CN1845745B - Use of TLR7 ligand and its prodrug in the preparation of medicines for treating hepatitis C virus infection - Google Patents

Abstract

The present invention relates to methods of treating or preventing hepatitis c virus infection in mammals using Toll-like receptor TLR7 ligands and prodrugs thereof. More particularly, the invention relates to methods of orally administering therapeutically effective amounts of one or more TLR7 ligand prodrugs for treating or preventing hepatitis c virus infection. Oral administration of these TLR7 immunomodulatory ligands and prodrugs thereof to mammals provides therapeutically effective amounts and reduces adverse side effects.

Description

Use of TLR7 ligand and its prodrug in the preparation of medicines for treating hepatitis C virus infection

This application claims the benefits of U.S. Provisional Patent Application 60/500,339, filed September 5, 2003, U.S. Provisional Patent Application 60/518,996, filed November 10, 2003, and U.S. Provisional Patent Application 60/518,997, filed November 10, 2003 priority.

1. Field of invention

The present application relates to methods of treating or preventing hepatitis C virus infection in mammals using Toll-like receptor (TLR) 7 ligands and prodrugs thereof. More specifically, the present invention relates to the oral administration of a therapeutically effective amount of one or more prodrugs of TLR7 ligands for the treatment or prevention of hepatitis C virus infection. Oral administration of these TLR7 immunomodulatory ligands and prodrugs thereof to mammals can provide therapeutically effective amounts with reduced adverse side effects.

2. Background of the invention

Small molecule immunomodulation is achieved by identifying compounds that bind and activate Toll-like receptors (TLRs). TLRs play a role in the innate immune response in mammals and are often the first line of defense against pathogens such as bacteria and viruses. The various TLRs are present in different amounts in different mammalian cell types, and the contents can also vary according to the molecular structure of binding TLRs and activating signaling pathways. These signaling pathways generate the spectrum of responses associated with innate immunity.

TLRs recognize PAMPs (pathogen-associated molecular models) and stimulate immune cells through the MyD88-dependent interleukin 1 receptor (IL-1R)-TLR signaling pathway, resulting in the activation of the transcription factor NF-κB2. Ten functional TLR family members (TLR1-TLR10) have been identified in humans. Akira S. et al., Nature Immunol., 2, 675-680 (2001). TLR2, TLR4 and TLR5 are critical for the recognition of peptidoglycan, lipopolysaccharide and flagellin. Hayashi, F. et al., Nature, 410, 1099-1103 (2001). TLR6 binds TLR2 and recognizes mycoplasma lipoproteins. Ozinsky, A., et al., Proc. Natl. Acad. Sci USA., 97, 13766-13771 (2000). TLR9 detects bacterial DNA containing unmethylated CpG motifs, and TLR3 activates immune cells in response to double-stranded RNA. Hemmi, H. et al., Nature, 408, 740-745 (2000).

Many compounds including guanosine analogs, substituted pyrimidines, imidazoquinolines have been reported as TLR7 ligands. For example, see Hemmi et al., Nature Immunol., 3, 196-200 (2002) (imiquimod and R-848 (resiquimod)); Jurk et al., Nat. Immunol., 3, 499 (2002) ( R-848); and Lee et al., Proc.Natl.Acad.Sci USA, 100,6646-6651 (2003) (wherein the guanosine analogue loxoribine, 7-thio-8-oxoguanosine (Isa Toribine) and 7-deazaguanosine (deazaguanosine), imidazoquinolines, imiquimod and R-848 (resiquimod) can selectively activate TLR7).

Prior to their use as potential TLR7 ligands, guanosine analogs and other D- and L-purine nucleotides have been extensively studied over the past 20 years. See, for example, Reitz et al., J.Med.Chem., 37, 3561-78 (1994); Michael et al., J.Med.Chem., 36, 3431-36 (1993) (7- and/or 8-positions have Immunomodulatory guanosine analogs of substituents); Patent 5,821,236 authorized to Krenitsky et al. (disclosing 6-alkoxy derivatives of arabinofuranosylpurine derivatives useful in tumor therapy); authorized to Robins et al. Patent 5,041,426 (disclosing certain pyrimido[4,5-d]pyrimidine nucleotides effective in the treatment of L1210 in BDF1 mice); Revankar et al., J.Med.Chem., 27, 1489-96 (1984 ) (3-deazaguanosine and nucleotides with important broad-spectrum antiviral activity against certain DNA and RNA viruses).

In recent years, many compounds known to have immunostimulatory activity have been identified in the literature as TLR7 ligands. For example, see Heil et al., Eur. J. Immunol., 33(11), 2987-97 (2003), Lore et al., J. Immunol., 171(8), 4320-8 (2003), Nagase et al., J. Immunol., 171(8), 3977-82(2003), Mohty et al., J.Immunol., 171(7), 3385-93(2003), Pinhal-Enfield, etc., Am.J.Pathol., 163( 2), 711-21 (2003), Doxsee et al., J. Immunol., 171 (3), 1156-63 (2003), Bottcher et al., Neurosci. Lett., 344 (1), 17-20 (2003), Kaisho et al., Curr.Mol.Med., 3(4), 373-85(2003), Okada et al., Eur.J. Immunol., 33(4), 1012-9(2003), Edwards et al., Eur.J .Immunol., 33(4), 827-33(2003), Akira et al., Immunol.Lett., 85(2), 85-95(2003), Ito et al., Hum.Immunol., 63(12), 1120 -5(2002), Rothenfusser et al., Hum. Immunol., 63(12), 1111-9(2002), Yamamoto et al., J.Immunol., 169(12), 6668-72(2002), Gibson et al., Cell Immunol., 218(1-2), 74-86(2002), Horng et al., Nature, 420(6913), 329-33(2002), Yamamoto et al., Nature, 420(6913), 324-9(2002) , Applequist et al., Int.Immunol., 14(9), 1065-74(2002), Sato et al., Int.Immunol., 14(7), 783-91(2002); Jurk et al., Nat.Immunol., 3 (6), 499(2002); Hornung et al., J. Immunol., 168(9), 4531-7(2002), Hemmi et al., Nat. Immunol., 3(2), 196-200(2002); Bruno et al., Eur.J.Immunol., 31(11), 3403-12(2001); Jarrossay et al., Eur.J.Immunol., 31(11), 3388-93(2001); Miettin en et al., Genes Immun., 2(6), 349-55(2001), Chuang et al., Eur.cytokine Netw., 11(3), 372-8(2000), and Du et al., Eur.cytokine Netw., 11(3), 362-71 (2000).

These TLR7 ligands are known to stimulate immune responses in vitro and in animals, which has led to research into the use of these compounds for several therapeutic applications, including antiviral and cancer treatment. These compounds are characterized as being analogs or derivatives of a) guanosine, b) imidazoquinoline and c) pyrimidine. See Akira, Current Opinion, 15, 5-11 (2003). One of the imidazoquinoline chemical classes (imiquimod) was found to be effective in the treatment of localized genital infections with papillomavirus. The second imidazoquinoline compound, resiquimod, can be used to treat HCV, but this compound has no anti-HCV effect at tolerated oral doses. Pockros et al., Gastroenterology, 124 (Suppl 1), A-766 (2003).

Thus, although TLR7 ligands have limited application in the treatment of immune diseases and viral infections; see, for example, US Patents 5,041,426 and 4,880,784 to Robins et al. (Semliki Forest) virally active 3-β-D-ribofuranosylthiazolo[4,5-d]pyrimidine); authorized to Averett et al., U.S. Patent Application Publication No.US 2003/0199461 and WO 03/045968 ( 3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidine nucleosides that are resistant to acute and chronic infection by RNA and DNA viruses; ligands that have so far proven ineffective in the treatment or prevention of hepatitis C virus.

Oral administration of many purine nucleoside analogs is known to suffer from difficulties, including poor absorption, poor dissolution, or degradation in the gastrointestinal tract due to acidic or alkaline conditions or the action of enzymes and/or combinations of these phenomena. Accordingly, there remains a need for purine nucleotide analogs with improved oral bioavailability and administration for use in modulating the immune system.

Furthermore, immunomodulatory nucleosides are poorly tolerated orally compared to the intravenous route. Also, there is a specific resistance barrier to immune agents in the gastrointestinal tract due to the large amount bound to the immune tissue of the intestinal wall (eg, intestine). Although this is an important biological mechanism to prevent the body from being attacked by intestinal flora, after oral administration of immunomodulatory compounds, immune tissues can also be preferentially infected due to the high local concentration of administered compounds in the intestine. This leads to adverse side effects, for example, gastroenteritis and local hemorrhage effects are observed for immune stimulators.

A solution to the problem of effective oral delivery of immunomodulators does not exist in the literature. Current evidence suggests that administered body concentrations of this class of drugs are limited by increased gastrointestinal toxicity following low oral doses. Thus, there remains a need for immunomodulatory TLR7 ligands with improved oral bioavailability and reduced gastrointestinal irritation.

3. Outline of the invention

3.1 TLR7 ligands

The present invention encompasses novel methods for treating or preventing hepatitis C virus infection, as well as pharmaceutical compositions utilizing TLR7 ligands or pharmaceutically acceptable salts, hydrates, metabolites or stereoisomers thereof.

In one embodiment, the present invention encompasses a method of treating or preventing hepatitis C virus infection in a patient in need thereof, said method comprising administering to the patient a therapeutically or prophylactically effective amount of a TLR7 ligand or a pharmaceutically acceptable salt thereof, Hydrates, metabolites or stereoisomers or pharmaceutically acceptable salts or hydrates of said stereoisomers.

In another embodiment, the present invention encompasses a method of treating or preventing hepatitis C virus infection in a patient in need thereof, said method comprising administering to the patient an effective or prophylactically effective amount of analogs and derivatives selected from TLR7 ligands: a) guanosine b) imidazoquinoline c) adenine and d) pyrimidine.

In another embodiment, the present invention encompasses a method of treating or preventing hepatitis C virus infection in a patient in need thereof, said method comprising administering to the patient a therapeutically or prophylactically effective amount of a TLR7 ligand selected from Acceptable salts, hydrates, metabolites or stereoisomers or pharmaceutically acceptable salts or hydrates of said stereoisomers:

in:

Each ^R is H, substituted or unsubstituted alkyl, alkenyl or alkynyl, which may be separated by one or more O, S or N heteroatoms, or substituted or unsubstituted aryl or hetero Aryl;

^R is H, OH, SH, halogen, substituted or unsubstituted alkyl, alkenyl, or alkynyl, which may be separated by one or more O, S, or N heteroatoms, or substituted or unsubstituted -O-(alkyl), -O-(aryl), -O-(heteroaryl), -S-(alkyl), -S-(aryl), -S-(heteroaryl), aryl or heteroaryl;

^R3 is H, OH, SH, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, -O-(alkyl), -O-(aryl), -O- (heteroaryl), -S-(alkyl), -S-(aryl), -S-(heteroaryl), -NH(alkyl), -NH(aryl), -NH(heteroaryl radical), -NH(R ⁴ )(alkyl), -NH(R ⁴ )(aryl), -NH(R ⁴ )(heteroaryl), wherein R ⁴ is substituted or unsubstituted alkyl;

X is O or S;

Y is H, halogen, OH, OR ⁴ , SH, SR ⁴ or substituted or unsubstituted alkyl or aryl;

Z is H, halogen, OH, OR ⁴ , SH or SR ⁴ ;

In another embodiment, the present invention encompasses a method of treating or preventing hepatitis C virus infection in a patient in need thereof, said method comprising administering to the patient a therapeutically or prophylactically effective amount of , Ie, If, Ig and Ih TLR7 ligands, wherein ^R is H or substituted or unsubstituted alkyl, alkenyl or alkynyl; ^R is H, OH, halogen, substituted or unsubstituted alkyl , alkenyl, alkynyl or -CH ₂ -O-(alkyl); R ³ is H, OH, SH, substituted or unsubstituted -O-(alkyl), -S-(alkyl) or - NH(alkyl); X is O or S; Y is H, halogen, OH, ^OR4 , SH or ^SR4 ; and Z is H, halogen, OH, ^OR4 , SH or ^SR4 .

In another embodiment, the present invention encompasses a method of treating or preventing hepatitis C virus infection in a patient in need thereof, said method comprising administering to the patient a therapeutically or prophylactically effective amount of a TLR7 ligand selected from Acceptable Salts, Hydrates, Metabolites or Stereoisomers:

and

In one aspect, the invention encompasses a method of treating or preventing hepatitis C virus infection in a mammal in need thereof, preferably a human in need thereof.

In an alternative embodiment, the present invention encompasses a method of treating or preventing hepatitis C virus infection in a patient in need thereof, said method comprising administering to the patient a therapeutically or prophylactically effective amount of a TLR7 ligand and a pharmaceutically acceptable adjuvant , carrier or medium.

In an alternative embodiment, the invention encompasses a method of treating or preventing hepatitis C virus infection in a patient in need thereof comprising administering orally, mucosally, topically or transdermally to the patient a therapeutically or prophylactically effective amount of TLR7 Ligand.

In a preferred embodiment, the present invention encompasses a method of treating or preventing hepatitis C virus infection in a patient in need thereof, said method comprising parenterally administering to the patient a therapeutically or prophylactically effective amount of a TLR7 ligand.

In a separate embodiment, the present invention encompasses a method of treating or preventing hepatitis C virus infection in a patient in need thereof, said method comprising administering to the patient a therapeutically or prophylactically effective amount of a TLR7 ligand and other therapeutic agent, preferably Other antiviral or immunomodulatory agents.

The present invention also encompasses pharmaceutical compositions suitable for parenteral administration to a patient comprising a therapeutically or pharmaceutically acceptable amount of a TLR7 ligand of the present invention in sterile form; comprising a therapeutically or pharmaceutically acceptable amount of a TLR7 ligand of the present invention A pharmaceutical composition suitable for oral administration to a patient, wherein the composition is formulated to reduce the contact between the subepithelial immune anatomical structure and the TLR7 ligand, and improve the body absorption of the TLR7 ligand; A pharmaceutical composition suitable for mucosal administration of a TLR7 ligand to a patient, wherein the composition is formulated to reduce exposure of the subepithelial immune anatomy to the TLR7 ligand and improve body absorption of the TLR7 ligand; and comprises therapeutically or pharmaceutically acceptable A pharmaceutical composition suitable for topical administration to a patient of an acceptable amount of a TLR7 ligand of the present invention, wherein the composition is formulated to reduce exposure of the subepithelial immune anatomy to the TLR7 ligand and improve body absorption of the TLR7 ligand. Depending on the particular tissue being treated, other ingredients such as penetration enhancers may be used prior to, concurrently with or after treatment with the active ingredients of the invention. In a preferred embodiment, each of said compositions is in the form of a single dosage unit containing an amount of the active ingredient sufficient to treat or prevent infection with hepatitis C virus in humans.

In a specific embodiment, the invention encompasses pharmaceutical compositions comprising a TLR7 ligand selected from a) guanosine, b) imidazoquinolines, c) adenine and d) analogs and derivatives of pyrimidine.

In another specific embodiment, the present invention encompasses a pharmaceutical composition comprising a TLR7 ligand selected from the group consisting of general formulas Ia, Ib, Ic, Id, Ie, If, Ig and Ih and pharmaceutically acceptable salts thereof, Hydrates, metabolites or stereoisomers or pharmaceutically acceptable salts or hydrates of said stereoisomers.

3.2 TLR7 ligand prodrugs

The present invention also encompasses novel methods of treating or preventing hepatitis C virus infection, as well as novel pharmaceutical compositions utilizing TLR7 ligand prodrugs or pharmaceutically acceptable salts, hydrates, metabolites or stereoisomers thereof.

The present invention also encompasses a method of treating a disease responsive to immunotherapy with an immunological agent, the method comprising orally administering a TLR7 ligand prodrug to a patient in need of immunotherapy, wherein the TLR7 ligand prodrug achieves TLR7 ligand treatment in the patient Effective plasma concentration.

In one embodiment, the invention encompasses a method of treating hepatitis C virus infection in a patient comprising orally administering a TLR7 ligand prodrug, or a pharmaceutically acceptable salt, hydrate, or stereoisomer thereof, to a patient in need thereof. Constructs, wherein, oral administration of TLR7 ligand prodrugs can achieve therapeutically effective plasma concentrations of TLR7 ligands while reducing adverse side effects associated with oral administration of TLR7 ligands. In preferred embodiments, the TLR7 ligand prodrug is a labeled TLR7 ligand prodrug.

In another embodiment, the present invention also encompasses a method of treating a disease responsive to responsive therapy while reducing side effects associated with immune agents, said method comprising orally administering a TLR7 ligand prodrug to a patient in need of immunotherapy, wherein the TLR7 ligand The prodrug can achieve therapeutically effective plasma concentrations of the TLR7 ligand in the patient. In preferred embodiments, the TLR7 ligand prodrug is a labeled TLR7 ligand prodrug.

In another embodiment, oral administration of a TLR7 ligand prodrug improves the in vivo bioavailability of the TLR7 ligand. In a preferred embodiment, oral administration of the TLR7 ligand prodrug results in an in vivo effective plasma concentration of the TLR7 ligand that is 10%-500% of the effective in vivo exposure obtained by oral administration of the TLR7 ligand alone. In another preferred embodiment, oral administration of the labeled TLR7 ligand prodrug achieves an effective plasma concentration of the TLR7 ligand in vivo that is 50%-200% of the effective in vivo exposure level obtained by oral administration of the TLR7 ligand alone.

In another embodiment, oral administration of TLR7 ligand prodrugs reduces side effects. In preferred embodiments, side effects include gastrointestinal irritation, wherein gastrointestinal irritation includes bleeding, injury and vomiting.

In another embodiment, the TLR7 ligand prodrug increases oral bioavailability by at least 25% and reduces gastrointestinal irritation by at least 50% in a patient compared to oral administration of the TLR7 ligand alone. In another embodiment, the TLR7 ligand prodrug increases oral bioavailability by at least 50% and reduces gastrointestinal irritation such that other toxicities in patients are limited compared to oral TLR7 ligand alone.

In preferred embodiments, the TLR7 ligand prodrug achieves therapeutically effective plasma concentrations of 25%-200% of the effective in vivo concentration of the TLR7 ligand administered orally to a patient with minimal gastrointestinal irritation.

In one embodiment, the method of the invention comprises administering to a patient in need thereof a therapeutically or prophylactically effective amount of a prodrug of a TLR7 ligand selected from a) guanosine, b) imidazoquinoline, c) adenine and d) pyrimidine analogues and derivatives.

In another embodiment, the method of the invention comprises administering to a patient in need thereof a therapeutically or prophylactically effective amount of a prodrug of a TLR7 ligand selected from a) guanosine, b) imidazoquinoline, c) adenine and d) Analogs and derivatives of pyrimidines, wherein the prodrugs are (a) amide, carbamate or amidine moieties converted from amine substituents of TLR7 ligands, (b) esters, carbonic acids converted from alcohol substituents of TLR7 ligands ester, carbamate, ether, imidate, acetal, aminal, or ketal moiety, (c) the acetal or ketal moiety after conversion of the ketone substituent of the TLR7 ligand, (d) The imide ester moiety after conversion of the carbonyl substituent of the amido substituent of the TLR7 ligand, (e) the deoxy moiety after conversion of the oxo substituent of the TLR7 ligand of pyrimidine or guanosine, or (f) the amine.

In another embodiment, the method of the present invention comprises administering to a patient in need thereof a therapeutically or prophylactically effective amount of a TLR7 ligand prodrug or a pharmaceutically acceptable salt, hydrate, metabolite or stereoisomer thereof or a TLR7 ligand prodrug selected from Pharmaceutically acceptable salts or hydrates of the above stereoisomers:

and

in:

Each ^R is H, substituted or unsubstituted alkyl, alkenyl or alkynyl, which may be separated by one or more O, S or N heteroatoms, or substituted or unsubstituted aryl or hetero Aryl;

^R is H, OH, SH, halogen, substituted or unsubstituted alkyl, alkenyl, or alkynyl, which may be separated by one or more O, S, or N heteroatoms, or substituted or unsubstituted -O-(alkyl), -O-(aryl), -O-(heteroaryl), -S-(alkyl), -S-(aryl), -S-(heteroaryl), aryl or heteroaryl;

^R3 is H, OH, SH, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, -O-(alkyl), -O-(aryl), -O- (heteroaryl), -S-(alkyl), -S-(aryl), -S-(heteroaryl), -NH(alkyl), -NH(aryl), -NH(heteroaryl radical), -NH(R ⁴ ) (alkyl), -NH(R ⁴ ) (aryl) or -NH(R ⁴ ) (heteroaryl);

R ⁴ is substituted or unsubstituted alkyl;

R ⁵ is independently H, -C(O)(C _1-18 alkyl), racemic, L- or D-amino-C(O)CHNH ₂ R ⁹ ;

R ⁶ is H, OR ¹⁰ or N(R ¹¹ ) ₂ ;

R ⁷ is independently H or substituted or unsubstituted -C (O) (C _1-18 alkyl) or -C (O) ₂ (C _1-18 alkyl);

R ⁸ is H, -OH, -O-(alkyl), -OCO ₂ (C _1-18 alkyl), -OC(O) (C _1-18 alkyl), racemic, L- or D -Amino-OC(O)CHNH ₂ R ¹ ;

R ⁹ is H, substituted or unsubstituted alkyl, C(O)CH(C _1-6 alkyl)NH ₂ or -C(O)CH(CH ₂ -aryl)NH ₂ ;

R ¹⁰ is independently H, C _1-6 alkyl, C _3-7 alkenyl, C _3-7 alkynyl, -(CR ¹² R ¹³ ) _t (C ₆ -C ₁₀ aryl), -(CR ¹² R ¹³ ) _t (C ₃ -C ₁₀ cycloalkyl), -(CR ¹² R ¹³ ) _t (C ₄ -C ₁₀ heterocyclyl), -(CR ¹² R ¹³ ) _t＞1 OH, -(CR ¹² R ¹³ ) _t>0 CO ₂ C _1-18 alkyl, -(CR ¹² R ¹³ ) _t>0 N(R ¹⁴ )CO ₂ C _1-18 alkyl and SO ₂ (aryl), wherein, unless Otherwise stated, t is an integer of 0-6, and the above-mentioned alkyl, alkenyl, alkynyl, aryl, cycloalkyl and heterocyclic groups are optionally substituted by groups independently selected from the following groups: halogen, cyanide radical, nitro, trifluoromethyl, trifluoromethoxy, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, hydroxyl, C ₁ -C ₆ alkoxy radical, -NH ₂ , -NH-alkyl, -N(alkyl) ₂ , -NH-aryl, -N(alkyl)(aryl), -N(aryl) ₂ , -NHCHO, -NHC (O)alkyl, -NHC(O)aryl, -N(alkyl)C(O)H, -N(alkyl)C(O)alkyl, -N(aryl)C(O)H , _-N (aryl)C(O)alkyl, -NHCO _2alkyl , -N(alkyl)CO 2alkyl, -NHC(O)NH ₂ , -N(alkyl)C(O)NH _2. -NHC(O)NH-alkyl, -NHC(O)N(alkyl) ₂ , -N(alkyl)C(O)NH-alkyl, N(alkyl)C(O)N( Alkyl) ₂ , -NHSO ₂ -alkyl, -N(alkyl)SO ₂ -alkyl, -C(O)alkyl, -C(O)aryl, -OC(O)alkyl, -OC (O)aryl, -CO ₂ -alkyl, -CO ₂ -aryl, -CO ₂ H, -C(O)NH ₂ , -C(O)NH-alkyl, -C(O)N( Alkyl) ₂ , -C(O)NH-aryl, -C(O)N(aryl) ₂ , -C(O)N(alkyl)(aryl), -S(O)alkyl, -S(O)aryl, _-SO2alkyl , -SO2aryl, _-SO2NH2 , _{-SO2NH -} alkyl and _-SO2N (alkyl _{) 2} ;

