteral administration in single or divided doses. Is administered.

The compounds of the present invention are used in pharmaceutical compositions in a therapeutically effective amount. The therapeutically effective amount of the compound of formula (I) will depend on the particular compound used, and a therapeutically effective amount of such compound ranging from about 1% to about 65% is readily incorporated into liquid or solid carrier delivery systems.

For medicinal use, the amount required for the compound of formula (I) to achieve a therapeutic effect will depend on the particular compound being administered, the route of administration, the condition of treatment in the mammal, and the particular disease in the disease of interest. Suitable systemic doses of compounds of formula (I) for mammals suffering from or likely to develop any condition as described herein generally range from about 0.1 to about 100 mg per kg body weight on a basis. The practitioner or veterinarian will be able to readily determine and prescribe the amount of the compound effective for the desired prophylactic or therapeutic treatment.

In proceeding in this way, the specialist or veterinarian may use an intravenous pill as desired, and then repeat the administration using an intravenous injection solution. In the method of the present invention, the compound may be used, for example, orally, parenterally, by inhalation spray, topically, into the rectum, into the nasal cavity, into the oral cavity, sublingually, into the vagina, into the ventricle, or by implantation. It can be administered as a dosage form containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles via a designated reservoir.

Parenteral administration includes, but is limited to, infusion techniques such as, for example, intravenous, subcutaneous, intramuscular, intrathecal, intraosseous, intraperitoneal, intrathecal, intraventricular, intrasternal, intracranial injection and subdural pumps. It does not become. It is preferred to be administered directly to invasive techniques, especially to damaged neuronal tissue. Although it is possible for the compound of formula (I) to be administered alone, it is preferred that it is provided as part of a pharmaceutical formulation.

In order to be therapeutically effective as a target of the central nervous system, the compound used in the method of the present invention must easily penetrate the blood-brain barrier when administered peripherally. However, compounds that cannot penetrate the blood-brain barrier can be effectively administered by the intraventricular route.

The compounds used in the methods of the present invention may be administered by a single dose, multiple individual doses or by continuous infusion. This compound is very suitable for continuous injection because it is small, readily diffuses and is relatively stable. Pump means, in particular subcutaneous or subdural pump means, are preferred for continuous infusion.

In the method of the present invention, any effective dosing regimen can be used that controls the timing and sequence of doses. The dosage of the present compound preferably comprises a pharmaceutical dosage unit comprising an efficacious amount of the active compound. By efficacious amount is meant an amount sufficient to provide an immune enhancing response and/or induce a desired effect by administering one or more pharmaceutical dosage units.

An exemplary daily dosage unit for a vertebrate host consists of an amount of about 0.001 mg/kg to about 50 mg/kg. In general, dosage levels on the order of about 0.1 mg to about 10,000 mg of the active ingredient compound are useful for the treatment of this condition, with a preferred level being about 0.5 mg to about 2,000 mg. Specific dosage levels for any particular patient may include the activity of the particular compound used, the age, weight, general health status, sex and diet of the patient; Time of administration; The rate of secretion of the compound in combination with other drugs; The severity of the particular disease being treated; And various factors including the dosage form and route. In general, the in vitro dose effect results provide a useful guide for suitable dosages for patient administration. Animal model studies can also be helpful. Considerations for determining suitable dosage levels are well known in the art.

The compounds and compositions of the present invention may be administered simultaneously with one or more therapeutic agents, either (i) together in a single dosage form, or (ii) separately in individual dosage forms designed for optimal release rates of each active agent. Each formulation contains from about 0.01% to about 99.99% by weight, preferably from about 3.5% to about 60% by weight of a compound of the present invention, as well as one or more pharmaceutical excipients such as wetting agents, emulsifying agents and pH buffering agents. It may contain. When a compound used in the methods of the present invention is administered in combination with one or more other therapeutic agents, the specific dosage level for such therapeutic agents will generally depend on considerations as identified above for the compositions and methods of the invention.

In the method of the present invention, an administration regimen that controls the timing and sequence of administration of the compound of the present invention may be used, and the treatment may be repeated as necessary. Such regimens may include pretreatment and/or co-administration with additional therapeutic agents.

The agents of the present invention can be prepared using reaction pathways and synthetic schemes as described below using general techniques known in the art using readily available starting materials. The synthesis of the non-exemplary compounds according to the present invention can be accomplished by modifications that are apparent to those skilled in the art, for example, by appropriately protecting an interfering group, by switching to other suitable reagents known in the art, or by customarily modifying the reaction conditions. It can be done successfully. Alternatively, it will be appreciated that other reactions described herein or generally known in the art may be applied to provide other compounds of the present invention.

Preparation of the compound

In the reaction scheme described below, unless otherwise specified, all temperatures are stated in degrees Celsius, and all parts and percentages are by weight. Reagents were purchased from commercial vendors such as Aldrich Chemical Company or Lancaster Synthesis Ltd., and were used without further purification unless otherwise specified. Tetrahydrofuran (THF) and N,N-dimethylformamide (DMF) were purchased from Aldrich as Sure Seal bottles, and were used as purchased. Unless otherwise specified, the following solvents and reagents were distilled under a blanket of dry nitrogen. THF and Et ₂ O were distilled from Na-benzophenone ketyl, CH ₂ Cl ₂ , diisopropylamine, pyridine and Et ₃ N were _{distilled from CaH 2} , MeCN was first distilled from _{P 2} O _{5, and then CaH 2} , MeOH was distilled from Mg, PhMe and EtOAc and i-PrOAc were _{distilled from CaH 2} and TFAA was purified via simple atmospheric distillation under anhydrous argon.

The reactions described below are generally carried out (unless otherwise specified) under positive pressure of argon at atmospheric temperature in anhydrous solvent, and the reaction flask is equipped with a rubber septum for introducing substances and reagents through a syringe. The glassware was oven dried and/or heat dried. The reaction was assayed by TLC and terminated judging by consumption of starting material. Analytical thin-film chromatography (TLC) was performed on an aluminum-supported silica gel 60 F ₂₅₄ 0.2 mm plate (EM Scienc), visualized with UV light (254 nm), and heated with conventional ethanol-based phosphomolybdic acid. Preparative thin layer chromatography (TLC) was performed on an aluminum supported silica gel 60 F ₂₅₄ 1.0 mm plate (EM Scienc) and visualized with UV light (254 nm).

Post-treatment was generally carried out by doubling the reaction volume with a reaction solvent and an extraction solvent, and washing with a specified aqueous solution using 25% by volume of the extraction volume unless otherwise specified. After drying the resulting solution over anhydrous Na ₂ SO ₄ and/or Mg ₂ SO ₄ , the solvent was filtered and evaporated under reduced pressure in a rotary evaporator, and the solvent was removed under vacuum. Column chromatography was completed under positive pressure using 230-400 mesh silica gel or 50-200 mesh natural alumina. The hydrocracking reaction was carried out at the pressure indicated in the examples or at atmospheric pressure.

¹ H-NMR spectra were recorded on a Varian Mercury-VX400 instrument operating at 400 MHz, and ¹³ C-NMR spectra were recorded on an instrument operating at 75 MHz. NMR spectra were obtained using chloroform (7.27 ppm and 77.00 ppm) as a comparative standard, using a _{solution of CDCl 3} (described in ppm), CD ₃ OD (3.4 and 4.8 ppm and 49.3 ppm), DMSO-D ₆ , or, as the case may be, internally. Obtained as tetramethylsilane (0.00ppm). Other NMR solvents were used as needed. When multiple peaks are described, the following abbreviations are used: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broadened), dd (doublet of doublets). , dt (doublet of tripelts). When presented, the coupling constant is written in Hertz (Hz).

Infrared (IR) spectra were recorded on an FT-IR spectrometer as neat oil, as KBr pellets, or as CDCl ₃ solution and expressed as wave number (cm ⁻¹ ). The mass spectra described are (+)-ES LC/MS performed by the Analytical Laboratory of Anadys Pharmaceuticals, Inc. Elemental analysis was performed by Atlantic Microlab, Inc. of Norcross, Georgia. The melting point (mp) was measured on an open capillary device and not calibrated.

A number of compound abbreviations are used in the described synthetic routes and experimental procedures, such as: THF (tetrahydrofuran), DMF (N,N-dimethylformamide), EtOAc (ethyl acetate), DMSO (di-methyl sulfoxide). Side), DMAP(4-dimethylaminopyridine), DBU(1,8-diazacyclo[5.4.0]undec-7-ene), DCM(4-dicyanomethylene)-2-methyl-6-( 4-Dimethylamino-styryl)-4H-pyran), MCPBA (3-chloroperoxybenzoic acid), EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride), HATU (O- 7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), HOBT (1-hydroxybenzotriazole hydrate), TFAA (trifluoroacetic anhydride) ), pyBOP (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate), DIEA (diisopropylethylamine), and the like.

Scheme 1 shows the general procedure for preparing the 5'-amino acid ester of 5-amino-3-β-D-ribofuranosylthiazolo[4,5-d]pyrimidine-2,7-dione:

Scheme 1

a) 2,2-dimethoxypropane, acetone, DMSO, MeSO ₃ H, O℃

b) BOC-NHCHR ⁴ CO ₂ H, EDC, DMAP, PhMe, 0℃-room temperature

c) anhydrous HCl, iPrOAc, iPrOH

In a typical synthetic route, the 2',3'-hyd of the β-D-ribose moiety of 5-amino-3-β-D-ribofuranosylthiazolo[4,5-d]pyrimidine-2,7-dione The hydroxyl group is first protected with an acetonide, preferably shown by formula (2). The free 5'-hydroxyl is then treated by various methods of esterification with N-protected amino acids to form formula (IIa). Thereafter, the nitrogen of the amino acid ester and the 2',3'-hydroxyl of the ribose unit are treated under various deprotection conditions, and preferably, salt formation of the free amine of the amino acid ester as shown by formula (II) is carried out at the same time. do.

Example 1: 5-Amino-3-(5'-OL - valinyl-β-D- ribofuranosyl )thiazolo[4,5-d] pyrimidine-2,7-dione dihydrochloride (3 )

Step 1: Preparation of 5-amino-3-(2',3'-O- isopropylidene -β-D- ribofuranosyl )thiazolo [4,5-d]pyrimidine-2,7-dione

At room temperature, compound 1 (5.37 g, 17.0 mmol) in acetone (40 mL) contained in a 250 mL Morton flask, according to the procedure described in U.S. Patent No. 5,041,426 (Example 2), which is incorporated herein by reference. Prepared), 2,2-DMP (6.26 mL, 50.9 mmol), DMSO (6.6 mL), and _{MeSO 3} H (220 µl, 3.39 mmol) were successively added at room temperature. When the reaction mixture was stirred vigorously, it became homogeneous and turned golden as the diol was consumed. TLC analysis (SiO ₂ , 10% MeOH-CHCl ₃ ) showed that the reaction was complete after 6 hours. The undissolved solid was removed through gravity filtration using fluted Whatman type 1 filter paper. When the filtrate was poured into 10 parts (~400 ml) of ice water, a precipitate of a white solid was immediately formed. After stirring for a short time, NaHCO ₃ (285 mg, 3.39 mmol) dissolved in water (10 mL) was added to neutralize _{MeSO 3 H.} Stirring vigorously continued for 15 minutes in the Morton reactor and filtered through a sintered glass funnel through the mixture. The solid was washed with ice water (100 ml), air-dried, and further dried under vacuum at 65° C. to give 5.36 g (88%) of acetonide (2) as a white solid: mp 280-81° C.; ¹ H (DMSO-d ₆ ) δ 1.28 (s, 3H), 1.47 (s, 3H), 3.43-3.55 (m, 2H), 3.95-3.99 (m, 1H), 4.77-4.80 (m, 1H) , 4.88-4.91 (m, 1H), 5.24-5.26 (m, 1H), 5.99 (s, 1H), 6.97 (br s, 2H), 11.25 (s, 1H).

Step 2: 5-Amino-3-(2',3'-O -isopropylidene -5'-N-tertiary -butoxycarbonyl -L - valinyl)-β -D-ribofuranosyl)thia Preparation of Zolo[4,5-d]pyrimidine-2,7-dione (4)

EDC (588 mg, 3.07 mmol) was added to a solution of N-butoxycarbonyl-(L)-valine ((671 mg, 2.81 mmol) in THF (9 mL) at 0° C. The formed homogeneous mixture was 45 at 0° C. After stirring for a minute, the solution became homogeneous at this time, and solid acetonide (2) (1.00 g, 2.81 mmol) from step 1 was added at once, and then solid DMAP (522 mg, 4.27 mmol) was added. Was brought to room temperature, stirred for an additional 5 h and concentrated via rotary evaporation to a yellow syrup at 25° C. The residue was dissolved in EtAOc (50 mL), partitioned with 1N HCl (10 mL), saturated NaHCO ₃ The acid was neutralized with (10 mL) aqueous solution The acidic aqueous phase was further extracted with EtOAc (2 x 50 mL) and then partitioned into the basic aqueous phase The combined organic phases were dried over _{Na 2} SO _{4 and SiO 2} short pad Filtered through and concentrated to give Boc-protected amino acid ester (4) (1.480 g, 96%) as a foam: mp 158° C. (decomposition); ¹ H (CDCl ₃ ) δ 0.86 (d, J= 7.0, 3H), 0.95 (d, J= 7.0, 3H), 1.35 (s, 3H), 1.44 (s, 9H), 1.56 (s, 3H), 1.75 (br s, 1H), 2.08-2.19 (m , 1H), 4.20-4.24 (m, 2H), 4.30-4.37 (m, 1H), 4.56 (dd, J=11.0, 5.9, 1H), 4.96 (dd, J= 6.2, 3.7, 1H), 5.11 ( br d, J=8.8, 1H), 5.29 (br d, J= 6.6, 1H), 5.88 (br s, 2H), 6.23 (s, 1H).

Step 3: 5-amino -3- (5'-OL- Bali carbonyl) - β-D- ribo furanyl nosil) thiazolo [4,5-d] blood limiter Dean-2,7-dione dihydrochloride (3 ) Of the manufacture

_{A stream of HCl gas was passed through a bubbler of concentrated H 2} SO ₄ until a saturated solution was obtained, and then in a 250 mL eggplant three-necked Morton flask containing anhydrous isopropyl acetate (80 mL) at 0°C ( Via a frited dispersion tube). To this was added a solution of the Boc-amino acid ester (5.53 g, 9.95 mmol) from step 2 above in isopropyl acetate (30 ml) and a white solid precipitate formed within 5 minutes. To this, 10% (v/v) IPA (11 mL) was added. After warming the reaction mixture to room temperature, it was stirred for 12 hours. The heterogeneous reaction mixture was diluted with anhydrous toluene (100 mL). Filtration using a medium pore sintered glass funnel under N _{2 gave a gray amorphous solid.} The solid was triturated in anhydrous THF, filtered, and dried in vacuo at 65° C. to give the title compound (3) (3.677 g, 81%) as a white solid: mp. 166-68° C. (decomposition); ¹ H (DMSO-d ₆ ) δ 0.90 (d, J = 7.0, 3H), 0.94 (d, J = 7.0, 3H), 2.14-2.18 (m, 1H), 3.83-3.85 (m, 1H), 3.96-4.00 (m, 1H), 4.23-4.28 (m, 2H), 4.42 (dd, J= 11.7, 3.4, 1H), 4.75 (dd, J= 10.3, 5.5, 1H), 5.81 (d, J= 4.4, 1H), 6.46 (br s, 3H), 7.23 (br s, 2H), 8.47 (s, 3H), 11.5 (br s, 1H).