R ¹¹ is independently H, C _1-6 alkyl, C ₃ -C ₁₀ cycloalkyl, or forms a 5- or 6-membered heterocyclic ring together with nitrogen;

R ¹² and R ¹³ are independently H, C _1-6 alkyl, C _2-6 alkenyl or C _2-6 alkynyl;

R ¹⁴ is H, C _1-6 alkyl or -CH ₂ -aryl;

X is O or S;

Y is H, halogen, OH, OR ⁴ , SH, SR ⁴ or substituted or unsubstituted alkyl or aryl; and

Z is H, halogen, OH, OR ⁴ , SH or SR ⁴ ;

In another embodiment, the present invention encompasses a method of treating or preventing hepatitis C virus in a patient in need thereof, said method comprising administering to the patient an effective amount of a compound selected from formula IIa, IIb, IIc, IId, IIe , IIf, IIg and IIh TLR7 ligands, wherein R ¹ is H or substituted or unsubstituted alkyl, alkenyl or alkynyl; R ² is H, OH, halogen, substituted or unsubstituted alkyl, chain Alkenyl or alkynyl or -CH ₂ -O-(alkyl); R ³ is H, OH, SH, substituted or unsubstituted -O-(alkyl), -S-(alkyl) or -NH( Alkyl); R ⁵ is independently H, -C(O)(C _1-18 alkyl), racemic, L- or D-amino-C(O)CHNH ₂ R ⁹ , wherein R ⁹ is not Substituted alkyl; R ⁶ is H or OR ¹⁰ , wherein R ¹⁰ is independently C _1-6 alkyl, C _3-7 alkenyl, C _3-7 alkynyl, -(CR ¹² R ¹³ ) _t (C ₆ -C ₁₀ aryl), -(CR ¹² R ¹³ ) _t (C ₄ -C ₁₀ heterocyclyl) and -(CR ¹² R ¹³ ) _t>0 N(R ¹⁴ )CO ₂ C _1-18 alkyl , wherein, unless otherwise specified, t is an integer of 0-4, and the above-mentioned alkyl, alkenyl, aryl and heterocyclic groups are optionally substituted by 1-3 groups independently selected from the following groups: halogen, Cyano, nitro, trifluoromethyl, trifluoromethoxy, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, hydroxyl, C ₁ -C ₆ alkane Oxygen, -CO ₂ -alkyl, -CO ₂ -aryl, -OC(O)alkyl and -OC(O)aryl, R ¹² and R ¹³ are independently H, C _1-6 alkyl or C _2-6 alkenyl; and R ¹⁴ is H, -CH ₃ or -CH ₂ CH ₃ ; R ⁷ is independently H, substituted or unsubstituted -C(O)(C _1-18 alkyl) or -C(O) ₂ (C _1-18 alkyl); R ⁸ is H, -OH, -O-(alkyl), -OCO ₂ (C _1-18 alkyl), racemic, L- or D-Amino-OC(O)CHNH ₂ R ¹ ; X is O or S; Y is H, halogen, OH, OR ⁴ , SH or SR ⁴ ; and Z is H, halogen, OH, OR ⁴ , SH or SR ⁴ .

In a specific embodiment, the present invention encompasses a method of treating or preventing hepatitis C virus infection in a patient in need thereof, said method comprising administering to the patient a therapeutically or prophylactically effective amount of a TLR7 ligand prodrug or a pharmaceutical agent thereof selected from A pharmaceutically acceptable salt, hydrate or stereoisomer or a pharmaceutically acceptable salt or hydrate of the stereoisomer.

和

and

In another preferred embodiment of the invention, the TLR7 ligand prodrug is an amino acid ester prodrug of a TLR7 ligand. In another preferred embodiment, the amino acid ester prodrug of a TLR7 ligand is a valyl ester.

In one embodiment of the invention, R ⁵ is either rac, L- or D-amino-C(O)CHNH ₂ R ⁹ . In another embodiment, when the TLR7 ligand prodrug is selected from compounds of general formula IIh, R ⁵ is whether racemic, L- or D-amino-C(O)CHNH ₂ R ⁹ .

In another alternative embodiment, the present invention encompasses a method of treating or preventing hepatitis C virus infection in a patient in need thereof, said method comprising administering to the patient a therapeutically or prophylactically effective amount of a prodrug of a TLR7 ligand and a pharmaceutical An acceptable excipient, carrier or vehicle.

In a separate embodiment, the present invention encompasses a method of treating or preventing hepatitis C virus infection in a patient in need thereof, said method comprising administering to the patient a therapeutically or prophylactically effective amount of a prodrug of a TLR7 ligand and other therapeutic agents, preferably other antiviral or immunomodulatory agents.

The present invention also encompasses a pharmaceutical composition suitable for parenteral administration to a patient comprising a therapeutically or pharmaceutically acceptable amount of a TLR7 ligand prodrug of the present invention in sterile form; comprising a therapeutically or pharmaceutically acceptable amount of a TLR7 of the present invention A pharmaceutical composition suitable for parenteral administration of a ligand prodrug to a patient; a pharmaceutical composition suitable for mucosal administration of a TLR7 ligand prodrug of the present invention comprising a therapeutic or pharmaceutically acceptable amount; and a pharmaceutical composition comprising a therapeutic or pharmaceutically acceptable A pharmaceutical composition suitable for topical administration to a patient of an acceptable amount of a TLR7 ligand prodrug of the invention. Depending on the particular tissue being treated, other ingredients such as penetration enhancers may be used prior to, concurrently with or after treatment with the active ingredients of the invention. In a preferred embodiment, each of said compositions is in the form of a single dosage unit containing an amount of the active ingredient sufficient to treat or prevent infection with hepatitis C virus in humans.

In a specific embodiment, the present invention encompasses a pharmaceutical composition comprising a TLR7 ligand prodrug selected from the group consisting of general formulas IIa, IIb, IIc, IId, IIe, If, IIg and IIh or a pharmaceutically acceptable salt, hydrate or stereoisomer thereof or a pharmaceutically acceptable salt or hydrate of said stereoisomer.

In another embodiment, depending on the particular tissue being treated, other components, including but not limited to penetration enhancers, may be used to target infection prior to, concurrently with, or after treatment with one or more TLR7 ligand prodrugs of the invention. Molecules of the region and molecules that reduce the toxicity of TLR7 ligand prodrugs in vivo.

TLR7 ligand prodrugs are useful as immune system enhancers and possess certain immune system properties including: modulating, mitogenic, augmenting and/or enhancing, or they are intermediates of compounds having these properties. After administration to a mammal, the compound is expected to exhibit an effect on at least one cell population of natural killer cells, macrophages, dendritic cells or lymphocytes in the host's immune system. Because of their properties, they are useful as anti-infective agents, including but not limited to antiviral and antitumor agents, or as intermediates for antiviral and antitumor agents. They can be used as active ingredients in suitable pharmaceutical compositions for the treatment of infected hosts.

In one aspect of the invention, TLR7 ligand prodrugs are used to treat overall viral diseases by administering to the mammal a therapeutically effective amount of the compound. Viral diseases contemplated to be treatable with TLR7 ligand prodrugs include acute and chronic infections caused by RNA and DNA viruses. While not limiting in any way the range of viral infections that can be treated, TLR7 ligand prodrugs are particularly effective in the treatment of diseases caused by the following viral infections: adenovirus, cytomegalovirus, hepatitis A virus (HAV), Hepatitis B virus (HBV), yellow fever virus including yellow fever virus and flavivirus family including hepatitis C virus (HCV), herpes simplex virus types 1 and 2, herpes zoster, human herpesvirus 6, human immunization Defective virus (HIV), human papillomavirus (HPV), influenza A virus, influenza B virus, measles, parainfluenza virus, pestivirus, poliovirus, poxviruses (including smallpox and monkeypox), Rhinoviruses, coronaviruses, respiratory syncytial virus (RSV), various viruses that cause hemorrhagic fever: including the arenavirus family (LCM, Junin, Machup, Guanarito, and Lassa fever), Buyaviruses (Hanta virus and Rift Valley fever) and filovirus family (Ebola and Marburg viruses); various viral encephalitis: including West Nile virus, LaCrosse virus, California encephalitis virus, Venezuelan equine encephalitis virus, eastern equine encephalitis virus, western equine encephalitis virus, Japanese equine encephalitis virus, Kysanur forest virus; and tickborne viruses such as Chrimean-Congo hemorrhagic fever virus.

In another aspect of the invention, TLR7 ligand prodrugs are used to treat bacterial, fungal and protozoan infections in a mammal by administering to the mammal a therapeutically effective amount of these compounds. A full range of pathogenic microorganisms, including but not limited to those organisms resistant to antibiotics, are contemplated to be treatable by administration of TLR7 ligand prodrugs. The ability of TLR7 ligand prodrugs to activate multiple components of the immune system's bypass tolerance machinery has generally been found to reduce susceptibility to antibiotics, thus treating mammals infected with such drug-resistant microorganisms by administering TLR7 ligand prodrugs Infections caused are a particular application of the invention.

In another aspect of the invention, a tumor in a mammal is treated by administering to the mammal a therapeutically effective amount of a TLR7 ligand prodrug. Tumors or cancers contemplated to be treatable include, but are not limited to, those caused by viruses, the effect of which involves the inhibition of transformation of virus-infected cells into a neobiotic state, inhibition of virus spread from transformed cells to other normal cells, and/or Prevents the growth of virus-transformed cells. TLR7 ligand prodrugs are expected to be useful against a broad spectrum of tumors, including but not limited to: carcinomas, sarcomas, and leukemias. Included in this class are: breast, colon, bladder, lung, prostate, stomach and pancreatic cancers and lymphoblastic and myeloid leukemias.

Another aspect of the present invention relates to a method of treating a mammal, said method comprising administering a therapeutically and/or prophylactically effective amount of a drug comprising a TLR7 ligand prodrug of the present invention. In this aspect, its role involves the modulation of certain parts of the mammalian immune system, in particular the modulation of Th1 and Th2 cytokine activity, including but not limited to: the interleukin family, such as IL-1 to IL-12, and Other cytokines, such as TNFα and interferon (including interferon alpha, interferon beta and interferon gamma) and their downstream effectors. Where modulation of Th1 and Th2 cytokines occurs, it is contemplated that the modulation may include: stimulation of Th1 and Th2, inhibition of Th1 and Th2, stimulation of Th1 or Th2 and inhibition of the other, or bimodal regulation (th1/Th2 at high concentrations) Levels produce one effect (eg, systemic inhibition) and at low concentrations produce another effect on Th1 or Th2 (eg, stimulate Th1 or Th2, and inhibit the other)).

In another aspect of the invention, a pharmaceutical composition comprising a TLR7 ligand prodrug is administered in a therapeutically effective amount to a mammal receiving an immunomodulatory drug that does not include a compound of the invention. In a preferred aspect of the invention, the dose of the immunomodulatory drug is reduced below its usual effective dose to reduce side effects. In a second preferred aspect, conventional doses of immunomodulatory drugs are used, but with improved therapeutic efficacy when administered with TLR7 ligand prodrugs.

In another aspect of the invention, a pharmaceutical composition comprising a TLR7 ligand prodrug is administered in a therapeutically effective amount to a mammal receiving an anti-infective drug that does not include a compound of the invention. In a preferred aspect of the present invention, the pharmaceutical composition containing the TLR7 ligand prodrug is administered in a therapeutically effective amount together with an anti-infective drug that acts directly on the infection medium to inhibit its growth or kill the infection medium.

4. Brief description of the drawings

Figure 1 shows plasma concentrations of exatoribine and interferon alfa in mice.

Figure 2 shows changes in viral load in HCV-infected patients receiving exatoribine.

5. Detailed Description of the Invention

5.1 Definition

The following terms used in the present invention have the following definitions:

The terms "comprises" and "including" are intended in an open, non-limiting manner.

The term "nucleoside" refers to any pentose sugar or modified pentose sugar bound to a specific position on a heterocycle or to the natural position of a purine (9-position) or pyrimidine (1-position) or an equivalent position of an analog. compound.

The term "purine" refers to a nitrogen-containing bicyclic heterocycle.

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

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

The term "immunomodulator" refers to natural or synthetic products that alter the normal or abnormal immune system by stimulation or suppression.

The term "NOAEL" refers to the concentration at which no side effects were observed (No Observed Adverse Event Level), which is a toxicology term used for the drug dose that causes no obvious toxicity under the conditions of specific dose level, frequency and duration in the selected type .

"Ligand" means a low molecular weight molecule capable of binding a biological receptor. Ligands can be agonists or antagonists, or have no effect.

"Agonist" refers to a ligand that, upon binding, stimulates a receptor to produce a biological effect consistent with the normal biological activity of the receptor.

"Antagonist" refers to a ligand that, upon binding, causes the receptor to cease to produce the normal biological activity of the receptor.

The term "mammal" includes animals and humans.

The term "prevention" refers to the ability of a compound or composition of the invention to prevent a mammal from being diagnosed with or at risk of a disease. The term also encompasses preventing the further development of a disease in a mammal already suffering from or having symptoms of such a disease.

The term "treatment" means:

(i) preventing the occurrence of a disease, disorder or condition in a mammal predisposed to developing the disease, disorder or condition but not yet diagnosed;

(ii) inhibit the disease, disorder or condition, i.e. prevent its development; and

(iii) Alleviation of a disease, disorder or condition, even if the disease, disorder and/or condition regresses.

The terms "α" and "β" refer to the specific stereochemical configuration of substituents on chiral carbon atoms in the drawn chemical structures.

The term "patient" or "subject" means an animal (such as cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, guinea pigs, etc.) or mammals, including hybrids and transgenic animals and mammals. In the treatment or prevention of HCV infection, the term "patient" or "subject" preferably means a monkey or a human, more preferably a human. In a specific embodiment, the patient or subject is infected or exposed to hepatitis C virus. In a certain embodiment, the patient is an infant (0-2 years), pediatric (2-17 years), adolescent (12-17 years), adult (18 years and older), or elderly (70 years and older) patient. Additionally, patients include immunocompromised patients, such as HIV positive patients, cancer patients, patients undergoing immunotherapy or chemotherapy. In a specific embodiment, the patient is a healthy individual, ie, a patient who does not display other symptoms of viral infection.

The term "therapeutically effective amount" refers to a TLR7 ligand or a TLR7 ligand of the present invention sufficient to treat or prevent a viral disease, delay or minimize symptoms associated with a viral infection or virus-induced disease, or cure or alleviate a disease or infection or its etiology Dosage of prodrug. Specifically, a therapeutically effective amount means an amount sufficient to produce a therapeutic effect in vivo. In relation to the use of an amount of a compound of the invention, the term preferably encompasses improvements in overall therapeutic effect, reduction or avoidance of symptoms or causes of disease, or enhancement of the therapeutic effect of another therapeutic agent or synergy with another therapeutic agent. Non-toxic amounts.

The term "prophylactically effective amount" refers to an amount of a compound of the present invention or other active ingredient sufficient to prevent infection, recurrence or spread of viral infection. A prophylactically effective amount may refer to an amount sufficient to prevent a primary infection or the recurrence or spread of an infection or a disease associated with an infection. In relation to the use of a certain amount of a compound of the present invention, the term preferably covers non-toxic substances that improve the overall prophylaxis or increase the prophylactic effect of another prophylactic or therapeutic agent or produce a synergistic effect with another prophylactic or therapeutic agent. Dosage.

The term "combination" refers to using more than one preventive and/or therapeutic agent simultaneously or sequentially, and making their respective effects add or synergize.

The term "pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic acids and bases and organic acids and bases. If the TLR7 ligand prodrug of the present invention is a base, the desired pharmaceutically acceptable salt can be prepared by any suitable method in the prior art, for example, treating the free base with a mineral acid such as hydrochloric acid, Hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or organic acids such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acids, such as glucuronic acid or galacturonic acid, alpha-hydroxy acids, such as citric acid or tartaric acid; amino acids, such as aspartic acid or glutamic acid; aromatic acids, such as benzoic acid or Cinnamic acid; sulfonic acids, such as p-toluenesulfonic acid or ethanesulfonic acid, etc. If the TLR7 ligand prodrug of the present invention is an acid, the desired pharmaceutically acceptable salt can be prepared by any suitable method, for example, treatment of the free acid with an inorganic or organic base, such as an amine ( primary, secondary or tertiary amines), alkali metal hydroxides or alkaline earth metal hydroxides, etc. Examples of suitable salts include: organic salts derived from amino acids such as glycine and arginine, ammonia, primary, secondary or tertiary amines, and cyclic amines such as piperidine, morpholine and piperazine; and those derived from sodium , calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium inorganic salts.

The term "prodrug" refers to a compound that, upon administration, can be converted by a metabolic reaction or solvolysis into another chemical entity that retains biological activity.

The term "TLR7 ligand prodrug" refers to a compound that, after administration, can be converted by a metabolic reaction or solvolysis into another chemical entity that retains biological activity and is a TLR7 ligand. TLR7 ligand prodrugs are themselves ligands for TLR7, or may be "masked" from being effective as ligands for TLR7.

The term "masked TLR7 ligand prodrug" refers to any compound that, after administration, can be converted by a metabolic reaction or solvolysis into another chemical entity that retains biological activity and is a TLR7 ligand Conversion or solvolysis yields compounds that are less effective TLR7 ligands.

The term "pharmaceutically acceptable active metabolite" refers to a pharmaceutically acceptable active product obtained through the metabolic reaction of a specific compound or its salt in vivo. Once inside the body, most drugs are substrates for chemical reactions that alter their physical and biological properties. These metabolic transformations often affect the polarity of TLR7 ligands, altering drug distribution and excretion pathways in vivo. In some cases, however, drug metabolism should produce a therapeutic effect. For example, many antimetabolite anticancer drugs must be converted to their active forms after being transported into cancer cells.

The term "alkyl", as used herein, unless otherwise stated, refers to a saturated straight or branched chain acyclic having 1-20 carbon atoms, preferably 1-10 carbon atoms, most preferably 1-4 carbon atoms hydrocarbons. Representative saturated straight chain alkyl groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, and n-decyl; saturated branched chain alkyl groups Including isopropyl, primary butyl, isobutyl, tert-butyl, isopentyl, 2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4 -Methylpentyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylbutyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylpentyl, 2,2 -Dimethylhexyl, 3,3-dimethylpentyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylpentyl, 3-ethylpentyl, 2- Ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, 2-methyl-4-ethylpentyl Base, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-methyl-4-ethylhexyl, 2,2-diethylpentyl, 3,3-di Ethylhexyl, 2,2-diethylhexyl, 3,3-diethylhexyl, etc. Alkyl groups can be unsubstituted or substituted.

The term "aryl" as used herein, unless otherwise stated, refers to a carbocyclic aromatic ring having 5-14 ring atoms. The ring atoms of a carbocyclic aryl group are all carbon atoms. Aromatic ring structures include compounds having one or more ring structures, such as mono-, bi- or tricyclic compounds, and benzo-fused carbocyclic moieties such as 5,6,7,8-tetrahydronaphthyl and the like. Preferably, aryl is monocyclic or bicyclic. Representative aryl groups include phenyl, tolyl, anthracenyl, fluorenyl, indenyl, azulenyl, phenanthryl and naphthyl. Carbocyclic aryl groups can be unsubstituted or substituted.

The term "substituted" refers to a specific group having one or more substituents. The term "unsubstituted" refers to a specified group having no substituents. "Substituted alkyl" or "substituted aryl" means substituted by one or more substituents selected from the group consisting of 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 (eg, -CF ₃ , -CH ₂ CF ₃ ), -O-haloalkyl (eg, -OCF ₃ , -OCHF ₂ ), etc.

As used herein, and unless otherwise indicated, the terms "optically pure" or "stereoisomerically pure" refer to a composition comprising one stereoisomer of a compound and substantially free of the other stereoisomer of that compound. For example, a stereomerically pure compound having one chiral center is substantially free of the opposite enantiomer of the compound. A typical stereomerically pure compound contains greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of the other stereoisomer of the compound. More preferably greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomer of the compound, even more preferably greater than about 95% by weight of one stereoisomer of the compound and less than About 5% by weight of the other stereoisomer of the compound, most preferably greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomer of the compound. Since many of the compounds of the present invention contain either D or L forms of polysaccharides, the present invention also encompasses D and L forms of sugars. For example, a stereomerically pure D sugar is substantially free of the L form. In alternative embodiments, the L-form TLR7 ligand is used substantially free of the D-form. Thus, the methods and compositions described herein include, in alternative embodiments, the use of levulose or polymers made therefrom.

The compounds of the present invention exhibit tautomerism. While formulas I and II cannot represent all possible tautomeric forms, it should be understood that formula I represents any tautomeric form of the compounds shown and is not limited to only one compound having the illustrated formula. For example, it is understood that regardless of whether the alcohol or ketone form has substituents, they represent the same compound (as shown in the examples of general formula Ha below).