Elemental analysis for C ₁₅ H ₂₁ N ₅ 0 ₇ S. 2HCl: calc: C, 36.89; H, 4.75; Cl, 14.52; N, 14.34; S, 6.57; Found: C, 37.03: H, 4.74; Cl, 14.26; N, 14.24; S, 6.42.

Example 2: 5-amino-3-(5'-OL -isoleucyl- β-D- ribofuranosyl )thiazolo[4,5-d] pyrimidine-2,7-dione 3/2 hydrochloride ( 5)

Step 1: 5-Amino-3-(2',3'-O -isopropylidene -5'-N-tertiary butoxycarbonyl -L - isoleucyl)-β-D-ribofuranosyl)thia Preparation of Zolo[4,5-d]pyrimidine-2,7-dione (6)

In a manner similar to step 2 of Example 1, 5-amino-3-(2',3'-O-isopropylidene-β-D-ribofuranosyl)thiazolo[4,5-d]pyrimidine- 5-amino-3-(2',3'-O-isopropylidene-5'-N-tertiary from 2,7-dione (2) and N-tert-butoxy-L-isoleucine (7) Butoxycarbonyl-L-isoleucyl)-β-D-ribofuranosyl)thiazolo[4,5-d]pyrimidine-2,7-dione (6) was prepared as a gray foam in 93% yield. : ¹ H NMR (400 MHz, d ₆ -DMSO) δ11.29 (s, 1H), 7.09 (d, J= 8.0, 1H), 7.02 (br s, 1H), 6.02 (s, 1H), 5.28 ( d, J = 6.2, 1H), 5.06 (br s, 1H), 4.16-4.22 (m, 2H), 3.85 (dd, J= 8.0, 6.6, 1H), 1.68 (br s, 1H), 1.47 (s , 3H), 1.34 (s, 9H), 1.29 (s, 3H), 0.71-0.89 (m, 5H).

Step 2: 5-Amino-3-(5'-OL -isoleucyl ) -β-D- ribofuranosyl )thiazolo[4,5-d] pyrimidine-2,7-dione dihydrochloride (5) Manufacture of

In a similar manner to step 3 of Example 1, the title compound was prepared from the intermediate as a white solid in 80% yield: mp 173-174° C. (decomposition); ¹ H NMR (400 MHz, d ₆ -DMSO) δ11.41 (br s, 1H), 8.41 (br s, 3H), 7.15 (br s, 2H), 5.82 (d, J= 4.8, 1H), 4.50 -5.00(m, 2H), 4.40 (dd, J= 11.7, 3.3, 1H), 4.21-4.30 (m, 2H), 3.91-4.0 (m, 2H), 1.84-1.91 (m, 1H), 1.37- 1.44 (m, 1H), 1.19-1.27 (m, 1H), 0.80-0.87 (m, 6H). C ₁₆ H ₂₃ N ₅ 0 ₇ S -3/2 Elemental analysis for HCl: calcd: C, 39.69; H, 5.10; N, 14.47; Cl, 10.98; S, 6.62; Found: C, 39.05; H, 5.13; N, 13.73; Cl, 11.08; S, 6.02.

Example 3: 5-Amino-3-(5'-O-[α-L-tertiary- butylglycinyl ]-β-D- ribofuranosyl ) thiazolo[4,5-d]pyrimidine -2,7-dione hydrochloride (8)

Step 1: 5-Amino-3-(2',3'-O -isopropylidene -5'-N-tertiary butoxycarbonyl -[α- L-tert-butylglycyl]-β-D Preparation of -ribofuranosyl)-thiazolo[4,5-d]pyrimidine-2,7-dione (9)

In a similar manner to step 2 of Example 1, 5-amino-3-(2',3'-O-isopropylidene-β-D-ribofuranosyl)-thiazolo[4,5-d]pyrimidine 5-amino-3-(2',3'-O-isopropylidene-5'-N-third moiety from -2,7-dione (2) and N-α-L-tertiary-butoxyglycine 66% of oxycarbonyl-[α-L-tert-butylglycyl]-β-D-ribofuranosyl)-thiazolo[4,5-d]pyrimidine-2,7-dione (9) Prepared as a gray foam in yield: ¹ H NMR (400 MHz, d ₆ -DMSO) δ 11.28 (br s, 1H), 6.70-7.40 (m, 3H), 6.02 (s, 1H), 5.30 (d, J= 6.2, 1H), 5.05 (br s, 1H), 4.17-4.24 (m, 3H), 3.77 (d, J= 8.4, 1H), 1.47 (s, 3H), 1.33 (s, 9H), 1.29 (s, 3H), 0.85 (s, 9H).

Step 2: 5-Amino-3-(5'-O-[α-L- tert -butylglycyl]-β-D- ribofuranosyl ) thiazolo[4,5-d]pyrimidine-2, Preparation of 7-dione (8)

In a similar manner to step 3 of Example 1, the title compound (8) was prepared as a white solid in a yield of 80% from the intermediate: mp 202-203° C. (decomposition); ¹ H NMR (400 MHz, d ₆ -DMSO) δ11.35 (br s, 1H), 8.31 (br s, 3H), 7.08 (br s, 2H), 5.83 (d, J= 4.0, 1H), 5.45 (br s, 1H), 5.21 (br s, 1H), 4.77-4.82 (m, 1H), 4.42 (dd, J= 11.4, 2.6, 1H), 4.23-4.28 (m, 1H), 3.96-4.04 ( m, 1H), 3.74 (s, 1H), 0.97 (s, 9H). C ₁₆ H ₂₃ N ₅ 0 ₇ S. Elemental analysis for HCl: calc: C, 41.25; H, 5.19; N, 15.03; Cl, 7.61; S, 6.88; Found: C, 40.41; H, 5.41; N, 14.16; Cl, 7.01; S, 6.23.

Example 4: 5-Amino-3-(5'-O-[α-LN- methylvalinyl ]-β-D- ribofuranosyl )thiazole [4,5-d]pyrimidine-2,7 -Dione hydrochloride (11)

Step 1: 5-Amino-3-(2',3'-O -isopropylidene -5'-N-tertiary butoxycarbonyl- [α- LN-methylvalinyl]-β-D-ribofura Preparation of nosyl)-thiazolo[4,5-d]pyrimidine-2,7-dione (12)

In a similar manner to step 2 of Example 1, 5-amino-3-(2',3'-O-isopropylidene-β-D-ribofuranosyl)-thiazolo[4,5-d]pyrimidine 5-amino-3-(2',3'-O-isopropylidene-5'-N from -2,7-dione (2) and N-tert-butoxy-LN-methylvalinyl (13) 63% of -tertiary butoxycarbonyl-[α-LN-methylvalinyl]-β-D-ribofuranosyl)-thiazolo[4,5-d]pyrimidine-2,7-dione (12) Prepared as a gray foam with a yield of: ¹ H NMR (400 MHz, d ₆ -DMSO) Rotamer carbamate δ 11.28 (br s, 1H), 7.00 (br s, 2H), 6.02 (s, 1H), 5.27 (d, J= 6.6, 1H), 5.04 (br s, 1H), 4.14-4.28 (m, 3H), 3.91 (d, J= 9.5, 1H), 2.79 (br s, 3H), 2.09 (br s, 1H), 1.46 (s, 3H), 1.36 (s, 4.5H), 1.32 (s, 4.5H), 1.28 (s, 3H), 0.78-0.89 (m, 6H).

Step 2: 5-Amino-3-(5'-O-[α-LN- methylvalinyl ]-β-D- ribofuranosyl )thiazolo [4,5-d]pyrimidine-2,7- Preparation of dione hydrochloride (11)

In a manner similar to step 3 of Example 1, the title compound (11) was prepared from the intermediate as a white solid containing some impurities in a yield of 60%: mp> 180° C. (decomposition); ¹ H NMR (400 MHz, d ₆ -DMSO) δ11.31 (br s, 1H), 9.05 (br s, 2H), 7.05 (br s, 2H), 5.83 (d, J= 4.4, 1H), 5.46 (br s, 1H), 5.21 (br s, 1H), 4.76-4.82 (m, 1H), 4.42-4.48 (m, 1H), 4.28-4.38 (m, 1H), 4.22-4.28 (m, 1H) , 3.94-4.04 (m, 2H), 2.54 (br s, 3H), 2.23 (br s, 1H), 0.98 (d, J = 7.0, 3H), 0.88 (d, J = 7.0, 3H). C ₁₆ H ₂₃ N ₅ 0 ₇ S. Elemental analysis for HCl: calc: C, 41.25; H, 5.02; N, 15.03; S, 6.88; Cl, 7.61; Found: C, 40.57; H, 5.37; N, 13.57; S, 6.16; Cl, 7.29.

Scheme 2

Scheme 2 shows 5-amino-7-methoxy-3-β-D-ribofuranosylthiazolo[4,5-d]pyrimidin-2-one and 5,7-diamino-3-β-D- The general procedure for preparing ribofuranosylthiazolo[4,5-d]pyrimidin-2-one is shown.

Example 5: 5-amino-β-D- ribo furanyl nosil -3-7-methoxy-thiazolo [4,5-d] pyrimidine -2-one (14)

Anhydrous compound (1) (2.0 g, 6.3 mmol) was dissolved in anhydrous pyridine under an argon atmosphere. The solution was cooled to 0° C., and TFAA (13.3 g, 63 mmol) was added dropwise to the mixture. After 5 minutes, the reaction was placed in an oil bath at 60° C. for 1.5 hours, and the formation of pyridinium cations was monitored by TLC (SiO ₂ ,20% MeOH-CHCl ₃ ). The starting material of 0.2 R _f was converted to a reference point that became blue fluorescence when exposed to 254 nm UV light. Upon conversion to the activated intermediate, a freshly prepared sodium methoxide (1.8 g Na, 78 mmol, 300 ml methanol) solution was added to the reaction at 0°C. The reaction was allowed to warm to room temperature and allowed to proceed for 2 days. Then, the mixture _{was quenched with 1M NH 4} Cl (100 mL) and extracted with 25% IPA-CHCl ₃ (5 x 100 mL). The crude mixture was filtered through a silica gel plug and then concentrated to obtain the title compound (14) (1.6 g, 75%). Analytical samples were obtained by preparative TLC (SiO ₂ ; water, methanol, ethyl acetate, 5:10:85) as a white solid: mp> 160° C. (decomposition); [M+H] ⁺ 330.9, [2M+H] ⁺ 661.1, [3M+H] ⁺ 991.0; R _f =0.6 (20% MeOH-CHCl ₃ ); mp 200.4°C-200.9°C; ¹ H NMR ( _{40OMHz, d 6} -DMS0) δ6.92 (s, 2H), 5.86 (d, J = 5.2, 1H), 5.28 (d, J = 5.6, 1H), 4.96 (d, J = 5.2, 1H), 4.78 (dd, J= 10.8, 5.6, 1H), 4.67 (t, J= 6.0, 1H), 4.07-4.10 (m, 1H), 3.91 (s, 3H), 3.70-3.80 (m, 1H) ), 3.55-3.60 (m, 1H), 3.40-3.45 (m, 1H). Elemental analysis for C ₁₁ H ₁₄ N ₄ 0 ₆ S: calcd: C, 40.00; H, 4.27; N, 16.96; S, 9.71; Found: C, 40.07; H, 4.43; N, 16.71; S, 9.53.

Example 6: 5,7 -diamino- 3-β-D- ribofuranosylthiazolo[4,5-d]pyrimidin- 2-one (15)

Anhydrous compound (1) (0.3 g, 0.9 mmol) was dissolved in anhydrous pyridine under an argon atmosphere. The solution was cooled to 0° C., and TFAA (1.2 mL, 9.5 mmol) was added dropwise to the mixture. After 5 minutes, the reaction was placed in an oil bath at 60° C. for 1.5 hours, and the formation of pyridinium cations was monitored by _{TLC (20% MeOH-CHCl 3 ).} The starting material of 0.2 R _f was converted to a reference point that became blue fluorescence when exposed to 254 nm UV light. Upon conversion to the activated intermediate, the reaction flask was placed in an ice bath. After equilibrating the temperature, 30% NH ₃ (25 ml) aqueous solution was added until the exothermic reaction stopped, and the rest was added. After a few minutes, the product was formed as indicated by _{preparative TLC R f} 0.25 (SiO ₂ , 20% MeOH-CHCl _{3 ).} After the flask was warmed to room temperature over 30 minutes, the aqueous solution was degassed under rotary vacuum _{and extracted with 25% IPA-CHCl 3} (5 x 100 mL). The product was introduced into flash chromatography (SiO ₂ , 10% MeOH-CHCl ₃ ) to obtain the title compound 15 (55 mg, 17%) containing some impurities. Analytical samples were obtained by preparative TLC (SiO ₂ ; water-MeOH-EtOAc, 5:10:85) as a white solid: mp> 155° C. (decomposition); [M+H] ⁺ 316.0; R _f = 0.25 (SiO ₂ , 20% MeOH-CHCl ₃ ); ¹ H NMR (40OMHz, d ₆ -DMSO) δ6.76 (s, 2H), 6.14 (s, 2H), 5.85 (d, J = 5.2, 1H), 5.22 (d, J = 4.8, 1H), 4.92 (d, J = 2.8, 1H), 4.70-4.83 (m, 2H), 4.05-4.10 (m, 1H), 3.65-3.80 (m, 1H), 3.52-3.62 (m, 1H), 3.40-3.50 (m, 1H). Elemental analysis for C ₁₀ H ₁₃ N ₅ 0 ₅ S -1/2 H ₂ 0: calc: C, 37.03; H, 4.35; N, 21.59; S, 9.89; Found: C, 37.27; H, 4.32; N, 20.43; S, 10.11.

Scheme 3

Example 7: 5-amino-7 -methylamino- 3-β-D- ribofuranosylthiazolo[4,5-d]pyrimidin -2-one (18)

Step 1: 5- Acetylamino- 3-(2',3',5'-tri-O-acetyl-β-D- ribofuranosyl ) thiazolo[4,5-d]pyrimidine-2,7( Preparation of 6H)-dione (16)

Anhydrous compound 1 (8.0 g, 39.5 mmol) was dissolved in anhydrous pyridine (65 mL). DMAP (3.1 g, 25.3 mmol) and acetic anhydride (19.1 mL 202.4 mmol) were then added. The reaction was allowed to proceed at room temperature for 2 hours _{, quenched with saturated NaHCO 3} (100 mL) and extracted with DCM (3 x 200 mL). The organic phase was concentrated and then triturated with ether. Thereby, 5-acetylamino-3-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)thiazolo[4] containing some impurities of 12.5 g (103%) ,5-d]pyrimidine-2,7(6H)-dione was obtained as a white solid (16): mp 246.7-248.1° C.; R _f =0.20 (SiO ₂ , 50% EtOAc-CHCl ₃ ); ¹ H NMR (40OMHz, d ₆ -DMSO) δ 12.23 (s, 1H), 11.85 (s, 1H), 5.97 (m, 2H), 5.48 (t, J= 6, 1H), 4.35-4.40 (m , 1H), 4.25-4.31 (m, 1H), 4.08-4.18 (m, 1H), 2.49 (s, 3H), 2.07 (s, 3H), 2.01 (s, 3H), 2.00 (s, 3H).