5.2 Identification of TLR7 ligands

Known TLR7 ligands include, but are not limited to, (1) guanosine analogs, such as 7-deazaguanosine and related compounds, including but not limited to Townsend, J.Heterocyclic Chem, 13, 1363 (1976) and Seela , et al., Chem.Ber., 114(10), 3395-3402(1981); 7-allyl, 8-oxo-guanosine (loxoribine) and related compounds, including but not limited to Reitz , etc., compounds described in J.Med.Chem, 37,3561-3578 (1994); 7-methyl, 9-deazaguanosine and related compounds, including but not limited to Girgis et al., J.Med.Chem. , 33, the compound described in 2750-2755 (1990); 8-bromoguanosine and other 8-halopurine compounds, including but not limited to the compound described in U.S. Patent 4,643,992; 6-amino-9-benzyl-2- Butoxy-9H-purin-8-ol and other 2,6,8,9-substituted purines, including but not limited to Hirota et al., J.Med.Chem., 45, 5419-5422 (2002), Henry et al., J. Med. Chem., 33, 2127-2130 (1990), Michael et al., J. Med. Chem., 36, 3431-3436 (1993), Furneaux et al., J. Org. Chem., 64 (22), 8411-8412 (1999), Barrio et al.; J.Org.Chem., 61, 6084-6085 (1996), U.S. Patent 4,539,205, U.S. Patent 5,011,828, U.S. Patent 5,041,426, U.S. Patent 4,880,784 and International Patent Application Publication No.WO 94 /07904 described compounds; (2) imidazoquinolines, including but not limited to 1-(4-amino-2-ethoxymethyl-imidazo[4,5-c]quinolin-1-yl)- 2-Methyl-propan-2-ol (imiquimoid), as described in International Patent Application Publication WO 94/17043; 1-isobutyl-1H-imidazo[4,5-c]quinolin-4-ylamine (resiquimoid), such as International Patent Application Publication WO 94/17043 and U.S. Patent Application No. 10/357,777 (U.S. Patent Application Publication No. US 2003/0195209), 10/357,733 (U.S. Patent Application Publication No. US 2003/0186949), 10 /358,017 (United States Patent Application Publication No.US2003/0176458), 10/357,995 (United States Patent Application Publication No.US 2003/0162806), 10/165,222 (United States Patent Application Publication No.US 20 03/0100764), 10/011,921 (U.S. Patent Application Publication No. US 2003/0065005) and 10/013,059 (U.S. Patent Application Publication No. US2002/0173655); U.S. Patent 5,395,937; International Patent Application Publication WO 98/17279 and (3) pyrimidine derivatives, including but not limited to 2-amino-6-bromo-5-phenyl-3H-pyrimidin-4-one (bropirimine) and similar substituted pyrimidines, including but not limited to Wierenga etc., J.Med.Chem, 23, 239-240 (1980), Fan et al., J.Heterocyclic Chem., 30, 1273 (1993), Skilnick et al., J.Med.Chem., 29, 1499-1504 (1986 ), Fried, et al., J. Med. Chem., 23, 237-239 (1980) and Fujiwara et al., Bioorg. Med. Chem. Lett., 10(12) 1317-1320 (2000). All patents, patent publications, and publications are incorporated herein by reference.

In addition to the TLR7 ligands described above, other TLR7 ligands can be readily identified by known screening methods. See, eg, Hirota et al., J. Med. Chem., 45, 5419-5422 (2002); and Akira S. et al., Immunology Letters, 85, 85-95 (2003). Using variants of one of these known screening methods (as described in Section 6.1), analogs and derivatives of adenine can also be identified as TLR7 ligands. Adenine derivatives are known in the art as described in European Patent Application Publication No. EP 1 035 123, EP 1 043 021 and EP 0 882 727; U.S. Patent 6,376,501; U.S. Patent 6,329,381; U.S. Patent 6,028,076 and U.S. Patent Application Publication No. US 2003/0162806.

TLR7 ligands of the general formulas Ia-Ih can be synthesized using methods known to those skilled in the art, in particular on the basis of the references and patents listed above.

5.3 Preparation of TLR7 Ligand Prodrugs

The preparation of the TLR7 ligand prodrug of the present invention is as follows: prepare (a) the amide, carbamate or amidine moiety after the TLR7 ligand amine substituent conversion, (b) the ester, carbonate after the TLR7 ligand alcohol substituent conversion , carbamate, ether, imide ester, acetal or ketal moiety, (c) acetal or ketal moiety after conversion of TLR7 ligand amine substituent, (d) carbonyl substitution of TLR7 ligand amide group (e) the deoxygenated product after conversion of the oxo substituent of the TLR7 ligand pyrimidine or guanosine, (f) amine. For example, TLR7 ligand prodrugs are prepared by (1) converting the hydroxyl (OH) substituent of the TLR7 ligand to an amino acid ester, or (2) converting the amine substituent of the TLR7 ligand to an amide or carbamate ester. Methods for preparing prodrugs are well known in the art, as described in Burger's Medicinal Chemistry and Drug Chemistry, 1, 172-178, 949-982 (1995). See also 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 (1992).

Schemes 1-18 show general preparations for the preparation of representative compounds of general formula II.

Schemes 1-6 describe how to synthesize 5'-amino acid esters from guanosine analogs and derivatives.

plan 1

Townsend, JHC, 13,

1976, 1363

Seela, et al. Chem Ber.,

114, 10, 1981, 3395-3402

a) 2,2-dimethoxypropane, acetone, DMSO, MeSO ₃ H, 0°C

b) BOC-NHCHR ¹ CO ₂ H, EDC, DMAP, PhMe, 0°C-rt

c) anh HCl, iPrOAc, iPrOH

Scenario 2

Reitz, et al., JMC, 37, 1994, 3561-3578

a) 2,2-dimethoxypropane, acetone DMSO, MeSO ₃ H, 0°C

b) BOC-NHCHR ¹ CO ₂ H, EDC, DMAP, PhMe, 0°C-rt

c) anh HCl, iPrOAc, iPrOH

Option 3

Girgis, et al., JMC, 33, 1990, 2750-2755

a) 2,2-dimethoxypropane, acetone DMSO, MeSO ₃ H, 0°C

b) BOC-NHCHR ¹ CO ₂ H, EDC, DMAP, PhMe, 0°C-rt

c) anh HCl, iPrOAc, iPrOH

Option 4

8-Bromoguanosine[303136-79-0]

commercially available

a) 2,2-dimethoxypropane, acetone DMSO, MeSO ₃ H, 0°C

b) BOC-NHCHR ¹ CO ₂ H, EDC, DMAP, PhMe, 0°C-rt

c) anh HCl, iPrOAc, iPrOH

In a typical synthetic route, acetonide is preferably used to first protect the 2', 3'-hydroxyl of the β-D-ribose moiety of the general formula Ia, Ib, Id, Ie or Ih, as shown in 2, 6, 10 or 14 . The free 5'-hydroxyl reacts with the N-protected amino acid under a variety of esterification conditions to form 3, 7, 11 or 15. The nitrogen of the amino acid ester and the 2',3'-hydroxyl of the ribose unit are reacted under various deprotection conditions, preferably simultaneously, and then the free amine of the amino acid ester forms a salt, as shown in 4, 8, 12 or 16.

Option 5

Reitz, et al. JMC, 37, 1994, 3561-3578

a) 2,2-dimethoxypropane, acetone, DMSO, MeSO ₃ H, 0°C

b) BOC-NHCHR ¹ CO ₂ H, EDC, DMAP, PhMe, 0°C-rt

c) anh HCl, iPrOAc, iPrOH

Option 6

Kini et al., JMC, 34, 1991, 3006-3010

a) 2,2-dimethoxypropane, acetone, DMSO, MeSO ₃ H, 0°C

b) BOC-NHCHR ¹ CO ₂ H, EDC, DMAP, PhMe, 0°C-rt

c) anh HCl, iPrOAc, iPrOH

In the synthetic pathways shown in Schemes 5 and 6, the 2',3'-hydroxyl groups of the β-D-ribose moieties of compounds 17 and 21 were first protected with acetonide to form 18 and 22, respectively. The free 5′-hydroxyl was then esterified with N-tert-butoxycarbonylvaline to form 19 and 23, respectively. Simultaneous deprotection of the nitrogen of the amino acid ester and the 2′,3′-hydroxyl of the ribose forms the hydrochloride salts shown in 20 and 24.

Schemes 7 and 8 describe how to synthesize carbamates and carbonates from adenine analogs and derivatives.

Option 7

a) HOBT, DMF, CH ₂ Cl ₂ , 0°C

b) TFA, CH ₂ Cl ₂ , 0°C-rt

In a typical synthetic route, the amino group of the general formula If is treated with a carbonate or chloroformate to form a carbamate. For 27, the amino acid ester N-terminal protected amine can be deprotected to form a salt such as 28.

Option 8

Kurimota, et al., Bioorg Med Chem,

12, 2004, p 1091-1099

a) C ₆ H ₁₃ OC(O)Cl, (iPr) ₂ NEt, CH ₂ Cl ₂ , MeOH, DMAP, 0-35°C

In Scheme 8, the hydroxyl group of adenine derivative 29 was esterified with n-hexyl chloroformate to give carbonate 30.

Schemes 9 and 10 describe how to synthesize carbamates from imidazoquinoline analogs.

Option 9

In a typical synthetic route, the amino group of an analogue of general formula Ic is reacted with a carbonate, pyrocarbonate or chloroformate under a variety of conditions to form a carbamate.

Scheme 10

a) [C ₅ H ₁₁ OC(O)] ₂ O, NEt ₃ , CHCl ₃ , 40°C

In Scheme 10, treatment of imidazoquinoline 31 with n-pentyl pyrocarbonate affords pentyl carbamate 34.

Schemes 11-12 describe how to synthesize carbamates and imide esters of pyrimidines of general formula Ig.

Scheme 11

Wierenga et al. JMC, 23, Fan et al., JHC, 30,

1980, 239-240 1993, 1273-1276

a) [EtO(CO)] ₂ O, NEt ₃ , DMF, 65°C

In a typical carbamate synthesis, the amino group of 35 is reacted with ethyl pyrocarbonate under the conditions described above to form carbamate 36.

Scheme 12

Wierenga et al., JMC, 23,

1980, 239-240

a) Polymer supported PPh ₃ , EtOH, DEAD, THF, rt

In a typical synthesis of imide esters, as shown above, the amino group of 35 is reacted with ethanol under Mitsunobu-type conditions to form the ethoxy derivative 37.

Scheme 13 describes how to synthesize imidazoquinoline analogs from imidazoquinoline analogs.

Scheme 13

In a typical synthetic route, the amino group of the derivative of general formula Ic reacts with carbonate, pyrocarbonate or chloroformate under various reaction conditions to form carbamate.

Scheme 14 shows the general preparation of 7-allyl-2-amino-9-β-D-ribofuranosyl-7,9-dihydro-purin-8-one.

Scheme 14

a) Ac ₂ O, DMAP, NEt ₃ , CH ₃ CN

b) POCl ₃ , 75°C

c) Zn-Cu, AcOH, 70°C

d) K ₂ CO ₃ , CH ₃ OH, rt

In a typical synthetic route, 7-allyl-2-amino-9-β-D-ribofuranosyl-7,9-dihydro-1H-purine-6,8-dione is preferably protected with an acyl group 17 2′,3′,5′-hydroxyl of β-D-ribose, as shown in 40, 7-allyl-2-amino-9-β-D-ribofuranosyl-7,9-dihydro -1H-purine-6,8-dione converts the C-6 carbonyl group into various groups that are easily reduced, including but not limited to halogen, under various reaction conditions, as shown in 41. After reduction under different or identical reaction conditions, the 2′, 3′, 5′-hydroxyl groups of the ribose unit were deprotected to form 43. Compound 43 can be further modified if desired.

Scheme 15 shows the general preparation of 7-allyl-2-amino-6-ethoxy-9-β-D-ribofuranosyl-7,9-dihydro-purin-8-one.

Scheme 15

a) Polymer supported PPh ₃ , EtOH, DEAD, THF, rt

b) K ₂ CO ₃ , CH ₃ OH, rt

In a typical synthetic route, 40 converts the carbonyl at C-6 to various imino-ethers, including but not limited to ethyl, under various conditions, as shown in 44. Then, the 2′, 3′, 5′-hydroxyl of the ribose unit is deprotected to form 45. Compound 45 can be further modified if desired.

Scheme 16 describes how to synthesize ethers from adenine analogs and derivatives.

Scheme 16

Kurimota, et al., Bioorg Med Chem,

12, 2004, p 1091-1099

a) Br ₂ , CH ₂ Cl ₂

b) NaOEt, EtOH

In a typical synthetic route, adenine derivatives can be halogenated at the C-8 position. The halogen can then undergo a displacement reaction with an appropriate alkoxide to form derivatives such as 64.

Scheme 17 shows the general preparation of 7-allyl-2-amino-6-substituted alkoxy-9-β-D-ribofuranosyl-7,9-dihydro-purin-8-one.

Scheme 17

a) Et ₃ SiCl, imidazole, DMF, rt

b) Polymer supported PPh ₃ , DEAD, THF, rt

c) HF·NEt ₃ , CH ₃ OH, rt

In a typical synthetic route, the hydroxyl group on ribose 17 is protected as a silyl ether. The carbonyl at the C-6 position of 69 was converted under a variety of conditions to various imino-ethers, including but not limited to 4-hydroxymethyl-5-methyl-[1,3]dioxo-2-one The ether, as shown in 70. Then, the 2′, 3′, 5′-hydroxyl of the ribose unit was deprotected to form 71.

Scheme 18 shows the general preparation of 7-allyl-2-amino-6-substituted alkoxy-9-β-D-ribofuranosyl-7,9-dihydro-purin-8-one.

Program 18

a) HOCH ₂ N(CH ₃ )CO ₂ Et, polymer supported PPh ₃ , DEAD, THF, rt

b) HF·NEt ₃ , CH ₃ OH, rt

The C-6 carbonyl of 69 was converted under a variety of conditions to various imino-ethers, including but not limited to ethers of N-methyl-N-(hydroxymethyl)carbamate, as shown in 72. Then, the 2′, 3′, 5′-hydroxyl of the ribose unit was deprotected to form 73. Compound 73 can be further modified if desired.

5.4 Treatment and prevention of hepatitis C virus infection

The present invention provides methods of treating or preventing hepatitis C virus infection in a patient in need thereof.

The present invention also provides the introduction of a therapeutically effective amount of a TLR7 ligand or prodrug thereof or a combination of these ligands and prodrugs into the patient's bloodstream during the treatment and/or prevention of hepatitis C virus infection.

The value of the TLR7 ligand or TLR7 ligand prodrug or its pharmaceutically acceptable salt, hydrate or stereoisomer in the treatment or prevention of acute or chronic infection of the present invention will depend on the nature and severity of the infection, Depending on the route of administration of the active ingredient. Dosage, and in some cases frequency of administration, will also vary depending on the infection being treated, the age, weight and individual response of the individual patient. Appropriate dosage regimens can be readily determined by those skilled in the art based on these factors.

The methods of the invention are particularly suitable for human patients. In particular, the methods and dosages of the present invention can be used in immunocompromised patients, including but not limited to: cancer patients, HIV-infected patients, and patients with immunodegenerative diseases. Additionally, these approaches could be used in immunocompromised patients who are currently in remission. The methods and dosages of the invention may also be used in patients undergoing other antiviral therapies. The prophylactic method of the present invention is particularly useful in patients at risk of being infected by a virus. These patients include, but are not limited to: health care workers, eg, doctors, nurses, hospice care workers; military personnel; teachers; child care workers; patients traveling or living abroad (especially third world), including social assistance Workers, missionaries and diplomatic envoys. Finally, the methods and compositions include patients who are refractory to management or patients who have developed resistance to therapy (eg, resistance to reverse transcriptase inhibitors, protease inhibitors, etc.).

dose

The toxicity and efficacy of the compounds of the present invention can be determined by standard pharmacological procedures in cell culture or experimental animals, such as determining the LD ₅₀ (the dose causing 50% of the population to die) and the ED ₅₀ (the dose that is therapeutically effective for 50% of the population) . The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as _LD50 / _ED50 .

The data obtained from cell culture assays and animal studies can be used in determining the dosage of the compound for use in humans. The dosage of such compounds lies preferably within a range of peripheral concentrations that include the _ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration. For any compound used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the _IC50 (i.e., the concentration of test compound that achieves a half-maximal inhibition of symptoms) determined in cellular assays; alternatively, formulated in animal models The dose of TLR7 ligand is such that the concentration range of TLR7 ligand in peripheral plasma is obtained in a one-to-one correspondence with the concentration required to obtain a fixed response magnitude. Such information can be used to more accurately determine useful doses in humans. Plasma concentrations can be determined, for example, by high performance liquid chromatography.

The regimens and compositions of the present invention are preferably tested in vitro for desired therapeutic or prophylactic activity before being used in humans, and then tested in vivo. For example, in vitro assays, including in vitro cell culture assays, in which cells responding to the action of a TLR7 ligand are exposed to the ligand and the magnitude of the response is measured by an appropriate technique, can be used to determine whether a particular treatment regimen needs to be administered. The potency of the TLR7 ligand prodrug was then evaluated in terms of the potency of the TLR7 ligand prodrug and the degree of conversion of the TLR7 ligand prodrug. The compounds used in the method of the present invention can be tested with appropriate animal models before being tested on humans, and the animal models include but are not limited to: rats, mice, chickens, cows, monkeys, rabbits, hamsters and the like. The compounds can then be used in appropriate clinical trials.

The TLR7 ligand of the present invention or its pharmaceutically acceptable salt, solvate, hydrate or stereoisomer, the prophylactic dose or the amount of therapeutic dose in the treatment or prevention of acute or chronic infection or disease will vary with the nature of the infection Depending on severity and route of administration of the active ingredient. Dosage, and possibly frequency of administration, will also vary with the infection being treated, the age, weight and individual response of the patient. Those skilled in the art can readily select an appropriate dosage regimen based on consideration of these factors. In a certain embodiment, the dosage administered is based on the particular compound used and the weight and condition of the patient. Moreover, the dose can also vary according to various specific TLR7 ligand prodrugs; the appropriate dose can be determined according to the aforementioned in vitro tests, especially tests using TLR7 ligands related to TLR7 ligand prodrugs, and based on animal studies. It is predicted that smaller doses will be used for those TLR7 ligand prodrugs that are shown to be effective at lower concentrations than other TLR7 ligand prodrugs when measured in the system described or as described herein. Generally, the daily dosage range is about 0.001-100 mg/kg, preferably about 1-25 mg/kg, more preferably about 5-15 mg/kg. For the treatment of hepatitis C virus infection, the dosage of about 0.1 mg-about 15 g/day is administered 1-4 times a day, preferably 100 mg/day-12 g/day, more preferably 100-8000 mg/day. In a preferred embodiment of a compound such as a 3-β-D-ribofuranosylthiazolo[4,5-d]pyrimidine prodrug, 200-8000 mg/day is administered in approximately 1-4 divided doses a day. Alternatively, the recommended daily dosage may be administered in a single formulation or in combined cycles with other therapeutic agents over a period of time. In one embodiment, the daily dose is a single dose or divided doses. In a related embodiment, the recommended daily dose may be administered once a week, twice a week, three times a week, four times a week, or five times a week.

In a preferred embodiment, the compounds of the invention are administered such that the compounds are distributed systemically in the patient. In a related embodiment, the compounds of the invention are administered to produce systemic effects in vivo.

In another embodiment, the compounds of this invention are administered orally, mucosally (including sublingually, buccally, rectally, nasally or vaginally), parenterally (including subcutaneously, intramuscularly, intravenously, intraarterially or Intravenous), transdermal or topical administration. In a specific embodiment, a compound of the invention is administered mucosally (including sublingually, buccally, rectally, nasally or vaginally), parenterally (including subcutaneously, intramuscularly, intravenously, intraarterially or Intravenous), transdermal or topical administration. In another specific embodiment, the compounds of the invention are administered orally. In another specific embodiment, the compounds of the invention are not administered orally.

Different therapeutically effective amounts may be used for different infections, as will be appreciated by those of ordinary skill in the art. Similarly, amounts sufficient to treat or prevent such infections, but insufficient to cause or reduce side effects associated with conventional treatments, are also encompassed within the above dosage ranges and dosing frequency regimens.

combination therapy

Particular methods of the invention also include the administration of other therapeutic agents (ie, therapeutic agents other than the compounds of the invention). In a particular embodiment of the invention, the compounds of the invention may be used in combination with at least one other therapeutic agent. Such therapeutic agents include, but are not limited to: antibiotics, antiemetics, antidepressants and antifungals, antiinflammatory agents, antiviral agents, anticancer agents, immunomodulators, beta-interferons, alkylating agents, hormones or cytokines. Preferred embodiments of the invention encompass the administration of other therapeutic agents that are HCV-specific or exhibit anti-HCV activity.

The TLR7 ligand prodrugs of the invention can be administered or formulated in combination with antibiotics. For example, they can be combined with macrolides (eg, tobramycin (Tobi ^_ )), cephalosporins (eg, cephalexin (Keflex ^_ ), cephradine (Velosef ^_ ), cefuroxime (Ceftin ^_ ), Cefprozil ( ^Cefzil_ ), Cefaclor ( ^Ceclor_ ), Cefixime ( ^Suprax_ ) or Cefadroxil ( ^Duricef_ )), clarithromycin (such as clarithromycin ( ^Biaxin_ )), erythromycin (eg, erythromycin (EMycin ^_ )), penicillin (eg, penicillin V (V-CillinK ^_ or PenVeeK ^_ )), or quinolones (eg, ofloxacin (Floxin ^_ ), ciprofloxacin ( Cipro ^_ ) or Noroxacin (Noroxin ^_ )), aminoglycoside antibiotics (eg, apramycin, arbekacin, bambemycin, butirocin, dibekacin, neomycin, undecylenate (salt), netilmicin, paromomycin, ribomycin, sisomicin, and spectinomycin), amphenicol antibiotics (eg, chloramphenicol azide, chloramphenicol, florfenicol, and thiamphenicol), ansamycin antibiotics (eg, riformide and rifampicin), carbacephems (eg, clocarbef), carbapenems (eg, , biapenem and imipenem), cephalosporins (e.g., cefaclor, cefadroxil, cefamandole, ceftrizime, cefoxizone, cefzoram, cefamizole, cefpiramide, and cephalosporins Piro), cephamycins (eg, cefbuperazone, cefmetazole, and cefminox), monobactamycins (eg, aztreonam, carumonam, and tigemonan), oxycephem Penicillins (e.g., fluoxyceph and latamoxef), penicillins (e.g., azamidine penicillin, azemidine penicillin pivoxil, amoxicillin, bahamicillin, benzyl penicillin acid, benzyl penicillin sodium, ipirin Penicillin, fenbecillin, flucloxacillin, penacillin, penicillin hydroiodide, penicillino-benethamine, penicillino-benethamine, penicillin O, penicillin V, benzathine penicillin V, halamine penicillin V, penicillin, and penicillin Potassium (phencihicillin potassium), lincosamides (eg, clindamycin and lincomycin), amphomycin, bacitracin, capreomycin, polymyxin E, duramycin, envinomycin, tetracycline (e.g., apicycline, chlortetracycline, clomocycline, and demeclocycline), 2,4-diaminopyridines (e.g., bromoprim), nitrofurans (e.g., levofuran ketones and furazolium chloride), quinolones and their analogs (e.g., cinoxacin, clinfloxacin, flumequine, and grepafloxacin), sulfonamides (e.g., acesulfame pyrazine, benzsulfonamide , noprosulfonamide, phthalosulfonamide, sulfacodine, and sulfoxetine), sulfones (eg, dithyristone, sulfone sodium, and phenylprosulfone), cycloserine, mupirocin, and potato globulin.

The TLR7 ligand prodrugs of the invention may be administered or formulated in combination with an antiemetic agent. Suitable antiemetics include, but are not limited to: metoclopramide, domperidone, prochlorperazine, promethazine, chlorpromazine hydrochloride, trimebenzine, ondansetron, granisetron, hydroxyzine, acetyl Leucine monoethanolamine, alipride, azasetron, benzquilamine, aminoalcohol acetophylline, bropride, bucrizine, clobopride, cyclizine, dimenhydrinate, Diphenidol, dolasetron, meclizine, mesaladol, metopperazine, nabilone, azolperazine, pipiperazine, scopolamine, sulpiride, tetrahydrocannabinol, thiophene Perazine, dapsoneperazine, tropisetron, and mixtures thereof.

The TLR7 ligand prodrugs of the present invention may be administered or formulated in combination with antidepressants. Suitable antidepressants include, but are not limited to: Benedarine, Caroxadone, Citalopram, Dimethoxan, Fencamamine, Indapine, Indolozine, Nefopam, Nomifensine , hydroxytryptophan, oxipetine, paroxetine, sertraline, thiazine, triadimefon, benzoprocarbazine, isoprochlorazine, iproniazid, isocarboxazid, nyalaamide , otamoxine, phenelzine, cotinine, rolipram, rolipram, maprotiline, metriindole, mianserin, mirtazepine, aldizolam, Amitriptyline, Oxyamitriptyline, Amoxapine, Butiline, Clomipramine, Demetriptyline, Desipramine, Diphenazepine, Dimethaline, Dolupine, Doxet Ping, triflupromazine, imipramine, imipramine N-oxide, iprindole, lofepramine, melitracen, metapramine, nortriptyline, noxitriptyline, oxitriptyline Pipmidol, phenthiazine, disopyrzepine, protriptyline, quinupramamine, tianeptine, trimipramine, atrafenib, benatizine, bupropion, butaxetine , Dioxadril, duloxetine, etoperazone, febabamate, femoxetine, fenpentyl glycol, fluoxetine, fluvoxamine, hematoporphyrin, hypericin, levofa Piperidate, medesamine, milnacipran, phendamorph, moclobemide, nefazodone, oxafluxan, pibelarine, prolintan, pyrisuximide, ritanser Lin, roxindole, rubidium chloride, sulpiride, tandospirone, tozalinone, tolfenacin, methylphenone, tranylcypromine, L-tryptophan, venlafaxine, vilo Sha Qin and Qi Meiding.

The TLR7 ligands or TLR7 ligand prodrugs of the present invention may be administered or formulated in combination with antifungal agents. Suitable antifungal agents include, but are not limited to: amphotericin B, itraconazole, ketoconazole, fluconazole, intrathecal, flucytosine, miconazole, butoconazole, clotrimazole, nystatin, Terconazole, tioconazole, ciclopirox, econazole, iodochlorophenyne ether, naftifine, terbinafine, undecylenate (salt), and griseofulvin.

The TLR7 ligands or TLR7 ligand prodrugs of the present invention may be co-administered or formulated with anti-inflammatory agents. Useful anti-inflammatory agents include, but are not limited to: NSAIDs such as salicylic acid, acetylsalicylic acid, methyl salicylate, diflunisal, salsalate, olsalazine, sulfasalazine Pyridine, acetaminophen, indomethacin, sulindac, etodolac, mefenamic acid, meclofenac sodium, tolmetin, ketorolac, dichlofenac, ibuprofen , naproxen, naproxen sodium, fenoprofen, ketoprofen, flurbiprofen, oxaprozin, piroxicam, meloxicam, ampiraxicam, droxicam, pivoxicam, for Noxicam, nabumetone, phenylbutazone, oxybuzone, antipyrine, aminopyrine, azapropazone, and nimesulide; leukotriene antagonists, including but not limited to: zileuton , aurothioglucose, aurothione disodium and auranofin; steroids, including but not limited to: aclomethasone dipropionate, amcinonide, beclomethasone dipropionate, betamethasone, betamethasone Tamethasone benzoate, betamethasone dipropionate, betamethasone sodium phosphate, betamethasone valerate, clobetasol dipropionate, chlorcotorone trimethyl acetate, hydrocortisone Dexonide, hydrocortisone derivatives, desonide, deoxymethasone, dexamethasone, flunisolide, flucoxinolide, fludrosulfone, clofloxone, medrison, methylprednisolone, methylprednisolone acetate , methylprednisolone sodium succinate, mometasone furoate, paramethasone acetate, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebuatate, prednisone , triamcinolone, triamcinolone acetonide, triamcinolone diacetate, and hextriamcinolone acetonide; and other anti-inflammatory drugs, including but not limited to: methotrexate, colchicine, allopurinol, probenecid, sulfinpyrazone and benzbromarone.

The TLR7 ligands or TLR7 ligand prodrugs of the present invention may be administered or formulated in combination with other antiviral agents. Useful antiviral agents include, but are not limited to, protease inhibitors, nucleoside reverse transcriptase inhibitors, and nucleoside analogs. Antiviral agents include, but are not limited to: zidovudine, acyclovir, ganciclovir, vidarabine, iodinidine, trifluridine, levovirin, viramidine, and Bavirin, and foscarnet, amantadine, rimantadine, saquinavir, indinavir, amprenavir, lopinavir, ritonavir, alpha-interferon, beta-interferon , Adefovir, Anti-Levudine, Entecavir, Pleconaril.

The TLR7 ligand or TLR7 ligand prodrug of the present invention can be administered or formulated in combination with an immunomodulator. Immunomodulators include, but are not limited to: methotrexate, leflunomide, cyclophosphamide, cyclosporine A, mycophenolate mofetil, rapamycin (sirolimus), mizoribine, deoxysperguanide Bacterines, buquinal, malononitriloamindes (such as leflunomide), T cell receptor modulators and cytokine receptor modulators, peptidomimetics, and antibodies (e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab or F(ab) ₂ fragments or epitope-binding fragments), nucleic acid molecules (eg, antisense nucleic acid molecules and triplexes), small molecules, organic and inorganic compounds. Examples of T cell receptor modulators include, but are not limited to: anti-T cell receptor antibodies (e.g., anti-CD4 antibodies (e.g., cM-T412 (Boeringer), IDEC- ^CE9.1_ (IDEC and SKB), mAB4162W94, Orthoclone and OKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies (eg, Nuvion (ProductDesignLabs), OKT3 (Johnson & Johnson) or Rituxan (IDEC)), anti-CD5 antibodies (eg, anti-CD5 ricin-linked immunoconjugated compound), anti-CD7 antibody (eg, CHH-380 (Novartis)), anti-CD8 antibody, anti-CD40 complex monoclonal antibody (eg, IDEC-131 (IDEC)), anti-CD52 antibody (eg, CAMPATH1H (Ilex)), anti-CD2 antibodies, anti-CD11a antibodies (eg, Xanelim (Genentech)) and anti-B7 antibodies (eg, IDEC-114 (IDEC)) and CTLA4-immunoglobulin. Cytokine receptor modulators Examples include, but are not limited to: soluble cytokine receptors (e.g., extracellular domain TNFα receptors or fragments thereof, extracellular domain IL-1β receptors or fragments thereof, and extracellular domain IL-6 receptors or fragments thereof), Cytokines or fragments thereof, (e.g., interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, TNF-α, interferon (IFN)-α, IFN-β, IFN-γ, and GM-CSF), anti-cytokine receptor antibodies (eg, anti-IFN receptor antibody, anti-IL-2 receptor antibody (eg, Zenapax (ProteinDesignLabs)), anti-IL-4 receptor antibody, anti-IL-6 receptor antibody, anti-IL-10 receptor antibody, and anti-IL -12 receptor antibody), anti-cytokine antibody (eg, anti-IFN antibody, anti-TNFα antibody, anti-IL-1β antibody, anti-IL-6 antibody, anti-IL-8 antibody (eg, ABX- IL-8 (Abgenix)) and anti-IL-12 antibody).

The TLR7 ligands or TLR7 ligand prodrugs of the present invention may be administered or formulated in combination with agents that inhibit viral enzymes. Such agents include, but are not limited to, inhibitors of HCV protease, such as BILN 2061, and inhibitors of NS5b polymerase, such as NM107 and its prodrug NM283 (Idenix Pharmaceuticals, Cambridge, MD, USA).

The TLR7 ligand prodrugs of the present invention can be combined with agents that inhibit HCV polymerase as described by Wu in Curr Drug Targets Infect Disord (2003; 3(3): 207-19) or with people such as BretnerM, in Nucleosides Nucleotides Nucleic Acids (2003; 22(5-8): 1531) Inhibition of the agent of virus helicalization function or with HCV specific targeting as described in Zhang X in Drugs (2002; 5(2): 154-8) Co-administration or formulation of inhibitors of drugs.

The TLR7 ligand or TLR7 ligand prodrug of the present invention can be administered or formulated in combination with an agent that inhibits virus replication.

The TLR7 ligands or TLR7 ligand prodrugs of the present invention may be administered or formulated in combination with cytokines. Examples of cytokines include, but are not limited to: Interleukin-2 (IL-2), Interleukin-3 (IL-3), Interleukin-4 (IL-4), Interleukin-5 (IL- 5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin -12 (IL-12), interleukin 15 (IL-15), interleukin 18 (IL-18), platelet-derived growth factor (PDGF), erythropoietin (Epo), epidermal growth factor ( EGF), fibroblast growth factor (FGF), granulocyte macrophage stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor, (M-CSF), lactation Stimulants and interferons (IFNs) (eg, IFN-α and IFN-γ).

The TLR7 ligand or TLR7 ligand prodrug of the present invention can be administered or formulated in combination with hormones. Examples of hormones include, but are not limited to: luteinizing hormone releasing hormone (LHRH), growth hormone (GH), growth hormone releasing hormone, ACTH, somatostatin, growth hormone, somatomodulin, parathyroid hormone, hypothalamus Release factor, insulin, glucagon, enkephalins, vasopressin, calcitonin, heparin, low molecular weight heparin, heparanoids, synthetic and natural opiates, insulin thyroid-stimulating hormone, and endorphins.

The TLR7 ligand or TLR7 ligand prodrug of the present invention can be administered or formulated in combination with β-interferon, and β-interferon includes but not limited to: interferon β-1a, interferon β-1b.

The TLR7 ligand or TLR7 ligand prodrug of the present invention can be administered or formulated in combination with α-interferon, including but not limited to: interferon α-1, interferon α-2a (roferon), interferon α -2b, intron, Peg-intron, Pegasys, synonymous interferon (ingergen) and albuferon.

The TLR7 ligand or TLR7 ligand prodrug of the present invention can be administered or formulated in combination with an absorption enhancer, especially an absorption enhancer targeting the lymphatic system, including but not limited to: sodium glycocholate; sodium caprate; N-Lauroyl-β-D-maltopyranoside; EDTA; mixed micelles; and substances reported by Muranishi in Crit. Rev. Ther. Drug Carrier Syst, 7-1-33, which is hereby incorporated in its entirety for reference. Other known absorption enhancers may also be used. Accordingly, the present invention also encompasses pharmaceutical compositions comprising one or more prodrugs of formula I, compounds of formula II and other compounds of the invention and one or more absorption enhancers.

The TLR7 ligands or TLR7 ligand prodrugs of the present invention may be administered or formulated in combination with an alkylating agent. Examples of alkylating agents include, but are not limited to: nitrogen mustards, aziridines, methylmelamines, alkyl sulfonates, nitrosoureas, triazenes, bischloroethylmethylamines, cyclophosphamide, isocyclic Phosphoramide, melphalan, chlorambucil, hexamethylmelaine, triamidophos, busulfan, carmustine, streptozotocin, dacarbazine, and temozolomide.

Compounds of the invention and other therapeutic agents may have additive or, more preferably, synergistic effects. In a preferred embodiment, a composition containing a compound of the invention is administered concurrently with another therapeutic agent, which may be the same or a different composition than a portion of the composition containing a compound of the invention. In another embodiment, the compound of the invention is administered before or after another therapeutic agent is administered. In a separate embodiment, a compound of the invention is administered to a patient who has previously been treated with another therapeutic agent, in particular an antiviral agent, or is currently no longer receiving treatment with another therapeutic agent.

In one embodiment, the methods of the invention comprise administering one or more TLR7 ligands or TLR7 ligand prodrugs of the invention without administration of another therapeutic agent.

Pharmaceutical Compositions and Dosage Forms

Pharmaceutical compositions and unit dosage forms comprising a TLR7 ligand or prodrug of the invention, or a pharmaceutically acceptable salt, hydrate or stereoisomer thereof, are also within the scope of the invention. The individual dosage forms of the present invention are suitable for oral, mucosal (including sublingual, buccal, rectal, nasal or vaginal administration), parenteral (including sublingual, intramuscular, intravenous bolus, intraarterial or intravenous administration) transdermal or topical administration. Pharmaceutical compositions and dosage forms of the invention typically also comprise one or more pharmaceutically acceptable excipients. Sterile formulations are also within the scope of the invention.

In an optional embodiment, the pharmaceutical composition therein comprises the TLR7 ligand or prodrug of the present invention, or a pharmaceutically acceptable salt, hydrate or stereoisomer thereof, and at least one other therapeutic agent. Examples of other therapeutic agents include, but are not limited to, those listed in Section 5.2.2 above.

The composition, shape and type of dosage form of the invention will typically vary with its use. For example, a dosage form used for the acute treatment of a disease or related diseases may contain greater amounts of one or more active ingredients than used for the chronic treatment of the same disease. Similarly, parenteral dosage forms may contain smaller amounts of one or more active ingredients than oral dosage forms used to treat the same disease or disorder.

Those skilled in the art will readily appreciate that certain dosage forms contemplated by the present invention may vary from one another in these and other ways. See, e.g., Remington's Pharmaceutical Science, 18th ed., Mack Publishing, Eston, PA, USA (1990). Examples of dosage forms include, but are not limited to: tablets; caplets; capsules, such as soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; ointments; Poutice; paste; powder; dressing; cream; plaster; solution; patch; aerosol (as nasal spray or inhalant); gel; suitable for oral or mucosal administration to a patient liquid dosage forms, including suspensions (e.g., aqueous or nonaqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to patients; and available Sterile solids (eg, crystalline or amorphous solids) to be reconstituted into liquid dosage forms suitable for parenteral administration to a patient.

Typical pharmaceutical compositions and dosage forms include one or more carriers, excipients or diluents. Suitable excipients are well known in the art of pharmacy, 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 various factors well known in the art, including, but not limited to, the route by which it is to be administered to a patient. For example, oral dosage forms such as tablets may contain excipients which are not suitable for parenteral dosage forms. The suitability of a particular excipient also depends on the particular active ingredient in the dosage form.

The invention also encompasses anhydrous pharmaceutical compositions and dosage forms containing active ingredients, since water can hasten the degradation of some compounds. For example, the addition of water (eg, 5% water) is generally accepted in the pharmaceutical arts as a means of simulating long-term storage to determine properties such as shelf-life or stability of a formulation over time. See, eg, Jens T. Carstensen, Drug Stability: Principles & Practice, 2nd ed., Marcel Dekker, New York, NY, USA, 1995, pp. 379-80. Water and heat can effectively accelerate the decomposition of some compounds. Thus, the effect of water on formulations can be dramatic due to the moisture and/or humidity typically encountered during manufacture, handling, packaging, storage, shipping and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms of the invention can be prepared using anhydrous or low humidity ingredients and under low humidity or low humidity conditions.

Anhydrous pharmaceutical compositions should be prepared and stored such that their anhydrous nature is maintained. Accordingly, anhydrous compositions are preferably packaged using water-resistant materials enabling their inclusion in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dosage containers (eg, vials), blister packs, and tape packs.

The invention also encompasses pharmaceutical compositions and dosage forms comprising one or more compounds that reduce the rate at which the active ingredient will decompose. Such compounds (hereinafter referred to as stabilizers) include, but are not limited to, antioxidants such as ascorbic acid, pH buffers or salt buffers.

As with the amount and type of excipients, the amount and specific type of active ingredient in a dosage form will vary according to various factors such as, but not limited to, the route of administration to a patient. However, typical dosage forms of the invention comprising a compound of the invention or a pharmaceutically acceptable salt or hydrate thereof comprise 0.1 mg to 1500 mg/unit to provide a dosage of about 0.01 to 200 mg/kg per day.

Oral dosage form

Pharmaceutical compositions of the present invention suitable for oral administration may be in discrete dosage forms such as, but not limited to, tablets (eg, chewable tablets), caplets, capsules, and liquids (eg, flavored slurries). Such dosage forms contain predetermined amounts of active ingredients and may be prepared by methods well known to those skilled in the art of pharmacy. See generally Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton, PA, USA (1990).

Typical oral dosage forms of this invention are prepared by intimately admixing the active ingredient with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take various excipient forms according to the desired form of preparation for administration. For example, suitable excipients for oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives and coloring agents. Examples of excipients suitable for solid oral dosage forms such as powders, tablets, capsules and caplets include, but are not limited to: starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders and disintegrants.

Because of their ease of administration, tablets and capsules represent the most preferred form of oral dosage form, in which case a solid excipient is used. Tablets may be coated, if desired, by standard aqueous or non-aqueous techniques. Such dosage forms may be prepared by any of the methods of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers, finely divided solid carriers or both, and, if necessary, shaping the product into the desired shape.

For example, a tablet may be prepared by compression or molding. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granules, optionally mixed with an excipient. Molded tablets may be made by molding in a suitable machine the powdered compound moistened with an inert liquid excipient.

Examples of excipients that may be used in oral dosage forms of the present invention include, but are not limited to, binders, fillers, disintegrants and lubricants. Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to: corn starch, potato starch or other starches, gelatin, such as acacia, sodium alginate, alginic acid, other alginate salts, powdered tragacanth, guarana Natural and synthetic gums of ear gels, cellulose and its derivatives (such as ethyl cellulose, cellulose acetate, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose), polyvinylpyrrolidone, methyl cellulose Vegetables, pregelatinized starch, hydroxypropyl methylcellulose (eg, Nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.

Fillers suitable for use in the pharmaceutical compositions and dosage forms herein include, but are not limited to: talc, calcium carbonate (eg, granules or powder), microcrystalline cellulose, powdered cellulose, dextrin, kaolin, mannitol, silicon Acids, sorbitol, starches, preformed starches and mixtures thereof. Binders or fillers in pharmaceutical compositions of the invention typically constitute about 50-99% by weight of the pharmaceutical composition or dosage form.

Suitable types of microcrystalline cellulose include, but are not limited to: trade names AVICEL-PH-101, AVICEL-PH-103 AVICEL RC-581, AVICEL-PH-105 (available from FMC Corporation, US Viscose Division, Avicel Sales , Marcus Hook, Pennsylvania, USA), and mixtures thereof. A particular binder is a mixture of microcrystalline cellulose and carboxymethyl cellulose available under the trade name AVICEL RC-581. Suitable anhydrous or low humidity excipients or additives include AVICEL-PH-103 ^™ and Starch 1500LM.

The disintegrant used in the composition of the invention causes the tablet to disintegrate when exposed to an aqueous environment. A tablet with too much disintegrant will disintegrate on storage, while a tablet with too little disintegrant will not disintegrate at the desired rate or under the desired conditions. Thus, neither too much nor too little disintegrant in sufficient amount to decisively alter the release of the active ingredient may be used to form the solid oral dosage forms of the present invention. The amount of disintegrant used may vary with the type of formulation, as is readily discernible to those of ordinary skill in the art. Typical pharmaceutical compositions contain from about 0.5 to about 15% by weight of disintegrant, especially from about 1 to 5% by weight of disintegrant.

Disintegrants that can be used in the pharmaceutical compositions and dosage forms of the present invention include, but are not limited to: agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrillin potassium , sodium starch glycolate, potato starch or tapioca starch, pregelatinized starch, other starches, clay, other algins, other celluloses, colloids and mixtures thereof.

Lubricants that can be used in the pharmaceutical compositions and dosage forms of the present invention include, but are not limited to: calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other Glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oils (such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, lauric acid Ethyl esters, agar and their mixtures. Other lubricants include, for example, silicate silica gel (AEROSIL 200, manufactured by W.R. Grace Co., Baltimore, Maryland, USA), synthetic silica gel (sold by Degussa Co., Plano, Texas, USA). ), CAB-O-SIL (pyrogenic silica product sold by Cabot Co., Boston, Massachusetts, USA) and mixtures thereof. When used, lubricants are combined in an amount of less than about 1% by weight of the pharmaceutical composition or dosage form.

sustained release dosage form

The active ingredients of the present invention may be administered by controlled release or using delivery devices known to those of ordinary skill in the art.例子包括但不限于：美国专利3,845,770；3,916,899；3,536,809；3,598,123；和4,008,719；5,674,533；5,059,595；5,591,767；5,120,548；5,073,543；5,639,476；5,354,556和5,733,566中所描述的那些，所有这些专利都纳入本文供参考。 Such dosage forms provide slow or controlled release of one or more active ingredients by using substances such as hydroxypropylmethylcellulose, other Polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or combinations thereof. Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the active ingredients of the present invention. The invention thus encompasses single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps and caplets for controlled release.

A general purpose shared by all controlled-release pharmaceutical products is to enhance the efficacy of the drug through its non-controlled counterpart. Ideally, the use of an optimally designed controlled-release formulation in drug therapy is characterized by the use of the minimum amount of drug required to cure or manage the condition for the shortest period of time. Advantages of controlled-release formulations include prolonged activity of the drug, reduced dosing 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 thus affect co-occurring side effects (eg, adverse reactions).

Most controlled-release formulations are designed to initially release a certain amount of drug (active ingredient) to rapidly produce the desired therapeutic effect, followed by gradual and sustained release of other amounts of drug to maintain the therapeutic or prophylactic effect over an extended period of time level. In order to maintain this steady blood drug level in vivo, the drug must be released from the dosage form at a rate that replaces the metabolized and excreted drug. Controlled release of the active ingredient can be stimulated by a variety of 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 intraarterial. Since their administration usually bypasses patients' natural defenses against contaminants, parenteral dosage forms are preferably sterile, or can be sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to: solutions ready for injection, anhydrous and/or lyophilized products (reconstitutable powders) ready to be dissolved or suspended in pharmaceutically acceptable excipients for injection , Suspensions and Emulsions for Injection.

Suitable vehicles for parenteral dosage forms of the invention 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 Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer injection; water-miscible excipients such as, but not limited to: ethanol, polyethylene glycol, and polypropylene glycol; non-aqueous excipients, such as but not limited to: corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate , Isopropyl myristate and Benzyl Benzoate.

Compounds that increase the solubility of one or more of the active ingredients disclosed herein can also be incorporated into the parenteral dosage forms of the invention.

transdermal dosage form

Transdermal dosage forms include "reservoir" or "matrix" patches which are applied to the skin and worn for a period of time to allow penetration of the active ingredient in the desired amount.

Suitable excipients (such as carriers and diluents) and other materials that can be used to provide transdermal and topical dosage forms of the invention are well known to those skilled in the pharmaceutical arts and can be determined according to the particular tissue to which the pharmaceutical composition or dosage form is administered. Certainly. With this in mind, typical excipients include, but are not limited to: water, acetone, ethanol, ethylene glycol, propylene glycol, butane-1,3-diol, isopropyl myristate, isopropyl palmitate, Mineral oils and their mixtures.

Depending on the particular tissue to be treated, additional compounds may be used before, during or after treatment with the active ingredients of the invention. For example, penetration enhancers can be used to assist in the penetration of the active ingredient into the tissue. Suitable penetration enhancers include, but are not limited to: acetone; various alcohols, such as ethanol, oleyl alcohol, and tetrahydrofuran; alkylsulfoxides, such as dimethylsulfoxide; dimethylacetamide; dimethylformamide; polyethylene glycol Alcohols; pyrrolidones such as polyvinylpyrrolidone: Kollidon grades (pyvidone, povidone); urea and various water-soluble or water-insoluble sugar esters such as Tween 80 (polysorbate 80) and Span 60 (Sorbitan Monostearate).