Step 2: 5- Acetylamino- 7-(2,4,6 -triisopropyl - benzenesulfonyloxy )-3- (2',3',5'-tri-O-acetyl-β-D-ribo Preparation of furanosyl)thiazolo[4,5-d]pyrimidin-2-one (17)

The intermediate (500 mg, 0.98 mmol) from step 1 above was dissolved in DCM (15 mL) at ambient temperature. After DMAP (7.3mg, 0.06mmol) and TEA (16ml, 11mmol) were added to the solution, 2,4,6-triisopropylbenzenesulfonyl chloride (454mg, 1.5mmol) was added. After 1 hour, after the reaction was completed, the crude mixture was concentrated, and then purified by flash chromatography (SiO ₂ , 10% EtOAc-CHCl ₃ ) to obtain 5-acetylamino-7-(2,4,6-triiso Propyl-benzenesulfonyloxy)-3-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)thiazolo[4,5-d]pyrimidin-2-one ( 690 mg, 92%) was obtained as a foamed white solid (17): 74.5-76.3° C.; R _f =0.7 (SiO ₂ , 20% EtOAc-CHCl ₃ ); ¹ H (40OMHz, d ₆ -DMSO) δ 10.83 (s, 1H), 7.39 (s, 2H), 6.03 (d, J= 4.0, 1H), 5.91-5.96 (m, 1H), 5.69 (t, J=6.4, 1H), 4.30-4.70 (m, 1H), 4.22-4.26 (m, 1H), 4.16-4.20 (m, 1H), 3.90-4.00 (m, 2H), 2.97-3.01 (m, 1H) ), 2.07 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H), 1.88 (s, 3H), 1.17-1.25 (m, 18H).

Step 3: 5- Acetylamino- 7 -methylamino- 3-(2',3',5'-tri-O-acetyl-β-D- ribofuranosyl )thiazolo[4,5-d]pyrimidine Preparation of -2-one (19)

The intermediate (1.7 g, 2.27 mmol) from step 2 above was dissolved in dioxane (20 mL) at ambient temperature. To this was added a 2.0M methylamine solution (3.4 mL, 6.8 mmol) in methanol. After 2 hours, the starting material was consumed. The reaction mixture was concentrated and then purified by flash chromatography (SiO ₂ , gradient elution, 20-80% EtOAc-CHCl ₃ ) to give the pure title compound (945 mg, 83%) as a yellow oil: [M +H] ⁺ 498.2, [2M+H] ⁺ 995.4; R _f = 0.55 (10% CH ₃ 0H-CHCl ₃ ); ¹ H NMR (40OMHz, d ₆ -DMSO) δ10.13 (s, 1H), 7.70 (d, J= 4.41, 1H), 5.95-6.02 (m, 2H), 5.69 (s, 1H), 4.35-4.39 (m, 1H), 4.16-4.23 (m, 2H), 2.90 (d, J = 4.8, 3H), 2.20 (s, 3H), 2.07 (s, 3H), 2.02 (s, 3H), 2.00 (s , 3H).

Step 4: Preparation of 5-amino-7 -methylamino- 3-β-D- ribofuranosylthiazolo[4,5-d]pyrimidin -2-one (18)

The intermediate (420mg, 0.85mmol) from step 3 was dissolved in dioxane (4mL), and 1M LiOH (8.5mL, 8.5mmol) was added to the solution. The O-acetyl group was removed within 40 minutes _{to obtain an intermediate in R f} = 0.15 (SiO ₂ , 5% MeOH-EtOAc). After 2 hours, N-acetyl was removed as indicated by _{TLC R f} = 0.20 (SiO _{2, 5% MeOH-EtOAc).} The reaction mixture was neutralized with a stoichiometric amount of acetic acid, _{extracted with 25% IPA-CHCl 3} and then concentrated to give 18 (195 mg, 70%). An analytical sample of the title compound (18) _{was obtained as a white solid by preparative TLC (SiO 2} ; water-MeOH-EtOAc, 10:20:70): [M+H] ⁺ 330.0; R _f =0.20 (5% MeOH-EtOAc); mp> 108 °C; ¹ H NMR (40OMHz, d ₆ -DMSO) δ7.06 (d, J= 3.6, 1H), 6.24 (s, 2H), 5.85 (d, J= 5.2, 1H), 5.22 (d, J= 4.8, 1H), 4.93 (d, J= 5.2, 1H), 4.70-4.80 (m, 2H), 4.07 (d, J= 4.8, 1H), 3.75 (d, J= 4.4, 1H), 3.5-3.6 (m , 1H), 3.40-3.50 (m, 1H), 2.82 (d, J= 4.4, 3H).

Example 8: 5-Amino-7-dimethylamino-3-β-D- ribofuranosylthiazolo[4,5-d] pyrimidine-2-one (20)

Step 1: 5- Acetylamino- 7-dimethylamino-3-(2',3',5'-tri-O-acetyl-β-D- ri bofuranosyl)-thiazolo[4,5-d] Preparation of pyrimidine-2-one

Example 7, in a manner similar to step 2, 5-acetylamino-7-dimethylamino-3-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-thiazolo [4,5-d]pyrimidin-2-one was obtained as a yellow oil in 80% yield: M ⁺ 511.14; R _f =0.70 (SiO ₂ , 10% MeOH-CHCl ₃ ); ¹ H NMR (40OMHz, d ₆ -DMSO) δ10.15 (s, 1H), 6.10-6.15 (m, 1H), 5.98-6.09 (m, 1H), 5.56-5.70 (m, 1H), 4.35-4.40 (m, 1H), 4.22-4.27 (m, 1H), 4.14-4.08 (m, 1H), 3.18 (s, 6H), 2.19 (s, 3H), 2.08 (s, 3H), 2.06 (s, 3H ), 1.99 (s, 3H).

Step 2: Preparation of 5-amino-7-dimethylamino-3-β-D- ribofuranosylthiazolo[4,5-d]pyrimidin -2-one (20)

In a similar manner to Example 7, step 3, the title compound (20) was obtained in a yield of 82%. An analytical sample was _{obtained as a white solid by preparative TLC (SiO 2} ; water-MeOH-EtOAc, 10:20:70): [M+H] ⁺ 344.0; [2M+H] ⁺ 687.4; mp> 112 °C; R _f =0.20 (5% MeOH-EtOAc); ¹ H NMR (400MHz, d ₆ -DMSO) δ6.27 (s, 2H), 5.91 (d, J= 4.8, 1H), 5.22 (d, J= 6.0, 1H), 4.93 (d, J= 5.2, 1H), 4.71-4.76 (m, 2H), 4.07-4.09 (m, 1H), 3.7-3.8 (m, 1H), 3.5-3.6 (m, 1H), 3.5-3.6 (m, 1H), 3.09 ( s, 6H). Elemental analysis for C ₁₂ H ₁₇ N ₅ 0 ₅ S: calcd: C, 41.98; H, 4.99; N, 20.40; Found: C, 41.32; H, 5.14; N, 18.59.

Example 9: 5-Amino-7- Cyclopropylamino -3-β-D- Ribofuranosylthiazolo [4,5-d]pyrimidin-2-one monohydrochloride salt (21)

Step 1: 5- Acetylamino- 7- cyclopropylamino- 3-(2',3',5'-tri-O-acetyl- β-D-ribofuranosyl)-thiazolo[4,5-d] Preparation of pyrimidine-2-one

Example 7, in a manner similar to step 2, 5-acetylamino-7-cyclopropylamino-3-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-thia Zolo[4,5-d]pyrimidin-2-one was obtained as a yellow oil in 80% yield: R _f =0.45 (SiO ₂ , 75% EtOAc-CHCl ₃ ); ¹ H NMR (400MHz, d ₆ -DMSO) δ10.11 (s, 1H), 7.87 (d, J= 2.8, 1H), 5.98-6.01 (m, 1H), 5.70-5.76 (s, 1H), 4.32 -4.39 (m, 1H), 4.16-4.30 (m, 2H), 3.85 (s, 1H), 2.87 (s, 1H), 2.25 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H ), 1.98 (s, 3H), 0.73-0.76 (m, 2H), 0.57-0.60 (m, 2H).