The pH of the pharmaceutical composition or dosage form, or the pH of the tissue to which the pharmaceutical composition or dosage form is administered, can also be adjusted to improve delivery of one or more active ingredients. Similarly, the polarity of a solvent carrier, its ionic strength or tonicity can be adjusted to improve delivery. Compounds such as stearic acid can also be added to pharmaceutical compositions or dosage forms to advantageously alter the hydrophilicity or lipophilicity of one or more active ingredients so as to improve delivery. In this regard, stearates can serve as a lipid vehicle for the formulation, as an emulsifying agent or surfactant, and as a delivery-enhancing or penetration-enhancing agent. Different salts, hydrates or solvates of the active ingredients can be used to further adjust the properties of the resulting composition.

Dosage Forms for Topical Administration

Topical dosage forms of the present invention include, but are not limited to, creams, lotions, ointments, gels, solutions, emulsions, suspensions, or other dosage forms known to those skilled in the art. See, eg, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton, PA, USA (1990); and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia, USA (1985).

Suitable excipients (e.g., carriers and diluents) and other materials useful in providing transdermal and topical dosage forms contemplated by the present invention are well known to those skilled in the pharmaceutical art, depending on the particular tissue to which the pharmaceutical composition or dosage form is administered. depends. With this in mind, typical excipients include, but are not limited to: water, acetone, ethanol, ethylene glycol, propylene glycol, butane-1,3-diol, isopropyl myristate, isopropyl palmitate, Mineral oil and other mixtures.

Depending on the particular tissue to be treated, additional components may be used before, during or after treatment with the active ingredients of the invention. For example, penetration enhancers can be used to assist in the penetration of the active ingredient into the tissue. Suitable penetration enhancers include, but are not limited to: acetone; various alcohols, such as ethanol, oleyl alcohol, and tetrahydrofuran; alkylsulfoxides, such as dimethylsulfoxide; dimethylacetamide; dimethylformamide; polyethylene glycol ; pyrrolidones, such as polyvinylpyrrolidone: Kollidon (Kollidon) grade (pyvidone, povidone); urea and various water-soluble or water-insoluble sugar esters, such as Tween 80 (polysorbate 80) and Span 60 (Sorbitan Monostearate).

For mucosal administration

Mucosal dosage forms of the present invention include, but are not limited to, ophthalmic solutions, sprays and aerosols or other forms known to those skilled in the art. See, eg, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton, PA, USA (1990); and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia, USA (1985). Dosage forms suitable for treating mucosal tissues within the oral cavity may be formulated as mouthwashes or oral gels. In a certain embodiment, the aerosol comprises a carrier. In another embodiment, the aerosol is carrier-free.

The TLR7 ligand or TLR7 ligand prodrug of the present invention can also be administered directly to the lungs by inhalation. For inhalation administration, the TLR7 ligands or TLR7 ligand prodrugs of the invention can be routinely delivered to the lung by a number of different devices. For example, a metered dose inhaler ("MDI") delivers a compound directly to the lungs using a small canister containing a suitable low boiling point propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorofluoromethane, Tetrafluoroethane, carbon dioxide or other suitable gases. MDI equipment is commercially available from a number of suppliers such as 3M Corporation, Aventis, Boehringer Ingleheim, Forest Laboratories, Glaxo-Wellcome, Schering Plow, and Vectura.

Alternatively, TLR7 ligands can be administered to the lungs using a dry powder inhaler (DPI) device (see, e.g., Raleigh et al., Proc. Amer. Assoc. Cancer Research Annual Meeting, 1999, 40, 397, which is hereby incorporated by reference in its entirety). DPI devices typically use a mechanism such as a gas burst to generate dry powder aerosols in a container, which is then inhaled by the patient. DPI devices are also well known in the art and are commercially available from a number of suppliers including, e.g., Fisons, Glaxo-Wellcome, Inhale Therapeutic Systems, ML Laboratories, Qdose, and Vectura. A popular variant is the multiple-dose DPI ("MDDPI") system, which delivers more than one therapeutic dose. MDDPI devices are commercially available from eg AstraZeneca, GlaxoWellcome, IVAX, Schering Plow, SkyePharma and Vectura. For example, gelatin capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch for use in these systems.

Another device that can be used to deliver the compounds of this invention to the lungs is a liquid spray device, such as that manufactured by Aradigm Corporation. Liquid spray devices use tiny nozzles to aerosolize liquid drug formulations, which can then be inhaled directly into the lungs.

In a preferred embodiment, a nebulizer device is used to deliver a TLR7 ligand or TLR7 ligand prodrug of the invention to the lung. Nebulizers create aerosols from liquid drug formulations by using, for example, ultrasonic energy to form fine particles that are easily inhaled (see, e.g., Verschoyle et al., British J. Cancer, 1999, 80, Suppl 2, 96, incorporated herein by reference) . Examples of nebulizers include Sheffield/Systemic Pulmonary Delivery Ltd. (see, Armer et al., U.S. Patent No. 5,954,047; van der Linden et al., U.S. Patent No. 5,950,619; van der Linden et al., U.S. Patent No. 5,970,974, incorporated herein by reference). Reference); devices from Aventis and Batelle Pulmonary Therapeutics.

In a particularly preferred embodiment, an electrohydrodynamic ("EHD") aerosol device is used to deliver a TLR7 ligand or TLR7 ligand prodrug of the invention to the lung. EHD aerosol devices use electrical energy to aerosolize aerosolized liquid drug solutions or suspensions (see, e.g., Noakes et al., U.S. Patent No. 4,765,539; Coffee, U.S. Patent No. 4,962,885; Coffee, PCT Application, WO 94/12285; Coffee, PCT Application, WO 94/14543; Coffee, PCT Application, WO 95/26234; Coffee, PCT Application, WO 95/26235; Coffee, PCT Application, WO 95/32807, incorporated herein by reference). The electrochemical properties of TLR7 ligands and TLR7 ligand prodrug formulations may be important parameters requiring optimization when using EHD aerosol devices to deliver drugs to the lungs, and such optimizations can be routinely performed by those skilled in the art. EHD aerosol devices can deliver drugs to the lungs more efficiently than existing pulmonary delivery technologies. Other methods of intrapulmonary delivery of the TLR7 ligands and TLR7 ligand prodrugs of the invention are known to those skilled in the art and are within the scope of the present invention.

Liquid pharmaceutical formulations suitable for use with nebulizers and liquid spray devices and EHD aerosol devices generally include a TLR7 ligand or TLR7 ligand prodrug and a pharmaceutically acceptable carrier. Preferably, the pharmaceutically acceptable carrier is a liquid such as alcohol, water, polyethylene glycol or perfluorocarbon. Optionally, another material may be added to alter the aerosol properties of the TLR7 ligand or prodrug solution or suspension of the TLR7 ligand. Preferably, the material is a liquid such as an alcohol, diol, polyol or fatty acid. Other methods of formulating liquid drug solutions or suspensions suitable for use in aerosol devices are known to those skilled in the art (see, e.g., Biesalski, U.S. Patent No. 5,112,598; Biesalski, 5,556,611, incorporated herein by reference). TLR7 ligands or TLR7 ligand prodrugs may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the aforementioned formulations, TLR7 ligands or TLR7 ligand prodrugs may also be formulated as depot formulations. Such long-acting formulations may be administered by implantation (eg, subcutaneously or intramuscularly) or by intramuscular injection. Thus, the compounds may be formulated with suitable polymeric or hydrophobic materials (eg, as emulsions in acceptable oils) or ion exchange resins, or as sparingly soluble derivatives, eg, as sparingly soluble salts.

Alternatively, other drug delivery systems can be used. Liposomes and emulsions are well known delivery vehicles that can be used to deliver the TLR7 ligands and TLR7 ligand prodrugs of the invention. Certain organic solvents such as dimethyl sulfoxide can be used, although generally more toxic. The TLR7 ligand or TLR7 ligand prodrug can also be delivered in a controlled release system. In one embodiment pump agents can be used (Sefton, CRC Crit. Ref Biomed Eng, 1987, 14, 201; Buchwald et al., Surgery, 1980, 88, 507; Saudek et al., N. Engl. J. Med., 1989, 321 , 574). In another embodiment polymeric materials can be used (see Medical Applications of Controlled Release, edited by Langer and Wise, CRC Press., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, edited by Smolen and Ball , Wiley, N.Y. (1984); Ranger and Peppas, J.Macromol.Sci.Rev.Macromol.Chem., 1983, 23, 61; see also Levy et al., Science, 1985, 228, 190; During et al., Ann. Neurol., 1989, 25, 351; Howard et al., 1989, J. Neurosurg. 71, 105). In another embodiment, the controlled release system can be located in the vicinity of the target of the compound of the invention, such as the lungs, so that only a fraction of the systemic dose is required (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, p. 2 Vol. 115 pp. (1984)). Other controlled release systems may also be used (see, eg, Langer, Science, 1990, 249, 1527).

Suitable excipients (such as carriers and diluents) and other materials that can be used to provide the mucosal administration dosage forms of the present invention are well known to those skilled in the pharmaceutical art and can be determined according to the particular site or method of administration of the composition or dosage form. Certainly. With this in mind, typical excipients include, but are not limited to: water, ethanol, ethylene glycol, propylene glycol, butane-1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil and their mixtures, the excipients are non-toxic and pharmaceutically acceptable. Examples of such additional components are well known in the art. See, eg, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton, PA, USA (1990).

The pH of the pharmaceutical composition or dosage form, or the pH of the tissue to which the pharmaceutical composition or dosage form is administered, can also be adjusted to improve delivery of one or more active ingredients. Similarly, the polarity of the solvent carrier, the ionic strength or tonicity of the solvent carrier can be adjusted to improve delivery. Compounds such as stearates can also be added to pharmaceutical compositions or dosage forms to advantageously alter the hydrophilicity or lipophilicity of one or more active ingredients so as to improve delivery. In this regard, stearates can serve as a liquid carrier for the formulation, as an emulsifying agent or surfactant, and as a delivery-enhancing or penetration-enhancing agent. Different salts, hydrates or solvates of the active ingredient can be used to further adjust the properties of the resulting composition.

Reagent test kit

The present invention provides a pharmaceutical pack or kit comprising one or more containers comprising a TLR7 ligand prodrug for use in the treatment or prevention of hepatitis C virus infection. In other embodiments, the present invention provides a pharmaceutical pack or kit comprising one or more containers containing a TLR7 ligand prodrug for use in the treatment or prevention of hepatitis C virus infection, and one or more containers containing other therapeutic agents, The other therapeutic agents include but are not limited to those listed in Section 5.2.2 above, especially antiviral agents, interferons, agents that inhibit viral enzymes or agents that inhibit viral replication, and other preferred therapeutic agents are HCV specific or exhibit anti-HCV activity.

The invention also provides a pharmaceutical pack or kit comprising one or more containers comprising one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such containers is an instruction set by a governmental agency in the form required for the manufacture, use or sale of the drug or biological product, which instructions show that the manufacture, use or sale of the drug or biological product for human administration has been Obtain a license from a government agency.

analyze

The activity of the TLR7 ligands, TLR ligand prodrugs, compositions and dosage forms of the invention is assayed in vitro or in vivo by a variety of methods known in the art. See, eg, the methods described below and the methods used throughout the Examples.

Various methods of assessing TLR7 activity are known, as described in the following publications, all of which are incorporated herein by reference: Hirota et al., J. Med. Chem., 45, 5419-5422 (2002); and Akira S. et al., Immunology Letters, 85, 85-95 (2003). For example, one system that can be used to analyze TLR7 ligands is to clone the TLR7 gene by methods known to those skilled in the art and transfect it into a suitable cell line that expresses TLR7 and couples to NFkB-luciferase Indicating plasmid. In this cell system, exposure of cells in culture to TLR7 ligands produces a measurable fluorescent signal. For example, see Lee et al., Proc.Nat.Acad.USA, 100, 6646-6651 (2003); Hemmi et al., Nat.Immunol., 3, 196-200 (2002); , 499 (2002) (of which Lee et al., Hemmi et al. and Jurk et al., the entire contents of which are incorporated herein by reference).

Another example of an in vitro method is to expose human peripheral blood mononuclear cells (PBMC) to a candidate TLR7 ligand prodrug for a predetermined interval (eg, 2-24 hours) and then measure immune activity. Immunological activity may include induction of cytokine synthesis, as determined by ELISA assays for cytokine proteins in culture supernatants, such as type 1 interferon (interferon alpha, interferon beta) or type 2 interferon (interferon gamma). Alternatively, PBMC can be collected after incubation with a candidate TLR7 ligand prodrug, PBMC RNA can be extracted, and the level of induction of immune system genes can be determined by performing an RNase protection assay on the extracted RNA. Commonly induced genes include 2'5'-OAS or interferon gamma, but a variety of cytokines can be measured. See, eg, Hirota et al., J. Med. Chem., 45, 5419-5422 (2002).

RNase Protection Assay (RPA) is a method known in the art in which single-stranded RNA is selectively degraded by first hybridizing a radiolabeled RNA sequence specific for the analyzed RNA to the RNA analyte for quantification, followed by enzymatic digestion. The samples are then subjected to gel electrophoresis under conditions that resolve the hybridized, protected double-stranded RNA. Next, autoradiography is performed on the gel, revealing the location and intensity of the electrophoretic bands. They can be quantified by methods known in the art. Multiple assay RNAs can be assayed simultaneously if the protective fragments differ enough that they can be separated by gel electrophoresis. The comparison of the levels of the analytical RNA and the control RNA species expressed at the same level in the cells is used as an internal standard, so that the level of the analytical RNA species can be detected even if the total amount of RNA changes. The procedure for this RNase protection assay is as follows:

RNA from PMBC was purified using the "RNAeasy" kit (Qiagen) according to the manufacturer's instructions. Sets of templates are available from PharMingen (BD Biosciences); a commercially available set of templates from this supplier contains the ability to analyze TNF-a, IL12p35, IP10, IL-1a, IL-1b, IL-6, interferon gamma, and control Substances of the RNA class L32 and GAPDH. This template set contains DNA suitable for the synthesis of appropriate RNA probes for each of the listed genes.

Buffer the probes using the PharMingen In Vitro Transcription Kit provided in the kit. The reaction contained RNase inhibitor; transcription buffer; 50 ng template set; 0.1375 mM each of rGTP, rCTP, rATP; 0.003 mM rUTP; The reaction mixture was incubated at 37°C for 1 hour, then 2 units of RNase-free DNase was added and the reaction was terminated by incubation at 37°C for an additional 30 minutes. The RNA probe synthesized in the above incubation was extracted once with 5.2 mM EDTA, 25 μl Tris-saturated phenol, 25 μL chloroform and 4 μg yeast tRNA, and then extracted again with 50 μL chloroform. 50 μL of 4M ammonium acetate and 250 μL of 100% ice ethanol were added to precipitate the RNA, and after incubation at -80°C for 30 minutes, the preparation was centrifuged at high speed for 30 minutes. Wash the centrifuge tubes in 100% ethanol, remove the ethanol, and resuspend the probes for RNase protection assays.

The RNase protection assay used the probe material prepared above and RNA extracted from PBMC. PBMC RNA was washed in 100% ethanol and quantified by absorbance at 260 nm. RPA was performed according to the protocol in the PharMingen RiboQuant kit. Mix 8 µL of PBMC RNA sample with 2 µL of the probe cover in a thin-walled tube, mix well, cover with mineral oil after a short centrifugation. Then, place the tubes in a 90°C PCR block, cool to 56°C, and incubate for 16 hours. The samples were then cooled to 37°C and RNase A and RNase T1 were added. The mixture was incubated at 30°C for 45 min, and the reaction was terminated with proteinase K and yeast tRNA. RNA was extracted with phenol-chloroform and precipitated from phenol-chloroform with ammonium acetate-ethanol. Vials were washed with ethanol and resuspended in buffer for electrophoresis. Samples were analyzed by gel electrophoresis according to methods well known in the field of molecular biology.

Many analytical methods can be used in the present invention to determine the antiviral activity of the compounds of the present invention, such as cell culture, animal models and human experiments. The assays described herein can be used to measure the growth of viruses over time to characterize the growth of viruses in the presence of compounds of the invention.

In another embodiment, a virus and a compound of the invention are administered to a subject animal susceptible to viral infection. Incidence, severity, duration, viral load, mortality, etc. of infection can be compared to the incidence, severity, duration, viral load, etc. , Mortality for comparison. The antiviral activity of the compounds of the present invention is manifested as a reduction in the incidence, severity, duration, viral load, mortality, etc. of infection in the presence of the compounds of the present invention. In a specific embodiment, the virus and the compound of the invention are administered to the animal subject simultaneously. In another specific embodiment, the virus is administered to the animal subject prior to administration of the compounds of the invention. In another specific embodiment, the compound of the invention is administered to the animal subject prior to administration of the virus.

In another embodiment, determination of viral growth rate can be performed by removing biological fluids/clinical samples (e.g., nasal Exhalation, throat swab, sputum, broncho-alveolar lavage, urine, saliva, blood or serum) and testing for virus levels. In particular embodiments, the analysis of virus growth rate can be carried out after the virus in the sample is grown in cell culture, after growth on growth medium, or after growth in a subject, using any method known in the art. The sample is analyzed for the presence of virus by methods such as, but not limited to, immunoassays (such as ELISA; for a discussion of ELISA see, for example, Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons , Inc., New York, 11.2.1), immunofluorescent staining or immunoblot analysis, using antibodies that immunospecifically recognize the virus to be analyzed or detecting virus-specific nucleic acids (e.g., by Southern blot or RT-PCR analysis, etc.).

In a specific embodiment, the determination of viral titer is carried out by obtaining a biological fluid/clinical sample from infected cells or infected subjects, preparing serial dilutions of the sample, and measuring Infect virus-susceptible monolayer cells (such as primary cells, transformed cell lines, patient tissue samples, etc.) at a dilution of the virus. The number of plaques was then calculated and virus titers were expressed as spot forming units/ml sample.

In a specific embodiment, the growth rate of the virus in the subject can be assessed by the titer of antibodies against the virus in the subject. Antibody serum titers can be determined by any method known in the art, for example, but not limited to, the amount of antibodies or antibody fragments in a serum sample can be quantified by, for example, ELISA.

Alternatively, the in vivo activity of a TLR7 ligand or TLR7 ligand prodrug can be determined by directly administering the compound to test animals and collecting biological fluids (e.g., nasal exhalations, throat swabs, sputum, broncho-alveolar lavage) , urine, saliva, blood or serum) and test the fluid for antiviral activity.

In embodiments where the sample to be analyzed for virus levels is a biological fluid/clinical sample (such as nasal exhalation, throat swab, sputum, broncho-alveolar lavage, urine, saliva, blood or serum), A sample may or may not contain intact cells. Samples from subjects containing intact cells can be processed directly, while isolates that do not contain intact cells can be first passed with or without incubation with susceptible cell lines (e.g., primary cells, transformed cell lines, patient tissue samples, etc.) or growth media (eg, LB broth/agar, YT broth/agar, blood agar, etc.). The cell suspension can be clarified by centrifugation (eg, 300 xg, 5 minutes at room temperature), followed by washing with PBS, pH 7.4 (Ca ⁺⁺ and Mg ⁺⁺ free) under the same conditions. Cell pellets can be resuspended in a small volume of PBS for analysis. Primary clinical isolates containing intact cells can be mixed with PBS and centrifuged at 300 x g for 5 min at room temperature. Mucus is removed from the interface with a sterile dropper tip, and the cell pellet can be washed again with PBS under the same conditions. Cell pellets were then resuspended in a small volume of PBS for analysis.

In another embodiment, a compound of the invention is administered to a human infected with a virus. The susceptibility, severity, duration, viral load, mortality, etc. of a human infected with the virus and administered a compound of the invention can be compared to the susceptibility observed in a human infected with the virus but not administered a compound of the invention or given a placebo , severity, duration, viral load, mortality, etc. The antiviral activity of the compounds of the invention can be shown by the reduction of the susceptibility, severity, duration, viral load, mortality of infection in the presence of the compounds of the invention. Any method known in the art can be used to determine antiviral activity in a subject (eg, those previously described).

Additionally, the in vivo activity of a TLR7 ligand or TLR7 ligand prodrug can be determined by directly administering the compound to an animal or human subject and collecting a biological fluid/clinical sample (e.g., nasal exhalation, throat swab, sputum, broncho- alveolar lavage, urine, saliva, blood or serum), and testing for antiviral activity in biological fluids/clinical samples (eg, adding compounds of the invention to cells in culture in the presence of virus).

The relevant and important features of the present invention have been described above. Those skilled in the art will appreciate that many changes and embodiments can be made. It is therefore to be understood that the appended claims cover all such changes and embodiments.

6. Example

The following examples are for illustration only and do not limit the scope of the invention.

6.1 TLR7 ligand detection

Three classes of TLR7 ligands are known: guanosines, imidazoquinolines and pyrimidines (see Section 5.2). As noted above, known screening methods have identified other TRL7 ligands. For example, the following screening procedure was used to identify adenine analogs and derivatives as TLR ligands, see Tables 1 and 2.

Stable HEK293-hTLR7 cell lines were obtained from Invivogen (San Diego, California) and transfected with selected pNiFty2-Luc, NF-kB-inducible luciferase indicator plasmids (Invivogen) and (dual) stable transfectants . The resulting dual (hTLR7/pNiFty2-Luc) cell lines were functionally assayed for response to loxoribine and exatoribine compared to no drug controls by a fold luciferase induction assay. The C23 line was chosen because of its satisfactory response and sensitivity profile to these (and other) TLR7 agonists. The biological rationale for matching TLR7 to NF-kB activation is widely accepted (for review, see Akira S. et al., Immunol. Lett., 85, 85-95 (2003)), thus, HEK293-TLR-NF-kB Inducible reporter systems are commonly used as standard assays for the analysis of TLR(7) agonists, either in transient or stable systemic modes. See, for example, Hemmi H. et al., Nat. Immunol., 3, 196-200 (2002); Jurk M. et al., Nat. Immunol., 3, 499 (2002); and Lee J. et al., Proc. Natl. Acad .Sci.USA, 100, 6646-51(2003).

For a typical C23 assay, cells are seeded in 96-well culture plates at 6 x ¹⁰⁴ cells/well and treated with various concentrations of compounds 4-24 hours later. After 2-48 hours of exposure, cell monolayers were lysed with passive lysis buffer (Promega) and firefly luciferase assays were performed with luciferase assay reagent (Promega) as described by the manufacturer. Relative luciferase activity is expressed as fold induction compared to no drug control. Induction over two-fold background was considered a true TLR7 agonist, thus showing a statistically significant increase.

Table 1: Exatoribine activates human TLR7 in HEK293 assay

Table 2: Adenine derivatives activate human TLR7 in the HEK293 assay

In Table 1, exatoribine was added to C23 cells and contacted for 48 hours, and then the cells were collected and analyzed for luciferase activity. Each time point was analyzed three times. Data shown represent fold induction compared to no drug control, with standard deviation in parentheses.

In Table 2, adenine derivative 29 was added to C23 cells for 24 hours, and then the cells were collected to analyze luciferase activity. Each time point was analyzed three times. Data shown represent fold induction compared to no drug control, with standard deviation in parentheses.

6.2 Testing TLR7 ligands as anti-HCV agents

Reduced HCV viral load

Exatoribine test drug was provided as a 1 mg/mL sterile saline solution contained in a 50 mL test tube. Exatoribine was administered to humans by intravenous infusion once daily for 7 days at doses of 200, 400, 600 or 800 mg/dose. All doses were given as a constant rate infusion over a 60-minute period, except the 800 mg dose, which was given over an 80-minute period. The flow rate for each dose was as follows: 3.33 mL/min for the 200 mg dose; 6.67 mL/min for the 400 mg dose; 8.33 mL/min for the 500 mg dose; and 10.0 mL/min for the 600 mg and 800 mg doses.

4-12 patients were enrolled in each dose group (200 mg, 400 mg, 600 mg and 800 mg/dose) administered intravenously once daily for 7 days. Before administration, blood was drawn from each patient to evaluate its HCV viral genotype.

For these daily (×7 days) dosing groups, baseline plasma HCV RNA was measured on days 2-7 prior to initiation of daily IV infusions of exatoribine (previous day or pretreatment and 2 pretreatment assays on day 1 average of). See Figure 2. Viral load was determined by branched DNA method (Versant ^™ v3.0bDNAassay, Bayer Diagnostics). For plasma HCV RNA, log-transformed values were used to estimate the maximum change from pretreatment baseline.

Plasma HCV RNA decreased during exatoribine treatment, with larger changes generally occurring in patients receiving higher daily doses (Figure 2). Eight of 12 patients receiving exatoribine 800 mg QD x 7 days showed greater than 0.5 log10 reduction in plasma viral load, with a mean change in 12 patients of -0.76 log10 units and a range of -2.85 to +0.21 log10 unit. This reduction in viral load in the 800 mg QD dose group was statistically significant (p=0.008). The decrease in plasma viral load was usually reversed during cessation of treatment.

Virus biological analysis based on HCV replicons

The HCV replicon has been shown to be highly sensitive to inhibition by interferon-alpha and interferon-gamma. The HCV replicon thus constitutes a very useful system for determining the amount of biologically active interferons in supernatants from human PBMCs stimulated with TLR7 agonists. Quantitative analysis was performed based on measuring the activity of the luciferase indicator gene integrated into the HCV replicon. By using this system, interferon from TLR7 agonist-treated PBMCs was assayed and its inhibitory activity was evaluated with a luciferase indicator replicon.

Human PBMCs isolated from healthy donors were placed in multi-cell culture wells (5×10 ⁶ cells/well). Incubate PBMCs without test compound for 24 hours at 37°C in a humidified environment containing 5% _CO2 to stabilize the culture conditions, then add TLR7 ligand or no drug control to cells containing TLR7 from the same donor. In duplicate wells of PBMC. TLR7 ligand concentrations can be varied to suit a particular assay, and PBMC cultures are then incubated for 8 hours at 37°C in a humidified atmosphere with 5% _CO2 . At the 8 hour time point (or 24 hour time point for loxoribine and its sampling), cell culture supernatants from TLR7 ligand-treated and control wells were sampled and analyzed by ELISA for interferon-α production. The supernatants from compound-treated cells and drug-free controls were diluted 1:10, 1:100, and 1:1000 in RPMI medium, and applied to 96 wells of Huh7 hepatocytes containing luciferase indicator replicons in the culture plate. Cells were incubated for 48 hours at 37°C in a tissue culture incubator.

After incubation, the 96-well plate was washed with 2×PBS and lysed with passive lysis buffer (Promega). Plates were shaken at room temperature for 20 minutes, standard luciferase assay reagent (Promega) was added to each well by injection, and read on an Lmax luminometer (Molecular Devices). Raw relative light units were converted to percent inhibition relative to no drug controls to determine the level of inhibition observed in the replicon assay. The estimated maximum concentration of interferon required to inhibit HCV replicon replication was determined as a 1:10 dilution of the supernatant of stimulated PBMC cells, which fell within the experimental range of serial dilutions. For all TLR7 ligands tested, 100% inhibition of the luciferase indicator replicon system was observed at a 1:10 dilution.

The data shown in Tables 3-8 represent the rate of inhibition of the HCV replicon system when PBMC cells were exposed to initial concentrations of compound for the indicated incubation times and diluted as specified in the first column ("PBMC-exposed compound"). Supernatants collected from PBMC cells, unexposed to compounds and diluted as indicated in the first column were used as controls ("blank supernatant"). PBMC cells were isolated from a single blood donor.

Table 3: Antiviral effect of exatoribine in in vitro HCV replicon bioassay

number 1

Incubation time: 8 hours

Starting concentration: 100 μM

Blood supply number: FL72035

1:1000 0 94

number 2

Incubation time: 8 hours

Starting concentration: 100 μM

Blood supply number: FL75287

number 3

Incubation time: 24 hours

Starting concentration: 100 μM

Blood supply number: FL75864

Table 4: Antiviral effect of loxoribine in in vitro HCV replicon bioassay

number 1

Incubation time: 24 hours

Starting concentration: 100 μM

Blood supply number: FL75864

Table 5: Antiviral effects of imiquimod in the in vitro HCV replicon bioassay

number 1

Incubation time: 8 hours

Starting concentration: 3.2 μM

Blood supply number: FL75287

number 2

Incubation time: 8 hours

Starting concentration: 3.2 μM

Blood supply number: FL75287

Table 6: Antiviral effect of resiquimod in in vitro HCV replicon bioassay

number 1

Incubation time: 8 hours

Starting concentration: 10 μM

Blood supply number: FL75287

number 2

Incubation time: 8 hours

Starting concentration: 10 μM

Blood supply number: FL75287

Table 7: Antiviral effect of bropirimine in in vitro HCV replicon bioassay

number 1

Incubation time: 8 hours

Starting concentration: 100 μM

Blood supply number: FL72035

number 2

Incubation time: 8 hours

Starting concentration: 100 μM

Blood supply number: FL72036

Table 8: Antiviral effects of adenine derivatives in in vitro HCV replicon bioassays

number 1

Incubation time: 8 hours

Starting concentration: 0.1 μM

Blood supply number: FL76418

Compound form: a TFA salt

number 2

Incubation time: 8 hours

Starting concentration: 0.1 μM

Blood supply number: FL76418

Compound form: TFA salt

6.3 Preparation of TLR7 Ligand Prodrug

Compounds of the invention were synthesized according to the methods described in Schemes 1-18 above. All temperatures are in degrees Celsius and all parts and percentages are by weight unless otherwise indicated. Reagents were purchased from commercial suppliers, such as Aldrich Chemical Company or Lancaster Synthesis Ltd., and were used without purification unless otherwise specified. Tetrahydrofuran (THF) and N,N-dimethylformamide (DMF) were purchased from Aldrich in safety-sealed bottles and used directly. Unless otherwise noted, the following solvents and reagents were distilled under dry nitrogen. THF and Et ₂ O were distilled in Na-benzophenone ketyl; CH ₂ Cl ₂ , diisopropylamine, pyridine and Et ₃ N were distilled in CaH ₂ ; in P ₂ O ₅ , This was followed by distillation of MeCN from _CaH2 ; MeOH from Mg; PhMe, EtOAc, and i-PrOAc from _CaH2 ; and purification of TFAA by simple atmospheric distillation under dry argon.

The following reactions were performed in anhydrous solvents under a positive pressure of argon at room temperature (unless otherwise specified) and with reaction flasks secured with rubber septa to facilitate introduction of substrates and reagents by syringe. Oven dry and/or heat dry glassware. Reactions were analyzed by TLC and terminated as judged by consumption of starting material. Analytical thin-layer chromatography (TLC) was performed on aluminum-backed silica gel 60F ₂₅₄ 0.2 mm plates (EM Science), visualized with UV light (254 nm), and then combined with commercially available alcoholic phosphomolybdic acid heating. Preparative thin layer chromatography (TLC) was performed on aluminum backed silica gel 60F ₂₅₄ 1.0 mm plates (EM Science) visualized with UV light (254 nm). HPLC was performed on a Waters Micromass ZQ system comprising a model 2525 binary gradient pump with an Alltech model 800 ELSD detector and a Waters model 996 photo(electrical) diode matrix detector.

Unless otherwise noted, workup was typically performed by doubling the reaction volume with either the reaction solvent or the extraction solvent, followed by washing with the indicated aqueous solution at 25% by volume of the extraction volume. The product solution was dried over anhydrous _Na2SO4 and/or _Mg2SO4 , then filtered and the solvent was _evaporated under reduced pressure on _a rotary evaporator and noted "Solvent removed in vacuo". Use 230-400 mesh silica gel or 50-200 mesh neutral alumina for column chromatography under positive pressure. The hydrogenolysis is carried out at the pressure specified in the examples or at ambient pressure.

¹ H-NMR spectra were recorded on a Varian Mercury-VX400 instrument operating at 400 MHz, while ¹³ C-NMR spectra were recorded at 75 MHz. NMR spectra were obtained from CDCl ₃ solution (in ppm), with chloroform as reference standard (7.27 ppm and 77.00 ppm), CD ₃ OD (3.4 and 4.8 ppm and 49.3 ppm) as appropriate, DMSO-d ₆ , Or internal standard tetramethylsilane (0.00ppm). Other NMR solvents were used as needed. When recording peak multiplicity, use the following abbreviations: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broadened peak), dd (paired doublet), dt (paired triplet). Coupling constants are reported in Hertz (Hz).

Infrared (IR) spectra were recorded on a Thermo Nicolet Avatar 370FT-IR as either neat oils or solids and are reported in wavenumbers (cm ⁻¹ ) when reporting the results obtained. Mass spectra were recorded as (+)-ESThermoFinnegan LCQ LC/MS performed by the Analytical Chemistry Department of Anadys Pharmaceuticals Inc. Elemental analyzes were performed by Atlantic Microlab, Inc. (Norcross, GA) or NuMega in San Diego, CA. Melting point (mp) determinations were made in open capillary devices without correction.

Many common chemical abbreviations are used in the synthetic pathways and experiments described: THF (tetrahydrofuran), DMF (N,N-dimethylformamide), EtOAc (ethyl acetate), DMSO (dimethyl sulfoxide), DMAP (4-dimethylaminopyridine), DBU (1,8-diazocyclo[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-ethyl Carbodiimide hydrochloride), HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), HOBT (1 -Hydroxybenzotriazole hydrate), TFAA (trifluoroacetic anhydride), pyBOP (benzotriazol-1-yloxy)tripyrrolidinyl-hexafluorophosphate), DIEA (diisopropylethylamine )etc.

Example 1: 7-allyl-2-amino-9-β-D-ribofuranosyl-7,9-dihydro-purin-8-one (43)

Step 1: Preparation of 7-allyl-2-amino-9-(2',3',5'-tri-O-acetyl-β-D-nucleofuranosyl)-7,9-dihydro- 1H-purine-6,8-dione (40)

7-Allyl-2-amino-9-β-D-ribofuranosyl-7,9-dihydro-1H-purine-6,8-dione 17 was stirred in anhydrous acetonitrile (25 mL) ( 1.00 g, 2.95 mmol, prepared according to Reitz et al., JMC, 37, 3561-3578 (1994)), a heterogeneous mixture of DMAP (0.036 mg, 0.29 mmol) and _NEt3 (2.05 mL, 14.74 mmol). Acetic anhydride (0.862 mL, 9.13 mmol) was slowly added to the suspension, and the reaction mixture was stirred at room temperature for 16 h. The solvent was removed in vacuo and the residue was dissolved in dichloromethane (DCM). The organic phase was then washed with saturated aqueous sodium bicarbonate (NaHCO ₃ ), brine, and dried over anhydrous magnesium sulfate (MgSO ₄ ). The solvent was concentrated in vacuo and dried under high vacuum at room temperature. Obtained 1.33 g of pale yellow solid 40 (97%): ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.12 (t, J = 6.0 Hz, 1 H), 6.01 (d, J = 3.6 Hz, 1 H), 5.89 (m , 1H), 5.82(t, J=6.0Hz, 1H), 5.39(br s, 2H), 5.21(m, 2H), 4.58(br s, 2H), 4.51(m, 1H), 4.32(m, 2H), 2.16(s, 3H), 2.15(s, 3H), 2.10(s, 3H); MS(+)-ES[M+H] ⁺ 466.2 m/z.

Step 2: Preparation of 7-allyl-2-amino-6-chloro-9-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-7,9 -Dihydro-purin-8-one (41)

Compound 40 (0.65 g, 1.39 mmol) was dissolved in phosphorus oxychloride (10 mL) and heated to 75° C. for 16 h. The reaction mixture was concentrated in vacuo and the crude product was dissolved in DCM. Then, the mixture was washed with _NaHCO3 solution, brine, dried ( _MgSO4 ) and filtered. The filtrate was concentrated in vacuo. Purification by flash chromatography was performed using a gradient of 10-50% ethyl acetate in hexanes. Removal of solvent afforded 280 mg (41%) of the desired product 41: ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.04 (d, J = 4.0 Hz, 1 H), 6.03 (t, J = 5.6 Hz, 1 H), 5.87(m, 1H), 5.86(t, J=5.6Hz, 1H), 5.18(m, 4H), 4.59(d, J=8.0Hz, 2H), 4.45(d, J=7.6Hz, 1H), 4.31 (m, 2H), 2.10 (s, 3H), 2.08 (s, 3H), 2.04 (s, 3H); MS(+)-ES[M+H] ⁺ 484.2 m/z.

Step 3: Preparation of 7-allyl-2-amino-9-(2',3',5'-tri-O-acetyl-β-D-nucleofuranosyl)-7,9-dihydro- Purin-8-one(42)

Compound 41 (0.27 g, 0.56 mmol) was dissolved in acetic acid, and Zn-Cu pair was added to the solution. The mixture was heated to 70 °C for 18 h. Suspended particles were filtered off, and the filtrate was concentrated in vacuo. Purification by flash chromatography was performed using a gradient of 10% to 100% ethyl acetate in hexane. Removal of solvent afforded 150 mg of beige solid (60%) 42: ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.05 (t, J = 4.0 Hz, 1 H), 6.03 (d, J = 4.0 Hz, 1 H), 5.87 (t, J=6.0Hz, 1H), 5.83(m, 1H), 5.48(br s, 2H), 5.33(s, 1H), 5.29(d, J=5.6Hz, 1H), 4.49(d, J =3.2Hz, 1H), 4.46(d, J=3.2Hz, 1H), 4.41(d, J=5.6Hz, 2H), 4.27(m, 2H), 2.12(s, 3H), 2.10(s, 3H ), 2.05(s, 3H); MS(+)-ES[M+H] ⁺ 450.0 m/z.

Step 4: Preparation of 7-allyl-2-amino-9-β-D-ribofuranosyl-7,9-dihydro-purin-8-one (43)

Solid K ₂ CO ₃ (0.024 g, 0.17 mmol) was added to a solution of 42 (0.13 g, 0.29 mmol) in methanol (4 mL), and the reaction solution was stirred at room temperature for 18 h. Amberlite CG-50 (0.5 g) was added to the cloudy liquid, stirred until neutral, and filtered. The filtrate was concentrated to obtain a beige solid, which was washed with water and dried under high vacuum to obtain a certain yield of 93.5 mg of pure 43 as a beige solid: ¹ H NMR (400MHz, d ₆ -DMSO) δ 7.88 (s, 1H), 6.33 (br s, 2H), 5.85(m, 1H), 5.66(d, J=6.0Hz, 1H), 5.30(d, J=5.6Hz, 1H), 5.20(s, 1H), 5.16(d, J=8.4 Hz, 1H), 5.01(d, J=4.8Hz, 1H), 4.89(q, J=5.6Hz, 1H), 4.75(br s, 1H), 4.35(d, J=5.2Hz, 2H), 4.10 (t, J=8.4Hz, 1) 3.80(q, J=3.6Hz, 1H), 3.57(m, 1H), 3.44(m, 1H).MS(+)-ES[M+H] ⁺ 324.1m /z.

Example 2: 7-allyl-2-amino-6-ethoxy-9-β-D-ribofuranosyl-7,9-dihydro-purin-8-one (45)

Step 1: Preparation of 7-allyl-2-amino-6-ethoxy-9-(2',3',5'-tri-O-acetyl-β-D-nucleofuranosyl)-7 , 9-dihydro-purin-8-one (44)

To a solution of 40 (0.30 g, 0.64 mmol) in anhydrous THF (15 mL) was added polymer-supported triphenylphosphine (0.89 g, 1.93 mmol) and EtOH (0.11 mL, 1.93 mmol) at room temperature. To the stirred mixture, diethyl azodicarboxylate (0.12 mL, 0.77 mmol) was added, and stirring was continued for 18 h. The spent polymer support was filtered off and the solvent was removed in vacuo. The residue was then purified by flash chromatography using a gradient of 10-50% ethyl acetate in hexanes. Removal of solvent gave 85 mg (26%) of the desired product 6 as a clear oil: ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.07 (d, J = 4.0 Hz, 1 H), 6.06 (d, J = 4.0 Hz , 1H), 6.01(d, J=3.6Hz, 1H), 5.96(t, J=6.0Hz, 1H), 5.87(m, 1H), 5.14(d, J=2.2Hz, 1H), 5.15(m , 1H), 4.80(br s, 2H), 4.46(m, 4H), 4.37(q, J=7.2Hz, 2H), 4.29(m, 2H), 2.09(s, 3H), 2.08(s, 3H ), 2.04(s, 3H), 1.35(t, J=7.6Hz, 3H); MS(+)-ES[M+H] ⁺ 494.1 m/z.

Step 2: Preparation of 7-allyl-2-amino-6-ethoxy-9-β-D-ribofuranosyl-7,9-dihydro-purin-8-one (45)

To a solution of 44 (0.084 g, 0.17 mmol) in methanol (4 mL), solid K ₂ CO ₃ (0.014 g, 0.17 mmol) was added, and the reaction solution was stirred at room temperature for 1 h. Amberlite CG-50 (0.5 g) was added to the cloudy liquid, stirred until neutral, and filtered. The filtrate was concentrated and purified by flash chromatography using 100% DCM-10% methanol in DCM. Removal of solvent afforded 62 mg of 7 (99%) as a clear oil: ¹ H NMR (400 MHz, CDCl ₃ ) δ 5.97 (d, J = 8.0 Hz, 1 H), 5.93 (m, 1 H), 5.25 (d, J=32.4, Hz, 1H), 5.21(s, 1H), 5.02(t, J=8.0Hz, 1H), 4.62(br s, 2H), 4.47(d, J=5.6Hz, 2H), 4.25- 4.45(m, 3H), 4.21(q, J=6.8Hz, 2H), 3.77(ABq, _ΔυAB =0.14, _JAB =12.4Hz, 2H), 1.37(t, J=6.8Hz, 3H), 1.27 (t, 7.6, 2H); MS(+)-ES[M+H] ⁺ 368.0 m/z.

Example 3: 5-Bromo-4-ethoxy-6-phenyl-pyrimidin-2-ylamine (37)

Step 1: Preparation of 5-bromo-4-ethoxy-6-phenyl-pyrimidin-2-ylamine (37)

In a manner similar to Step 2 in Example 2, the title compound was prepared as a white solid from 2-amino-5-bromo-6-phenyl-3H-pyrimidin-4-one 35 (Wierenga, et al., JMC, 23, 239-240 (1980)), yield 13%: ¹ H NMR (400MHz, CDCl ₃ ) δ7.61 (m, 2H0, 7.42 (m, 3H), 5.15 (br s, 2H), 4.23 (q, J =7.2Hz, 2H), 1.44(t, 6.8Hz, 3H); MS(+)-ES[M] ⁺ 294.1[M+2] ⁺ 296.0m/z. Elemental Analysis C ₁₂ H ₁₂ BrN ₃ O: Calculated Values: C, 49.00; H, 4.11; N, 14.29; Found: C, 48.94; H, 4.18; N, 14.01.

Example 4: 4-(2-amino-5-bromo-6-phenyl-pyrimidin-4-yloxymethyl)-5-methyl-[1,3]dioxol-2-one (dioxol-2-one)(46)

Step 1: Preparation of 4-(2-amino-5-bromo-6-phenyl-pyrimidin-4-yloxymethyl)-5-methyl-[1,3]dioxol-2-one (46)

The title compound was prepared in 4% yield as a white solid from 2-amino-5-bromo-6-phenyl-3H-pyrimidin-4-one 35 in a manner similar to step 2 in Example 2: ¹ H NMR (400MHz, CDCl ₃ )δ7.62(m, 2H), 7.45(m, 3H), 5.18(s, 2H), 5.07(s, 2H), 2.26(s, H); MS(+)-ES[ M] ⁺ 378.2[M+2] ⁺ 380.1 m/z. Elemental Analysis C ₁₅ H ₁₂ BrN ₃ O ₄ : Calculated: C, 47.64; H, 3.20; N, 11.11; Found: C, 46.98; H, 3.23; N, 10.70.

Example 5: 5-Bromo-4-phenyl-pyrimidin-2-ylamine (48)

Step 1: Preparation of 4-phenyl-pyrimidin-2-ylamine (47)

To a solution of bromobenzene (4.43 mL, 42.06 mmol) in anhydrous THF (100 mL) was added BuLi (394 mL, 63.08 mmol) at -78 °C, and the mixture was stirred at -78 °C for 2 h. To this mixture was added a solution of 2-aminopyrimidine (2.0 g, 21.03 mmol) in hot toluene (80 mL) over a period of 15 minutes. The mixture was refluxed for 16 h, cooled to room temperature, and quenched carefully with aqueous NaHCO ₃ . The mixture was filtered, and the filtrate was concentrated in vacuo. The residue was then dissolved in DCM, washed with aqueous NaHCO ₃ , brine, and dried (MgSO ₄ ). Removal of solvent afforded 350 mg of pale yellow solid 47 (10%): ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.32 (d, J = 4.8 Hz, 1H), 7.97 (m, 2H), 7.45 (m, 3H ), 7.02 (J=4.8Hz, 1H).5.27 (br s, 2H); MS(+)-ES[M+H] ⁺ 172.2m/z.