Step 2: Preparation of 5-amino-7- cyclopropylamino -3-β-D- ribofuranosylthiazolo[4,5- d]pyrimidin-2-one

Example 7, in a manner similar to step 3, 5-amino-7-cyclopropylamino-3-β-D-ribofuranosylthiazolo[4,5-d]pyrimidin-2-one in 79% yield Obtained as. Analytical samples were obtained by preparative TLC (SiO ₂ ; water-MeOH-EtOAc, 10:20:70) as a white solid: R _f = 0.20 (5% MeOH-EtOAc); mp>100°C; [M+H] ⁺ 356.0; ¹ H (400MHz, d ₆ -DMSO) δ7.24 (s, 1H), 6.28 (s, 2H), 5.86 (d, J= 5.6, 1H), 5.22 (d, J= 6, 1H), 4.92 ( d, J= 5.2, 1H), 4.70-4.80 (m, 2H), 4.05-4.10 (m, 1H), 3.7-3.8 (m, 1H), 3.5-3.6 (m, 1H), 3.45-3.50 (m) , 1H), 2.8 (s, 1H), 0.68-0.70 (m, 2H), 0.54-0.57 (m, 2H).

Step 3: Preparation of 5-amino-7- cyclopropylamino- 3-β-D- ribofuranosylthiazolo[4,5- d]pyrimidin-2-one hydrochloride salt (21)

Prepared by adding the solid prepared in step 2 above to 4M HCl in dioxane which was stirred vigorously to give the title compound as a white solid: mp> 99 °C; ¹ H NMR (40OMHz, d ₆ -DMSO) δ7.25 (d, 1H, J = 2.8, 1H), 6.23 (s, 2H), 5.87 (d, J= 5.2, 1H), 5.21 (bs, 1H) , 4.98 (bs, 1H), 4.73-4.79 (m, 2H), 4.09 (t, J= 5.6, 1H), 3.72-3.79 (m, 1H), 3.55-3.60 (m, 1H), 3.45-3.37 ( m, 1H), 2.75-2.82 (m, 1H), 0.72-0.79 (m, 2H), 0.55-0.63 (m, 2H). C ₁₃ H ₁₇ N ₅ 0 ₅ S • Elemental analysis for HCl: calcd: C, 39.85; H, 4.63; N, 17.87; Cl, 9.05; Found: C, 39.66; H, 4.85; N, 16.57; Cl, 8.13.

Example 10: 5-Amino-7- Cyclopentylamino -3-β-D- Ribofuranosylthiazolo [4,5-d]pyrimidin-2-one (22)

Step 1: 5- Acetylamino- 7 -pyrrolidino- 3-(2',3',5'-tri-O-acetyl-β-D- ribofuranosyl )-thiazolo[4,5-d] Preparation of pyrimidine-2-one

Example 7, in a manner similar to step 2, 5-acetylamino-7-pyrrolidino-3-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-thia Zolo[4,5-d]pyrimidin-2-one was obtained in a yield of 70%. Analytical samples were obtained as white solids by preparative TLC (SiO ₂ ; water-MeOH-EtOAc, 10:20:70): mp> 108° C. (decomposition); R _f =0.80 (10% water and 20% methanol in ethyl acetate); [M+H] ⁺ 384.0; ¹ H NMR (400MHz, d ₆ -DMSO) δ7.00 (d, J= 7.2, 1H), 6.17 (s, 2H), 5.18 (d, J= 5.2, 1H), 5.21 (d, J= 5.6, 1H), 4.92 (d, J= 5.6, 1H), 4.74-4.80 (m, 2H), 4.30-4.35 (m, 1H), 4.05-4.10 (m, 1H), 3.70-3.80 (m, 1H), 3.55-3.60 (m, 1H), 3.30-3.45 (m, 1H), 1.40-2.0 (m, 8H).

Step 2: Preparation of 5-amino-7 -cyclopentylamino -3-β-D- ribofuranosylthiazolo[4,5-d] pyrimidin-2-one

In a similar manner to Example 7, Step 3, the title compound (22) was obtained in a yield of 70%. Analytical samples were obtained by preparative TLC (SiO ₂ ; water-MeOH-EtOAc, 10:20:70) as a white solid: mp> 108° C. (decomposition); R _f =0.80 (10% water and 20% methanol in ethyl acetate); [M+H] ⁺ 384.0; ¹ H NMR (40OMHz, d ₆ DMSO) δ7.00 (d, J= 7.2, 1H), 6.17 (s, 2H), 5.18 (d, J= 5.2, 1H), 5.21 (d, J= 5.6, 1H ), 4.92 (d, J= 5.6, 1H), 4.74-4.80 (m, 2H), 4.30-4.35 (m, 1H), 4.05-4.10 (m, 1H), 3.70-3.80 (m, 1H), 3.55 -3.60 (m, 1H), 3.30-3.45 (m, 1H), 1.40-2.0 (m, 8H).

Example 11: 5-Amino-7 -pyrrolidino -3-β-D- ribofuranosylthiazolo[4,5-d]pyrimidin -2-one (23)

Step 1: 5- Acetylamino- 7 -pyrrolidino- 3-(2',3',5'-tri-O-acetyl-β-D- ribofuranosyl )-thiazolo[4,5-d] Preparation of pyrimidine-2-one

Example 7, in a manner similar to step 2, 5-acetylamino-7-pyrrolidino-3-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-thia Zolo[4,5-d]pyrimidin-2-one was obtained as a yellow oil in 79% yield: [M+H] ⁺ 538.1; R _f =0.80 (SiO ₂ ; water-MeOH-EtOAc, 10:20:70); ¹ H (40OMHz, d ₆ -DMSO) δ10.04 (s, 1H), 5.97-6.02 (m, 2H), 5.68 (s, 1H), 4.38 (dd, J = 11.6, 3.6, 1H), 4.15- 4.23 (m, 2H), 3.58 (s, 4H), 2.23 (s, 3H), 2.08 (s, 3H), 2.05 (s, 3H), 1.98 (s, 3H), 1.89 (s, 4H).

Step 2: Preparation of 5-amino-7 -pyrrolidino -3-β-D- ribofuranosylthiazolo[4,5-d]pyrimidin -2-one

In a similar manner to Example 7, Step 3, the title compound (23) was obtained in a yield of 81%. Analytical samples were obtained by preparative TLC (SiO ₂ ; water-MeOH-EtOAc, 10:20:70) as a white solid: mp> 112.4° C. (decomposition); [M+H] ⁺ 370.3; ¹ H NMR (400MHz, d ₆ -DMSO) δ6.22 (s, 2H), 5.90 (d, J = 4.8, 1H), 5.23 (d, J= 5.2, 1H), 4.94 (d, J= 4.4, 1H), 4.68-4.75 (m, 2H), 4.08 (d, J= 4.8, 1H), 3.71-3.76 (m, 1H), 3.55 (bs, 5H), 3.38-3.54 (m, 1H), 1.87 ( s, 4H).