Step 2: Preparation of 5-bromo-4-phenyl-pyrimidin-2-ylamine (48)

Compound 47 (0.30 g, 1.75 mmol) was dissolved in glacial acetic acid (15 mL), heated to 45°C, and Br ₂ (0.09 mL, 1.75 mmol) was added slowly. Then, the resulting mixture was stirred at room temperature for 3 h. The solvent was removed in vacuo to give a solid residue. Then, the residue was transferred to a filter funnel, washed with DCM and then with water. Then, the residual solid was dried under high vacuum for 16 h to obtain 197 mg of pale yellow solid 13 (45%): ¹ H NMR (400 MHz, d ₆ -DMSO) δ8.40 (s, 1H), 7.61 (m, 2H), 7.45 (m, 3H), 6.96(s, 2H); MS(+)-ES[M] ⁺ 250.0[M+2] ⁺ 252.0m/z. Elemental analysis for C ₁₀ H ₈ BrN ₃ : Calculated: C, 48.02 H, 3.22; Br, 31.95; N, 16.80; Found: C, 47.91; H, 3.28; Br, 32.15;

Example 6: (5-Bromo-6-oxo-4-phenyl-1,6-dihydro-pyrimidin-2-yl)-ethyl carbamate (36)

Step 1: Preparation of ethyl (5-bromo-6-oxo-4-phenyl-1,6-dihydro-pyrimidin-2-yl)-carbamate (36)

To a solution of 35 (0.25 g, 0.94 mmol) in DMF (8 mL), _NEt3 (0.14 mL, 0.99 mmol) and diethylpyrocarbonate (0.27 mL, 1.89 mmol) were added. The reaction mixture was kept at 65 °C for 20 h. The solvent was removed and the residue was treated with DCM. The resulting mixture was filtered to remove residual starting material 35 and the filtrate was washed with aqueous _NaHCO3 , brine and dried ( _MgSO4 ). The filtrate was concentrated and purified by HPLC (Thomson ODS-A 100A 5 μl 50×21.2 mm column; flow rate = 30 ml/min; CH ₃ CN with 0.05% TFA (A), water with 0.05% TFA (B); make-up pump flow rate = 0.9 ml/min; supplementary pump mobile phase; MeOH containing 0.05% TFA using the following gradient system: t=0; 15% A, 85% B; t=3.0 min; 15% A, 85% B; t=9.5 min ; 70% A, 30% B; t = 10.0 minutes; 100% A, 0% B; t = 12.0 minutes; 100% A, 0% B; t = 12.5 minutes; 15.0 minutes; 15% A, 85% B.), 54 mg of clear oil 36 (17%) was obtained: ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.66 (m, 1H), 7.44 (m, 3H), 4.26 (q, J=7.6Hz, 2H), 1.32t, J=6.8Hz, 3H); MS(+)-ES[M] ⁺ 338.1[M+2] ⁺ 340.0m/z. Elemental Analysis C ₁₃ H _{12 BrN3O3} : Calculated: C, 46.17; H, 3.58; N, _12.43 ; Found: C, 46.43; H, 3.74; N, 11.95.

Example 7: (5-Bromo-6-oxo-4-phenyl-1,6-dihydro-pyrimidin-2-yl)-amylcarbamate (49)

Step 1: Preparation of (5-Bromo-6-oxo-4-phenyl-1,6-dihydro-pyrimidin-2-yl)-amylcarbamate (49)

The title compound of clear oil was prepared from 35 and dipentyl pyrocarbonate in a manner similar to Step 1 in Example 6, and purified by HPLC (Thomson ODS-A 100A 5 μl 50×21.2 mm column; flow rate=30 ml/min; containing 0.05% _CH3CN in TFA (A), water with 0.05% TFA (B); make-up pump flow rate = 0.9 ml/min; make-up pump mobile phase; MeOH with 0.05% TFA, gradient system as follows: t=0; 35% A, 65% B; t=3.0 minutes; 35% A, 65% B; t=10 minutes; 100% A, 0% B; t=12.0 minutes; 100% A, 0% B; t=12.5 minutes; 35% A, 65% B; t = 15.0 min; 35% A, 65% B.) Yield 9%: ¹ H NMR (400MHz, CDCl ₃ ) δ7.69 (br s, 1H), 7.67 ( m, 2H), 7.43(d, J=2.0Hz, 3H), 4.17(t, J=7.2Hz, 2H), 1.64(t, J=6.8Hz, 2H), 1.34(m, 4H), 0.92( t, J=6.4Hz, 3H); MS(+)-ES[M] ^+380.1 [M+2] ^+382.1 m/z.

Example 8: (1-isobutyl-1H-imidazo[4,5-c]quinolin-4-yl)-amylcarbamate (34)

Step 1: Preparation of (1-isobutyl-1H-imidazo[4,5-c]quinolin-4-yl)-amylcarbamate (34)

1-isobutyl-1H-imidazo[4,5-c]quinolin-4-ylamine 31 (0.15 g, 0.62 mmol, prepared according to the method described in WO94/17043) in _CHCl3 (5 mL) To the suspension, NEt ₃ (0.09 mL, 0.65 mmol) and dipentyl dicarbonate (0.231 g, 0.94 mmol) were added. The mixture was stirred at 40 °C for 60 h. The reaction mixture was washed with aqueous NaHCO ₃ , brine and dried over MgSO ₄ . The filtrate was concentrated and purified by flash chromatography using a gradient of 10%-70% ethyl acetate in hexanes to afford 50.5 m g of white solid 34 (23%): ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.31 (brs, 1H), 8.15(t, J=8.0Hz, 2H), 7.85(t, J=7.2Hz, 1H), 7.77(t, J=8.0Hz, 1H), 4.43(d, J=7.6Hz, 2H) , 4.36(t, J=7.2Hz, 2H), 2.31(m, 1H), 1.75(t, J=6.8Hz, 2H), 1.36(m, 4H), 1.06(d, J=6.4Hz, 6H) , 0.89 (t, J = 6.8 Hz, 2H); MS(+)-ES[M+H] ⁺ 355.3 m/z.

Example 9: (1-isobutyl-1H-imidazo[4,5-c]quinolin-4-yl)-ethyl carbamate (50)

Step 1: Preparation of ethyl (1-isobutyl-1H-imidazo[4,5-c]quinolin-4-yl)-urethane (50) In a manner similar to Step 1 in Example 8, from 31 and diethylpyrocarbonate to prepare the title compound as a white solid in 67% yield: ¹ H NMR (400MHz, CDCl ₃ ) δ9.32 (br s, 2H), 8.2 (d, J=8.0Hz, 2H) 8.12 (d, J=8.0Hz, 1H), 7.83(t, J=7.2Hz, 1H), 7.74(t, J=8.0Hz, 1H), 4.43(m, 4H), 2.35(m, 1H), 1.39( t, J=7.2Hz, 3H), 1.08 (d, J=6.4Hz, 6H); MS(+)-ES[M+H] ⁺ 313.2m/z.

Example 10: Ethyl 6-amino-9-benzyl-2-(2-methoxy-ethoxy)-9H-purin-8-yl carbonate (51)

Step 1: Preparation of ethyl 6-amino-9-benzyl-2-(2-methoxy-ethoxy)-9H-purin-8-yl carbonate (51)

6-Amino-9-benzyl-2-(2-methoxy-ethoxy)-9H-purin-8-ol, 29 (11.75 mg, 0.027 mmol, according to Kurimota, et al., Bioorg.Med. Chem., 12, 1091-109 (2004)) was suspended in CH ₂ Cl ₂ (0.6 mL) and cooled to 0°C. Then DIEA (11.96 μL, 0.068 mmol) and ethyl chloroformate (3.86 mg, 0.036 mmol, added as a 10% by volume solution in dichloromethane) were added to the suspension. The reaction mixture was stirred at 0°C for 10 minutes, then warmed to room temperature for 15 minutes. TLC of the reaction mixture showed the presence of starting material. The reaction mixture was heated to 35 °C and DMAP (catalytic amount), methanol (60 μL portion) was added to dissolve 29, followed by ethyl chloroformate (3.86 mg, 0.036 mmol, added as a 10 vol% solution in dichloromethane) until the reaction is complete. The crude mixture was purified by flash chromatography using a gradient of 0-100% ethyl acetate in hexanes. The desired peaks were collected and concentrated in vacuo to give 8.5 mg (80%) of compound 51 as a white solid: ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.45 (d, J=Hz, 2H), 7.27 (m, 3H), 4.98 (s, 2H), 4.46 (m, 4H), 3.74 (m, 2H), 3.42 (s, 2H), 1.46 (t, 3H); MS [M+H]+m/z 388.3.

Embodiment 11-20 is according to the method described in embodiment 10, from 6-amino-9-benzyl-2-(2-methoxy-ethoxy)-9H-purin-8-alcohol, 29 and suitable Prepared from chloroformate.

Example 11: 6-Amino-9-benzyl-2-(2-methoxy-ethoxy)-9H-purin-8-yl ester propyl carbonate (52)

White solid in 86% yield: ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.45 (d, J = 6.4 Hz, 2H), 7.27 (m, 3H), 4.98 (s, 2H), 4.46 (m, 4H ), 3.74 (t, J = 5.2 Hz, 2H), 3.42 (s, 3H), 1.46 (t, J = 7.6 Hz, 3H); MS [M+H] ⁺ m/z 402.2.

Example 12: 6-Amino-9-benzyl-2-(2-methoxy-ethoxy)-9H-purin-8-yl ester isobutyl carbonate (53)

White solid in 92% yield: ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.45 (d, J = 6.4 Hz 2H), 7.27 (m, 3H), 4.99 (s, 2H), 4.45 (m, 2H), 4.17(d, J=7.4Hz, 2H), 3.73(t, J=4.8Hz, 2H), 3.4(s, 3H), 2.15(m, 1H), 1.06(d, J=6.8Hz, 6H); MS [M+H]+m/z 416.3.

Example 13: 6-Amino-9-benzyl-2-(2-methoxy-ethoxy)-9H-purin-8-yl ester pentyl carbonate (54)

White solid in 92% yield: ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.45 (d, J = 6.4 Hz, 2H), 7.27 (m, 3H), 4.99 (s, 2H), 4.45 (t, J =4.8Hz, 2H), 4.39(t, J=7.2Hz, 2H), 3.73(t, J=5.2Hz, 2H), 3.4(s, 3H), 2.15(m, 1H), 1.82(m, 2H ), 1.40 (m, 4H), 0.93 (t, J=6.8Hz, 3H); MS [M+H]+m/z 430.2.

Example 14: Allyl carbonate 6-amino-9-benzyl-2-(2-methoxy-ethoxy)-9H-purin-8-yl ester (55)

White solid in 93% yield: ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.46 (d, J = 6.0 Hz, 2H), 7.25 (m, 3H), 6.0 (m, 1H), 5.5 (m, 1H ), 5.35(m, 1H), 4.99(s, 2H), 4.89(d, J=2.4Hz, 2H), 4.46(t, J=4.8Hz, 2H), 3.74(d, J=5.2Hz, 2H ), 3.42 (s, 3H); MS [M+H]+m/z 400.2.

Example 15: 6-Amino-9-benzyl-2-(2-methoxy-ethoxy)-9H-purin-8-yl carbonate 4-chloro-butyl ester (56)

White solid in 98% yield: 1H ¹ H NMR (400MHz, CDCl ₃ ) δ7.44(d, J=6.0Hz, 2H), 7.25(m, 3H), 4.98(s, 2H), 4.45(m, 4H), 3.63 (t, J = 5.2 Hz, 2H), 3.42 (s, 3H), 1.99 (m, 4H); MS [M+H]+m/z 450.2.

Example 16: Butyl 6-amino-9-benzyl-2-(2-methoxy-ethoxy)-9H-purin-8-yl carbonate (57)

100% yield of white solid: ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.45 (d, J = 6.0 Hz, 2H), 7.27 (m, 3H), 4.99 (s, 2H), 4.46 (m, 2H ), 4.41(t, J=4.4Hz, 2H), 3.73(t, J=7.2Hz, 2H), 3.42(s, 3H), 1.79(m, 2H), 1.48(m, 2H), 0.96(t , J=7.6Hz, 3H); MS [M+H]+m/z 416.2.

Example 17: 6-Amino-9-benzyl-2-(2-methoxy-ethoxy)-9H-purin-8-yl ester phenyl carbonate (58)

White solid in 100% yield: ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.52 (d, J = 6.8 Hz, 2H), 7.41 (m, 2H), 7.30 (m, 3H), 7.25 (m, 3H ), 4.99(s, 2H), 4.47(t, J=4.8Hz, 2H), 3.75(t, J=4.8Hz, 2H), 3.43(s, 3H); MS[M+H]+m/z 436.2.

Example 18: 6-Amino-9-benzyl-2-(2-methoxy-ethoxy)-9H-purin-8-yl carbonate 2,2-dimethyl-propyl ester (59)

100% yield of white solid: ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.43 (d, 2H), 7.25 (m, 3H), 4.99 (s, 2H), 4.47 (t, 2H), 4.08 (s , 2H), 3.75(t, 2H), 3.42(s, 3H), 1.07(s, 9H); MS[M+H]+m/z 430.2.

Example 19: 6-Amino-9-benzyl-2-(2-methoxy-ethoxy)-9H-purin-8-yl ester heptyl carbonate (60)

White solid in 100% yield: ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.45 (d, J = 6.0 Hz, 2H), 7.27 (m, 3H), 4.99 (s, 2H), 4.46 (t, J =5.2Hz, 2H), 4.39(t, J=7.2Hz, 2H), 3.74(t, J=5.2Hz, 2H), 3.42(s, 3H), 1.8(m, 2H), 1.4(m, 2H ), 1.3(m, 6H)0.87(t, J=7.2Hz, 3H); MS[M+H]+m/z 458.3.

Example 20: 6-amino-9-benzyl-2-(2-methoxy-ethoxy)-9H-purin-8-yl ester hexyl carbonate (30)

White solid in 74% yield: ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.45 (d, J = 5.6 Hz, 2H), 7.27 (m, 3H), 4.98 (s, 2H), 4.45 (t, J =4.8Hz, 2H), 4.41(t, J=6.8Hz, 2H), 3.73(t, J=4.4Hz, 2H), 3.42(s, 3H), 1.81(m, 2H), 1.34(m, 2H ), 1.31 (m, 2H), 1.26 (m, 2H), 0.89 (t, J=2Hz, 3H); MS [M+H]+m/z 444.4.

Examples 22 and 23 were obtained from 9-benzyl-2-(2-methoxy-ethoxy)-9H-purin-6-ylamine, 62, via 9-benzyl-8-bromo-2-( 2-methoxy-ethoxy)-9H-purin-6-ylamine, 63 and sodium ethoxide or sodium methoxide, each of which was prepared according to Kurimota et al., Bioorg.Med.Chem., 12, 1091-1099 ( 2004) prepared by the method described.

Example 21: 9-Benzyl-2-(2-methoxy-ethoxy)-9H-purin-6-ylamine (62)

Brown solid in 100% yield: ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.59 (s, 1H), 7.27 (m, 5H), 5.83 (s, 2H), 5.26 (s, 2H), 4.49 (t , J = 4.8 Hz, 2H), 3.75 (t, J = 5.2 Hz, 2H), 3.43 (s, 3H); MS [M+H] ⁺ m/z 300.2.

Example 22: 9-Benzyl-8-ethoxy-2-(2-methoxy-ethoxy)-9H-purin-6-ylamine (64)

Brown solid in 91% yield: ¹ H NMR (400 MHz, d ₆ -DMSO) δ 7.24 (m, 2H), 7.24 (m, 3H), 5.00 (s, 2H), 4.42 (m, 2H), 4.27 (t, J=4.8Hz, 2H), 3.58(t, J=4.8Hz, 2H), 3.26(s, 3H), 1.32(t, J=7.2Hz, 3H); MS[M+H] ⁺ m /z344.1.

Example 23: 9-Benzyl-8-methoxy-2-(2-methoxy-ethoxy)-9H-purin-6-ylamine (65)

Brown solid in 91% yield: ¹ H NMR (400 MHz, d ₆ -DMSO) δ 7.28 (m, 2H), 7.22 (m, 3H), 6.86 (s, 2H), 5.01 (s, 2H), 4.26 (t, J = 4.4 Hz, 2H), 4.02 (s, 3H), 3.58 (t, J = 4.8 Hz, 2H), 3.25 (s, 3H); MS [M+H] ⁺ m/z 330.2.

Example 24: 7-allyl-2-amino-9-(5'-O-L-valyl-β-D-ribofuranosyl)-7,9-dihydro-1H-purine-6,8- Diketone(68)

Step 1: Preparation of 7-allyl-2-amino-9-(2',3'-O-isopropylidene-β-D-ribofuranosyl)-7,9-dihydro-1H-purine -6,8-dione(66)

Compound 17 (0.17 g, 0.49 mmol) was dissolved in DMF (4.0 mL), and acetone (3.0 mL) was added to the solution. 2,2-Dimethoxypropane (0.18 mL, 1.47 mmol) and MeSO ₃ H (0.02 mL, 0.05 mmol) were added to the mixture. The reaction mixture was stirred with saturated aqueous NaHCO ₃ for 20 h at room temperature. _The aqueous phase was then extracted with _CH2Cl2 (4x). The combined organic phases were dried (MgSO ₄ ), filtered, and concentrated in vacuo to afford 130 m g of white solid 66 in 70% yield: ¹ H NMR (400 MHz, CD ₃ OD) δ 5.97 (d, J = 2.4 Hz, 1 H ), 5.93(m, 1H), 5.34(dd, J=4.4, 2.0Hz, 1H), 5.15(m, 1H), 5.12(dd, J=7.6, 1.2Hz, 1H), 4.98(m, 1H) , 4.52(d, J=5.6, 2H), 4.16(m, 1H), 3.71(m, 2H), 1.56(s, 3H), 1.36(s, 3H); MS(+)-ES[M+H ] ⁺ 380.0m/z.

Step 2: Preparation of 7-allyl-2-amino-9-(2',3'-O-isopropylidene-5'-N-tert-butoxycarbonyl-L-valeryl)-β-D- ribofuranosyl)-7,9-dihydro-1H-purine-6,8-dione (67)

66 (0.13 g, 0.34 mmol), BOC-valine (0.08 g, 0.36 mmol), EDC (0.07 g, 0.38 mmol), DMAP (0.05 g, 0.38 mmol) in THF (8.0 mL) The mixture with pyridine (0.8 mL) was stirred at room temperature under N ₂ for 16 h. The solvent was removed in vacuo, and the residue was dissolved in EtOAc. The organic phase was washed with saturated aqueous NaHCO ₃ , brine, dried (MgSO ₄ ) and filtered. The filtrate was concentrated in vacuo and purified by flash chromatography using a gradient of 2%-5% MeOH in _CH2Cl2 to afford 180 mg of 67 (91%) as _a light yellow solid: ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.07 (s , 1H), 5.80(m, 1H), 5.56(br s, 2H), 5.41(d, J=5.6Hz, 1H), 5.15(m, 3H), 4.97(br s, 1H), 4.53(br s , 2H), 4.46(m, 1H), 4.32(m, 2H), 4.20(m, 1H), 2.11(m, 1H), 1.56(s, 3H), 1.44(s, 9H), 1.36(s, 3H), 0.93 (d, J = 6.8 Hz, 3H), 0.84 (d, J = 6.8 Hz, 3H); MS(+)-ES[M] ⁺ 578.9 m/z.

Step 3: 7-allyl-2-amino-9-(5'-O-L-valyl-β-D-ribofuranosyl)-7,9-dihydro-1H-purine-6,8-di Ketones(68)

To a solution of 67 (0.18 g, 0.311 mmol) in MeOH (10 mL) was added AcCl (0.86 mL, 12.07 mmol) under _N2 . After the reaction mixture was stirred at room temperature for 18 h, it was carefully neutralized with saturated aqueous NaHCO ₃ . Silica gel was added to the mixture, and concentrated in vacuo. The residue was purified by flash chromatography using a gradient of 10%-20% MeOH in _CH2Cl2 to afford 80 m g of white solid 68 (59% ₎ : ¹ H NMR (400 MHz, d ₆ -DMSO) δ 6.72 (br s, 2H), 5.84(m, 1H), 5.59(d, J=4.8Hz, 1H), 5.43(d, J=5.6Hz, 1H), 5.15(brs, 1H), 5.07(d, J=12Hz, 1H ), 5.0(d, J=18.8Hz, 1H), 4.77(q, J=4.8Hz, 1H), 4.36(m, 3H), 4.26(t, J=4.4Hz, 1H), 4.20(m, 1H ), 3.93(m, 1H), 3.57(br s, 1H), 2.01(m, 1H), 0.86(d, J=4.4Hz, 3H), 0.85(d, J=5.2Hz, 3H); MS( +)-ES[M+H] ⁺ 439.1m/z.

Example 25: 7-allyl-2-amino-9-β-D-ribofuranosyl-6-(5-methyl-2-oxo-[1,3]dioxol-4 -yl (dioxolyl)methoxy)-7,9-dihydro-purin-8-one (71)

Step 1: Preparation of 7-allyl-2-amino-9-(2',3',5'-tri-O-triethylsilyl-β-D-ribofuranosyl)-7,9 -Dihydro-1H-purine-6,8-dione (69)

To a solution of 17 (0.46 g, 1.36 mmol) and imidazole (0.93 g, 13.64 mol) in DMF (13 mL), chlorotriethylsilane (0.92 mL, 5.46 mmol) was added dropwise and stirred at room temperature 2.5h. The reaction mixture was treated with saturated aqueous NaHCO ₃ and the resulting two phases were separated. The aqueous phase was washed with diethyl ether (2x). The organic phases were combined and washed with water, dried ( _MgSO4 ), filtered and concentrated. Purification by flash chromatography using a 2%-10% gradient of MeOH in _CH2Cl2 gave 830 mg of 69 (89%) as _a pale yellow oil: ¹ H NMR (400 MHz, CDCl ₃ ) δ 5.92 (m, 1H) , 5.85(d, J=6.8Hz, 1H), 5.3(m, 1H), 5.15-5.23(m, 2H), 5.09(br s, 2H), 4.54(d, J=4.4Hz, 2H), 4.33 (m, 1H), 3.98(m, 1H), 3.67-3.80(m, 2H), 0.85-1.02(m, 26H), 0.48-0.71(m, 19H): MS(+)-ES[M] ⁺ 682.6m/z.

Step 2: Preparation of 7-allyl-2-amino-9-(2',3',5'-tri-O-triethylsilyl-β-D-ribofuranosyl)-6-( 5-methyl-2-oxo-[1,3]dioxol-4-ylmethoxy)-7,9-dihydro-purin-8-one (70)

In a manner similar to step 1 of Example 2, from compound 69 and 4-hydroxymethyl-5-methyl-[1,3]dioxol-2-one (according to Alepegiani, Syn.Comm., 22(9), prepared by the method described in 1277-82 (1992)) to prepare compound 70 with a yield of 5%, HPLC purification (Thomson ODS-A 100A 5u50×21.2mm column; flow rate=30 ml/min; containing 0.05% TFA CH ₃ CN (A), water containing 0.05% TFA (B); make-up pump flow rate = 1.0 ml/min; make-up pump mobile phase; MeOH containing 0.05% TFA, the gradient system is as follows: t=0; 50% A , 50% B; t=2.0 minutes; 50% A, 50% B; t=5.0 minutes; 100% A, 0% B; t=9.5 minutes; 100% A, 0% B; t=10.0 minutes; 50 %A, 50%B; t=13.0 min; 50%A, 50%B). White solid obtained after purification: MS(+)-ES[M] ⁺ 794.1 m/z.