Scheme 4

a) 2,2-dimethoxypropane, acetone, DMSO, MeSO ₃ H, O℃

b) BOC-L-valine, EDC, DMAP, PhMe, 0℃-room temperature

c) anhydrous HCl, iPrOAc, iPrOH

Example 12: 5-Amino-7-cyclopentylamino -3- (5'-OL- Bali carbonyl) -β-D- ribo furanyl nosil) thiazolo [4,5-d] pyrimidin-2-one hydrochloride Chloride (24)

With vigorous stirring, intermediate B was dissolved in anhydrous hydrogen chloride solution in isopropyl acetate at 0° C. and allowed to warm to room temperature. To this heterogeneous mixture, additional isopropyl acetate was added. The reaction mixture was stirred for an additional 12 hours. Toluene was added and the product was filtered and dried under vacuum to give the desired di-HCl salt (24).

The following intermediates were prepared:

5-Amino-7-cyclopentylamino-3-(2',3'-O-isopropylidene-β-D-ribofuranosyl)thiazolo[4,5-d]pyrimidin-2-one (A )

Compound (A) is 5-amino-7-cyclopentylamino-3-β-D-ribofuranosylthiazolo[4,5-d]pyrimidin-2-one (22 ) And acetone, DMSO, methanesulfonic acid and excess dimethoxypropane were prepared by stirring at 0° C. until the starting material was consumed. The reaction mixture was added to ice water and NaHCO ₃ Neutralized to pH 7 with a saturated solution, and extracted with EtOAc. The organic layer was concentrated and added to column chromatography on silica to give the 2',3'-protected diol product.

5-Amino-7- Cyclopentylamino -3-(5'-O-(N-(t -butoxycarbonyl )-L- valinyl )- 2',3'-O-isopropylidene-β- D-ribofuranosyl)thiazolo[4,5-d]pyrimidin-2-one (B)

To a solution of 1.0 equivalent of N-(t-butoxycarbonyl)-L-valine in THF at 0° C. was added 1.1 equivalents of EDC. After stirring for 30 minutes, 1.0 equivalent of 5-amino-7-cyclopentyl-3-(2',3'-O-isopropylidene-β-D-ribofuranosyl)thiazolo[4,5-d ] Pyrimidine-2-one, compound (A) and 1.5 equivalents of DMAP were added. The reaction mixture was warmed to room temperature and stirred for 5 hours to concentrate. The residue was dissolved in EtOAc and partitioned into 1 N HCl to NaHCO ₃ Neutralized with saturated aqueous solution (10 ml). The aqueous phase was further extracted with EtOAc. The combined organic layers were dried over Na ₂ SO ₄ , filtered and evaporated under vacuum to give intermediate (B) and purified by column chromatography on silica.

Biological test

The efficacy of a compound of formula (I) that exhibits good oral delivery properties and induces an immune response when administered by a given route has been readily demonstrated by experiments in mice and beagle dogs. By comparing the above measurement results for the compound of formula (I) with similar experimental results with the compounds described in the documents cited herein (e.g., U.S. Patent Nos. 5,041,426 and 4,880,784), pharmacokinetic and pharmacodynamic properties The advantages of the compound of formula (I) on can be revealed.

Interferon alpha in mice (Mu- IFN -α) concentration

Normal mice are a useful system for assaying the extent to which the present invention described herein improves substances in oral delivery of compound (1) (isatoribin). The experimenter could not only measure the plasma concentration of isatoribine induced by the oral administration of the drug, but extensive immunological studies induced in mice have shown that interferon alpha, which reflects one of the desired biological activities of isatoribine. In other words, it provides a reagent suitable for measuring the level of cytokines.

The present inventors found that compound (3), that is, the 5'-valine ester of compound (1) (val-isatoribin), induces a substantially improved interferon response compared to the interferon response induced from administration of isatoribine itself. The murine system was used in a series of experiments demonstrating sikim.

Table 1 records the results of the assay for murine interferon alpha in mouse plasma, administered twice by oral route at a level of 50 mg/kg of isadoribine formulated with bicarbonate.

Table 1

Interferon Alpha (Mu-IFN-α) Plasma Concentration (pg/mL) in Mice Following Two Oral Administrations of 50mg/kg Isatoribin at 4 Hour Intervals

^{BQL n} -below the elevated quantifiable limit <n pg/mL.

Table 2 records the results of the assay for murine interferon alpha in mouse plasma, which was administered orally at a level of 50 mg/kg of isatoribine formulated as bicarbonate 4 hours after the first administration of bicarbonate. Interferon has been reported as interferon in plasma in 4 mice, including 2 mice administered a bicarbonate vehicle dose. All values reported in this experiment were low, and the reported interferon levels were not consistently reported for all 3 mice tested at each time point, so these results were the date of the fabrication resulting from measurements near the lower limit of the assay. Suggests that you can.

Table 2

Interferon alpha (Mu-IFN-α) plasma concentration (pg/mL) in mice following oral administration of one vehicle dose and one 50 mg/kg dose of isatoribine 4 hours later

^{BQL n} -below the elevated quantifiable limit <n pg/mL.

NR-not reportable

Table 3 reports the results of the assay for murine interferon alpha in plasma of mice administered orally at the same amount as 50/mg/kg of isatoribine on a molar basis of val-isotoribin dissolved in bicarbonate. . In this experiment, it is demonstrated that interferon can be easily measured at 1.0 hour, 1.5 hours, and 2.0 hours after administration. Interferon was detected in all mice tested at a given time point, showing confidence in the effect after administration of val-isatoribin. Therefore, single administration of val-isotoribin was superior to single administration or repeated administration of isatoribine.

Table 3

Plasma concentration (pg/mL) of interferon alpha (Mu-IFN-α) in mice according to Val-isatoribin 73.0mg/kg once dose

BQL-below quantifiable limit <12.5 pg/mL

^{BQL n} -below the elevated quantifiable limit <n pg/mL.

NR-not reportable

The data shown in Tables 1, 2 and 3 can be considered in terms of the frequency of measurable interferon levels. Interferon was detected in only 4 plasmas of 114 mice used in the study of isatoribine, and interferon was detectable in 10 plasmas of 30 mice administered with val-isatoribin. Therefore, the prodrug increased the proportion of mice exhibiting interferon responses from 4% to 30%, and the magnitude of the mean and maximal responses doubled.

In another experiment, plasma levels of isatoribine and interferon alpha were measured in mice administered isatoribine by the intravenous route, and these levels were measured for isatoribine and interferon alpha induced after oral administration of val-isatoribin. Compared with the level. These data are summarized in Figure 1. In this figure, the level of interferon alpha induced by oral val-isatoribin ("val-isator") (in 50 mg/kg isatoribine molar equivalents) is intravenously 25 mg/kg isatoribin ("isator"). It was similar to the level of interferon alpha from. Thus, oral val-isatoribin provides isatoribine and interferon levels that are about 50% of the concentration observed after intravenous administration of isatoribine itself.

beagle dog

The effect of the prodrug (val-isatoribin, 3) on the systemic exposure of isatoribine (1) after oral administration to beagle dogs was investigated. Isatoribine was prepared with sodium bicarbonate solution. val-isotoribin and isatoribine were prepared as the following formulations selected to ensure solubility:

Formulation 1: Isatoribin, 1 and 4 mg/mL in sodium bicarbonate solution.

Formulation 2: val-isotoribin, 1.62 and 6.48 mg/mL in phosphate buffered saline, equal to 1 and 4 mg/mL isatoribine on a molar basis.

Four male and four magnetic beagle dogs weighing 15 to 27 kg and 1 to 2 years of age were used in the study. The animals were divided into 2 groups, each of 2 males and 2 males. The test substance was administered orally on the 1st and 8th days, and a washing period of 7 days was provided between administrations. Blood samples (2 mL) were collected from each animal into lithium heparin tubes before administration and at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours and 10 hours after each administration. I did. Plasma was frozen at -70°C until analysis. Plasma was analyzed for isatoribine by HPLC-MS/MS assay.

The pharmacokinetic parameters for isatoribine derived from isatoribine or val-isatoribin in each dog are summarized in Tables 4 and 5. Table 6 summarizes the ratios for the key pharmacokinetic parameters that define the total exposure as measured by the maximum concentration (Cmax) and the area under the time-concentration curve (AUC) for a 50 mg/kg dose of prodrug and bicarbonate solution. For prodrug (3), the Cmax ratio was 2.98±0.695 and the AUC ratio was 2.38±0.485. These results indicate that the 50 mg/kg dose of the prodrug val-isatoribin provided substantially higher Cmax and greater bioavailability than isatoribine in the bicarbonate solution.

The ratio of Cmax and AUC of the prodrug to the bicarbonate solution at a dose of 10 mg/kg is summarized in Table 7. For prodrugs, the Cmax ratio was 2.24±0.249 and the AUC ratio was 1.82±0.529. These results indicate that the prodrug val-isatoribin at the 10 mg/kg dose provided higher Cmax and greater bioavailability than isatoribine in the bicarbonate solution.

Thus, the maximum concentration of isatoribine achieved after oral administration of the prodrug val-isatoribin is more than twice as high as compared to isatoribine itself at both the 10 and 50 mg/kg doses, Was improved about twice as much.

Table 4

Pharmacokinetic parameters of isatoribine in dogs administered at 50 mg/kg

Table 5

Pharmacokinetic parameters of isatoribine in dogs administered at 10 mg/kg

Table 6

Percentage of pharmacokinetic parameters of isatoribine in dogs administered at 50 mg/kg

Table 7

Percentage of pharmacokinetic parameters of isatoribine in dogs administered at 10 mg/kg

Prodrugs are desirable for several reasons. First, prodrugs are easily formulated so that the active agent is provided in high proportions. This phenomenon makes the capsule size smaller for a given dose and is advantageous for oral products. Second, prodrugs provide the effect of masking active structures while the agent passes through the lymphoid tissue under the intestine, thereby minimizing activation of such tissues and enhancing oral tolerance. Finally, at the doses tested, val-isatoribin provided plasma levels of isatoribine within the desired range for biological effects after oral administration, which was not the case with isatoribine itself.

The exemplary compounds described above can be formulated into pharmaceutical compositions according to the following general examples.

Example 1: Parenteral composition

To prepare a parenteral pharmaceutical composition suitable for administration by injection, 100 mg of a water-soluble salt of the compound of formula (I) was dissolved in DMSO and then mixed with 10 mL of 0.9% sterile saline. The mixture was incorporated into dosage units suitable for administration by injection.

Example 2: oral composition

To prepare a pharmaceutical composition for oral delivery, 100 mg of the compound of formula (I) was mixed with 750 mg of lactose. The mixture was incorporated into oral dosage units such as hard gelatin capsules suitable for oral administration.

The present invention provides a pharmaceutical composition containing 3-β-D-ribofuranosylthiazolo[4,5-d]pyridine nucleoside and such compounds having immunomodulatory activity. The present invention also provides therapeutic or prophylactic uses of such compounds and compositions and methods of treating diseases and disorders described herein by administering an effective amount of such compounds.

It should be understood that the above description is for the purpose of implementation and description and is intended to illustrate the invention and its preferred embodiments. Through routine experimentation, those skilled in the art can recognize obvious changes and changes that can be changed and changed without departing from the spirit of the present invention. Accordingly, it is intended to limit the present invention not by the above description, but by the appended claims and their equivalents.

Claims (11)

A compound of formula (I) or a pharmaceutically acceptable salt thereof:

Where R ¹ is independently H, —C (O) R ³ , or racemic L- or D-amino acid group —C (O) CHNH ₂ R ⁴ , wherein R ³ is optionally substituted alkyl and R ⁴ is H or substituted or unsubstituted alkyl); R ² is H, OR ⁵ , or N (R ⁶ ) ₂ , wherein R ⁵ is independently H or alkyl, and R ⁶ is independently H, substituted or unsubstituted alkyl, cycloalkyl, or with nitrogen Forms a substituted or unsubstituted heterocycloalkyl ring); When R ² is -OH, at least one of the R ¹ groups is a racemic L- or D-amino acid group -C (O) CHNH ₂ R ⁴ . The compound of claim 1, wherein at least one of the R ¹ groups is a racemic L- or D-amino acid group —C (O) CHNH ₂ R ⁴ , wherein R ⁴ is substituted or unsubstituted alkyl, and the remaining R ¹ is H, R ² is OR ⁵ or N (R ⁶ ) ₂ , wherein R ⁵ is independently selected from H or alkyl, and R ⁶ is independently H, substituted or unsubstituted alkyl, cycloalkyl , Together with nitrogen, forms a substituted or unsubstituted heterocycloalkyl ring) or a pharmaceutically acceptable salt thereof. The compound of claim 2, wherein at least one of the R ¹ groups is a racemic L-amino acid group —C (O) CHNH ₂ R ⁴ , wherein R ⁴ is substituted or unsubstituted alkyl, and the remaining R ¹ groups are H And R ² is OR ⁵ or N (R ⁶ ) ₂ , wherein R ⁵ is substituted alkyl and R ⁶ is independently H or substituted or unsubstituted alkyl, or a pharmaceutical thereof Acceptable salts. The method of claim 3, wherein at least one of the R ¹ groups is a racemic L-amino acid group —C (O) CHNH ₂ R ⁴ , wherein R ⁴ is —CH (CH ₃ ) ₂ and the remaining R ¹ groups are H and R ² is OH, or a pharmaceutically acceptable salt thereof. The method of claim 1,

Compounds or pharmaceutically acceptable salts thereof, characterized in that selected from the group consisting of. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of formula (I) or a pharmaceutically acceptable salt thereof:

Where R ¹ is independently H, —C (O) R ³ , or racemic L- or D-amino acid group —C (O) CHNH ₂ R ⁴ , wherein R ³ is optionally substituted alkyl and R ⁴ is H or substituted or unsubstituted alkyl); R ² is H, OR ⁵ , or N (R ⁶ ) ₂ , wherein R ⁵ is independently H or alkyl, and R ⁶ is independently H, substituted or unsubstituted alkyl, cycloalkyl, or with nitrogen Forms a substituted or unsubstituted heterocycloalkyl ring); When R ² is -OH, at least one of the R ¹ groups is a racemic L- or D-amino acid group -C (O) CHNH ₂ R ⁴ . The compound of claim 6, wherein at least one of the R ¹ groups is a racemic L- or D-amino acid group —C (O) CHNH ₂ R ⁴ , wherein R ⁴ is substituted or unsubstituted alkyl, and the remaining R ¹ group is H, R ² is OR ⁵ or N (R ⁶ ) ₂ , wherein R ⁵ is independently selected from H or alkyl, and R ⁶ is independently H, substituted or unsubstituted alkyl, cycloalkyl Or, together with nitrogen, form a substituted or unsubstituted heterocycloalkyl ring. The compound of claim 7, wherein at least one of the R ¹ groups is a racemic L-amino acid group —C (O) CHNH ₂ R ⁴ , wherein R ⁴ is substituted or unsubstituted alkyl, and the remaining R ¹ groups are H And R ² is OR ⁵ or N (R ⁶ ) ₂ , wherein R ⁵ is substituted alkyl and R ⁶ is independently H or substituted or unsubstituted alkyl. The compound of claim 8, wherein at least one of the R ¹ groups is a racemic L-amino acid group —C (O) CHNH ₂ R ⁴ , wherein R ⁴ is —CH (CH ₃ ) ₂ and the remaining R ¹ groups are H and R ² is OH. The method of claim 6,

Pharmaceutical composition, characterized in that selected from the group consisting of. Providing a compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof, A method of modulating immune cytokine activity in a patient, comprising treating the patient in need thereof with such a compound or a pharmaceutically acceptable salt thereof.

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