Step 3: Preparation of 7-allyl-2-amino-9-β-D-ribofuranosyl-6-(5-methyl-2-oxo-[1,3]dioxol-4 -ylmethoxy)-7,9-dihydro-purin-8-one (71)

To a solution of 70 (9.0 mg, 0.012 mmol) in MeOH (1.5 mL), 3HF·NEt ₃ (0.01 mL, 0.07 mmol) was added and stirred at room temperature for 16 h. The solvent was removed in vacuo and the residue was purified by flash chromatography. The desired product was eluted with a gradient of 2%-5% _MeOH in _CH2Cl2 to afford 3.26 m g of white solid 71 (64%): ¹ H NMR (400 MHz, CDCl ₃ ) 5.96 (d, J = 7.6 Hz, 1H), 5.85(m, 1H), 5.17(m, 4H), 4.96(t, J=7.2Hz, 1H), 4.47(d, J=6.0Hz, 2H), 4.25(J=5.2Hz, 1H) , 4.24(s, 1H), 3.81(ABq, ΔυAB=0.17, JAB=11.6Hz, 2H), 2.22(s, 3H): MS(+)-ES[M+H] ⁺ 452.4m/z.

Example 26: (7-allyl-2-amino-9-β-D-ribofuranosyl-8-oxo-8,9-dihydro-7H-purin-6-yloxymethyl)-methanol Base-urethane (73)

Step 1: Preparation of (7-allyl-2-amino-9-(2'3'5'-tri-O-triethylsilyl-β-D-ribofuranosyl)-8-oxo- 8,9-Dihydro-7H-purin-6-yloxymethyl)-methyl-urethane (72)

Compound 72 was prepared from compound 69 and N-methyl-N-(hydroxymethyl)urethane (Kelper, JOC, 52, 1987, p.453-455) in a manner similar to Example 2, step 1, Yield 4%, white solid after HPLC purification: ¹ H NMR (400MHz, CDCl ₃ ) δ 5.94 (m, 1H), 5.82 (d, J=6.4Hz, 1H), 5.53 (br s, 1H), 5.23 -5.29(m, 2H), 5.15(t, J=9.6Hz, 1H), 4.57(d, J=5.2Hz, 2H), 4.35(br s, 1H), 4.21(m, 2H), 3.96(br s, 1H), 3.73(m, 2H), 3.06(s, 3H), 1.30(m, 4H), 0.87-1.01(m, 24H), 0.57-0.68(m, 19H): MS(+)-ES [M+H] ⁺ 797.7m/z.

Step 2: Preparation of (7-allyl-2-amino-9-β-D-ribofuranosyl-8-oxo-8,9-dihydro-7H-purin-6-yloxymethyl)-methanol Base-urethane (73)

In a manner similar to step 3 in Example 25, compound 73 was prepared in a 64% yield and purified by HPLC (Thomson ODS-A 100A 5 μ 50×21.2 mm column; flow rate=30 ml/min; CH ₃ CN containing 0.05% TFA (A), water containing 0.05% TFA (B); make-up pump flow rate = 1.0 ml/min; make-up pump mobile phase; MeOH containing 0.05% TFA, the gradient system is as follows: t=0; 50%A, 50%B ; t=2.0 minutes; 50% A, 50% B; t=5.0 minutes; 100% A, 0% B; t=9.5 minutes; %B; t = 13.0 min; 50% A, 50% B) followed by a white solid. ¹ H NMR (400MHz, CDCl ₃ ) δ5.92(m, 1H), 5.92(d, J=7.6Hz, 1H), 5.51(m, 2H), 5.22(d, J=15.6Hz, 1H), 5.16 (d, J=9.2Hz, 1H), 4.92(t, J=7.2Hz, 1H), 4.57(d, J=6.0, 2H), 4.40(d, J=6.0, 1H), 4.21(m, 3H ), 3.79(ABq, ΔυAB=0.178, JAB=14.0Hz, 2H), 3.06(s, 3H), 1.31(t, J=7.2Hz, 3H): MS(+)-ES[M] ⁺ 455.4m/ z.

Example 27: 5-Amino-3-(5'-O-L-valyl-β-D-ribofuranosyl)thiazolo[4,5-d]pyrimidine-2,7-dione dihydrochloride (24)

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

In a 250 mL Morton flask, a heterogeneous mixture of acetone (40 mL) in 21 (5.37 g, 17.0 mmol, prepared according to the method described in U.S. Patent 5,041,426 (Example 2), the contents of which are incorporated herein by reference) 2,2-DMP (6.26 mL, 50.9 mmol), DMSO (6.6 mL) and MeSO ₃ H (220 μL, 3.39 mmol) were added successively at room temperature. The reaction mixture was stirred vigorously and became homogeneous and golden as the diketone was consumed. TLC analysis ( _SiO2 , 10% MeOH- _CHCl3 ) indicated complete reaction after 6 hours. Undissolved solids were gravity filtered using fluted Whatman type 1 filter paper. Then, the filtrate was poured into 10 volumes of ice water (-400 mL), allowing the immediate precipitation of a white solid. After stirring briefly, _NaHCO3 (285 mg, 3.39 mmol) dissolved in water (10 mL) was added to neutralize the _MeSO3H . Vigorous stirring was continued in the Morton reactor for 15 minutes and the mixture was filtered through a coarse sintered peeling funnel. The solid material was washed with ice water (100 mL), air dried, then further dried under high vacuum at 65 °C to give 5.36 g (88%) of ketal 22 as a white solid: mp 280-81 °C; ¹ H(DMSO-d ₆ ) 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: Preparation of 5-amino-3-(2',3'-O-isopropylidene-5'-N-tert-butoxycarbonyl-L-valeryl)-β-D-ribofuranosyl) -Thiazolo[4,5-d]pyrimidine-2,7-dione (23)

To a solution of N-butoxycarbonyl-(L)-valine (671 mg, 2.81 mmol) in THF (9 mL) at 0°C was added EDC (588 mg, 3.07 mmol). After the resulting homogeneous mixture was stirred at 0°C for 45 min, the mixture became heterogeneous and a portion of the solid acetonide 2 from step 1 above (1.00 g, 2.81 mmol) was added. Then solid DMAP (522 mg, 4.27 mmol) was added. The reaction mixture was returned to room temperature, stirred for another 5 hours, and concentrated by rotary evaporation at 25° C. to obtain a yellow syrup. The residue was dissolved in EtOAc (50 mL), partitioned with 1N HCl (10 mL), and the acid was neutralized with saturated aqueous NaHCO ₃ (10 mL). The acidic aqueous phase was further extracted with EtOAc (2 x 50 mL), then partitioned with basic aqueous phase. The combined organic phases were dried over _Na2SO4 , filtered through a short plug of _SiO2 and concentrated to give 1.480 g of Boc _- protected amino acid ester 23 as a foam (96%): melting point (mp) 158 °C (dec); ¹ H (CDCl ₃ ) 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(brs , 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: Preparation of 5-amino-3-(5'-O-L-valyl-β-D-ribofuranosyl)thiazolo[4,5-d]pyrimidine-2,7-dione dihydrochloride (24)

After HCl gas passed through the concentrated _H2SO4 bubbler, it was introduced into a 250 _mL three-necked Morton flask containing anhydrous isopropyl acetate (80 mL) at 0 °C (through a glass separation tube) until a saturated solution was obtained. To this solution, a solution of Boc-amino acid ester (5.53 g, 9.95 mmol) from step 2 above in isopropyl acetate (30 mL) was added and a white solid precipitated within 5 minutes. 10% (v/v) IPA (11 mL) was added. The reaction mixture was warmed to room temperature and stirred for an additional 12 hours. The heterogeneous reaction mixture was diluted with anhydrous toluene (100 mL). Filtration with a medium pore scintered glass funnel under _N2 afforded a beige amorphous solid. The solid was triturated in anhydrous THF, filtered, and dried under vacuum at 65°C to afford 3.677 g of the title compound 24 as a white solid (81%): mp 166-68°C (dec); ¹ H(DMSO-d ₆ ) 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 O ₇ S 2HCl: Calculated: 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 28: 5-Acetylamino-3-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)thiazolo[4,5-d]pyrimidine-2 , 7(6H)-diketone (74)

Step 1: Preparation of 5-acetylamino-3-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)thiazolo[4,5-d]pyrimidine-2 , 7(6H)-diketone (74)

Anhydrous 21 (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 added sequentially. After 2 h at room temperature, the reaction was quenched with saturated aqueous NaHCO ₃ (100 mL), extracted with DCM (3×200 mL). The organic phase was concentrated and triturated with ether. 12.5 g (103%) of 5-acetylamino-3-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)thiazolo-[4, 5-d] Pyrimidine-2,7(6H)-dione 74: mp 246.7-248.1 °C; R _f =0.20 (SiO ₂ , 50% EtOAc-CHCl ₃ ); ¹ H NMR (400 MHz, d ₆ -DMSO) 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).

Example 29: 5-Amino-3-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)thiazolo[4,5-d]pyrimidine-2,7 (6H)-Diketone (75)

Step 1: Preparation of 5-amino-3-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)thiazolo[4,5-d]pyrimidine-2,7 (6H)-Diketone (75)

To a suspension of 21 (5.00 g, 15.8 mmol) in acetonitrile (160 mL) at 0°C, Et ₃ N (11.0 mL, 79.0 mmol), DMAP (195 mg, 1.59 mmol) and Ac ₂ O (4.47 mL, 47.4 mmol). After stirring the reaction mixture at room temperature for 2 hours, it was concentrated to give a brown syrup. The residue was purified by flash column chromatography (silica, MeOH/CHCl ₃ =1-10%) to give 6.22 g (89%) of the triacetate salt of 75 as a white solid: mp 198-199 °C; ¹ H (400 MHz , d ₆ -DMSO). 11.34 (s, 1H), 7.02 (br s, 2H), 5.90 (m, 2H), 5.51 (t, J=6.0Hz, 1H), 4.36 (dd, J=12.4, 3.2 Hz, 1H), 4.21(m, 1H), 4.08(q, J=6.0Hz, 1H), 2.06(s, 3H), 2.06(s, 3H), 2.00(s, 3H); MS(+)- ES [M+H] ⁺ m/z 443.3.

Example 30: 5-Amino-7-ethoxy-3-β-D-ribofuranosyl-thiazolo[4,5-d]pyrimidin-2-one (77)

Step 1: Preparation of 5-acetylamino-7-ethoxy-3-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-thiazolo[4, 5-d]pyrimidin-2-one (76)

In a manner similar to Example 2, Step 1, a white foam 76 was prepared from 74 and ethanol in 72% yield: MS(+)-ES[M+H] ⁺ m/z 513. R _f =0.45 (75% acetic acid ethyl ester-CHCl ₃ ).

Step 2: Preparation of 5-amino-7-ethoxy-3-β-D-ribofuranosyl-thiazolo[4,5-d]pyrimidin-2-one (77)

In a similar manner to Example 2, step 2, the title compound was prepared as a white solid from 76 in 65% yield: ¹ H NMR (400 MHz, d ₆ -DMSO) 6.87 (s, 2H), 5.85 (d, J= 4.8Hz, 1H), 5.27(d, J=5.6Hz, 1H), 4.96(d, J=5.2Hz, 1H), 4.78(m, 1H), 4.66(m, 1H), 4.36(m, 2H) , 4.09(m, 1H), 3.74(m, 1H), 3.58(m, 1H), 3.40(m, 1H), 1.29(m, 3H); MS(+)-ES[M+H] ⁺ m/ z 445, [2M+H] ⁺ m/z 689. R _f = 0.2 (50% THF-CHCl ₃ ). Elemental analysis for C ₁₂ H ₁₆ N ₄ O ₆ S·0.25 H ₂ O: Calculated: C, 41.31; H , 4.77; N, 16.06; S, 9.19. Found: C, 41.24; H, 4.71; N, 15.89; S, 9.06.

Example 31: 5-Amino-7-methoxy-3-β-D-ribofuranosyl-thiazolo[4,5-d]pyrimidin-2-one (79)

Step 1: Preparation of 5-acetylamino-7-methoxy-3-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-thiazolo[4, 5-d]pyrimidin-2-one (78)

In a manner similar to Example 2, step 1, 77 was prepared as a white foam from 74 and methanol in 65% yield: MS(+)-ES [M+H] ⁺ 499. _Rf = 0.5 (75% ethyl acetate ester - CHCl ₃ ).

Step 2: Preparation of 5-amino-7-methoxy-3-β-D-ribofuranosyl-thiazolo[4,5-d]pyrimidin-2-one (79)

In a similar manner to Example 2, Step 2, the title compound was prepared as a white solid from 78 in 78% yield: ¹ H NMR (400 MHz, d ₆ -DMSO) 6.91 (s, 2H), 5.86 (d, J= 5.2Hz, 1H), 5.28(d, J=5.2Hz, 1H), 4.96(d, J=5.2Hz, 1H), 4.77(m, 1H), 4.66(m, 1H), 4.09(m, 1H) , 3.90(s, 3H), 3.75(m, 1H), 3.56(m, 1H), 3.43(m, 1H); MS(+)-ES[M+H] ⁺ 331.R _f =0.2(50% THF-CHCl ₃ ). Elemental Analysis C ₁₁ H ₁₄ N ₄ O ₆ S·0.25H ₂ O: Calculated: C, 39.46; H, 4.37; N, 16.73; S, 9.58. Found: C, 39.59; H , 4.17; N, 16.55; S, 9.52.

Example 32: (5-Amino-2-oxo-3-β-D-ribofuranosyl-2,3-dihydro-thiazolo[4,5-d]pyrimidin-7-yloxymethyl)- Urethane(82)

Step 1: Preparation of 5-amino-3-(2',3',5'-tri-O-triethylsilyl-β-D-ribofuranosyl)-thiazolo[4,5-d] Pyrimidine-2,7-dione(80)

To a suspension of 21 (1.00 g, 3.16 mmol) in DMF (20 mL) at room temperature, imidazole (753 mg, 11.06 mmol), DMAP (39 mg, 0.32 mmol) and chlorotriethyl Monosilane (1.64 mL, 9.80 mmol). After the reaction mixture was stirred at room temperature for 2 hours, it was quenched with saturated aqueous NaHCO ₃ (20 mL). The mixture was extracted with _CHCl3 (3 x 20 mL), dried over _MgSO4 and concentrated. The residue was purified by flash column chromatography (silica, MeOH/CHCl ₃ =1-5%) to obtain 1.91 g of compound 80 as a white solid (92%): ¹ H (400 MHz, d ₆ -DMSO).5.99( s, 1H), 5.62 (br s, 2H), 5.19 (dd, J=4.4, 6.0Hz, 1H), 4.35 (dd, J=2.8, 4.4Hz, 1H), 3.99 (m, 1H), 3.77 ( dd, J=7.6, 10.8Hz, 1H), 3.68(dd, J=4.8, 10.4Hz, 1H), 1.10(t, J=7.1Hz, 3H), 0.96(t, J=7.1Hz, 3H), 0.89(t, J=7.1Hz, 3H), 0.68(q, J=7.1Hz, 2H), 0.61(q, J=7.1Hz, 2H), 0.54(m, 2H); MS(+)-ES[ M+H] ⁺ m/z 660.0.

Step 2: Preparation of 5-amino-3-(2',3',5'-tri-O-triethylsilyl-β-D-ribofuranosyl)-2,3-dihydro-thiazolo [4,5-d]pyrimidin-7-yloxymethyl)-urethane (81)

In a manner similar to step 1 in Example 2, compound 81 was prepared as a white solid from 80 and N-ethylcarbamate, yield 31%: [M+H] ⁺ 760.5; ¹ H NMR (400MHz, CDCl ₃ ) δ6.43(br s, 2H), 6.09(t, J=7.6Hz, 1H), 5.94(d, J=6.0Hz, 1H), 5.31(d, J=4.8Hz, 2H), 5.19( dd, J=6.0, 4.8Hz, 1H), 4.35(dd, J=4.8, 2.8Hz, 1H), 4.19(q, J=6.4Hz, 2H), 3.98(m, 1H), 3.76(dd, J =10.8, 7.6Hz, 1H), 3.68(dd, J=10.4, 4.8Hz, 1H), 1.29(t, J=6.8Hz, 3H), 1.02(t, J=8.0Hz, 3H), 0.96(t , J=7.6Hz, 3H), 0.90(t, J=8.0Hz, 3H), 0.69(q, J=8.0Hz, 2H), 0.61(q, J=8.0Hz, 2H), 0.55(m, 2H ); [M+H] ⁺ 760.5.

Step 3: Preparation of (5-amino-2-oxo-3-β-D-ribofuranosyl-2,3-dihydro-thiazolo[4,5-d]pyrimidin-7-yloxymethyl)- Urethane(82)

A solution of 81 (244 mg, 321 μmol), 5M HF in pyridine (321 μL, 1.60 mmol) and THF (3.20 mL) was stirred at room temperature for 5 hours. The solvent was removed in vacuo and the resulting residue was purified by flash chromatography ( _SiO2 , 10% MeOH- _CHCl3 ) to give 82 as a white solid (119 mg, 90%): ¹ H NMR (400 MHz, d ₆ -DMSO) δ8. 43(br s, 1H), 7.76(br s, 2H), 5.82(d, J=5.2Hz, 1H), 5.78(s, 2H), 5.32(d, J=5.6Hz, 1H), 5.24(dd , J=6.0, 4.8Hz, 1H), 5.00(d, J=5.6Hz, 1H), 4.82(q, J=5.6Hz, 1H), 4.68(t, J=6.0, 1H), 4.11(q, J=5.2Hz, 1H), 4.09(q, J=7.2Hz, 2H), 3.78(q, J=5.6Hz, 1H), 3.60(m, 1H), 3.46(m, 1H), 1.21(t, J=7.2Hz, 3H); [M+H] ⁺ 418.2.

Example 33: (5-Amino-2-oxo-3-β-D-ribofuranosyl-2,3-dihydro-thiazolo[4,5-d]pyrimidin-7-yloxymethyl)- Methyl-urethane (84)

Step 1: Preparation of (5-amino-2-oxo-3-(2',3',5'-tri-O-acetyl-β-D-nucleofuranosyl)-2,3-dihydro-thiazole And[4,5-d]pyrimidin-7-yloxymethyl)-methyl-urethane (83)

In a manner similar to Example 2, step 1, compound 83 was prepared as a white solid from 75 and N-methyl-N-(hydroxymethyl)urethane in 24% yield: R _f =0.4 (33% EtOAc-CHCl ₃ ); ¹ H NMR (400MHz, CDCl ₃ ) δ 11.49 (br s, 1H), 6.08 (d, J=4.0Hz, 1H), 5.75 (t, J=6.0Hz, 1H), 5.53 (s, 2H), 4.49(dd, J=13.5, 8.4Hz, 1H), 4.30(m, 5H), 3.62(q, J=7.2Hz, 2H), 2.30(s, 3H), 2.12(s, 3H), 2.09(s, 3H), 2.08(s, 3H), 1.36(t, J=6.8Hz, 3H), 1.20(t, J=6.8Hz, 3H); [M+H] ⁺ 614.2.

Step 2: Preparation of (5-amino-2-oxo-3-β-D-ribofuranosyl-2,3-dihydro-thiazolo[4,5-d]pyrimidin-7-yloxymethyl)- Methyl-urethane (84)

In a similar manner to Example 1, Step 4, the title compound was prepared as a white solid from 83 in 20% yield: ¹ H NMR (400 MHz, d ₆ -DMSO) δ 7.86 (br s, 2H), 5.82 (d , J=4.8Hz, 1H), 5.47(s, 2H), 5.31(d, J=5.2Hz, 1H), 5.00(d, J=5.6Hz, 1H), 4.82(q, J=5.2Hz, 1H ), 4.67(q, J=5.6Hz, 1H), 4.18(q, J=6.4Hz, 2H), 4.12(m, 1H), 3.78(q, J=6.0Hz, 1H), 3.60(m, 1H ), 3.47(m, 1H), 3.30(s, 3H), 1.27(t, J=6.8Hz, 3H); [M+H] ⁺ 432.3.

Example 34: 5-amino-7-(5-methyl-2-oxo-[1,3]dioxol-4-ylmethoxy)-3-(2',3',5 '-tri-O-acetyl-β-D-ribofuranosyl)-thiazolo[4,5-d]pyrimidin-2-one (85)

Step 1: Preparation of 5-amino-7-(5-methyl-2-oxo-[1,3]dioxol-4-ylmethoxy)-3-(2',3',5 '-tri-O-acetyl-β-D-ribofuranosyl)-thiazolo[4,5-d]pyrimidin-2-one (85)

To a solution of 75 triacetate (1.55 g, 3.50 mmol) in THF (50 mL) was added polymer-supported-triphenylphosphine (4.95 g, 10.50 mmol, Argonaut) at 0°C. To this mixture was added 4-hydroxymethyl-5-methyl-[1,3]dioxolan-2-one (0.91 g, 7.00 mmol) (according to Alepegiani, Syn.Comm., 22 (9), prepared by the method described in 1277-82 (1992). Then diethyl azodicarboxylate (0.73 mL, 4.60 mmol) was added dropwise. The resulting mixture was stirred at room temperature for 48 hours, filtered and washed with MeOH and _CHCl3 . The filtrate was concentrated and purified by flash column chromatography (silica, acetone/CHCl ₃ =10-20%) to obtain the dioxolanone derivative 85 (1.38 g, 71%) as a white solid: ¹ H (400 MHz , d ₆ -DMSO); 7.06(s, 2H), 6.00(d, J=4.0Hz, 1H), 5.92(dd, J=6.6, 4.4Hz, 1H), 5.56(t, J=6.4Hz, 1H ), 5.30(s, 2H), 4.38(dd, J=11.6, 3.6Hz, 1H), 4.25(t, J=3.6Hz, 1H), 4.10(q, J=6.0Hz, 1H), 2.23(s , 3H), 2.08(s, 3H), 2.07(s, 3H), 2.00(s, 3H); MS(+)-ES[M+H] ⁺ m/z 555.3. Elemental analysis for C ₂₁ H ₂₂ N ₄ O ₁₂ S Me ₂ CO, calculated: C, 47.06; H, 4.61; N, 9.15; S, 5.23. Found: C, 47.25; H, 4.37; N, 9.53 ; S, 5.52.

Example 35: 5-Amino-7-(5-methyl-2-oxo-[1,3]dioxol-4-ylmethoxy)-3-β-D-nucleofuranosyl -Thiazolo[4,5-d]pyrimidin-2-one (87)

Step 1: Preparation of 5-amino-7-(5-methyl-2-oxo[1,3]dioxol-4-ylmethoxy)-3-(2',3',5' -Tri-O-triethylsilyl-β-D-nucleofuranosyl)-thiazolo[4,5-d]pyrimidin-2-one (86)

In a similar manner to Example 34, Compound 86 was prepared as a white solid from 80 and 4-hydroxymethyl-5-methyl-[1,3]dioxol-2-one in 45% yield: ¹ H NMR (400MHz, CDCl ₃ ) δ6.06 (d, J=6.0Hz, 1H), 5.21 (dd, J=6.0, 4.8Hz, 1H), 5.18 (d, J=3.2Hz, 2H), 4.94 (br s, 2H), 4.38(dd, J=4.8, 2.8Hz, 1H), 4.00(m, 1H), 3.79(dd, J=11.2, 8.0Hz, 1H), 3.69(dd, J=10.8, 5.2Hz, 1H), 2.23(s, 3H), 1.02(t, J=8.0Hz, 3H), 0.96(t, J=7.6Hz, 3H), 0.89(t, J=8.4Hz, 3H), 0.70 (q, J = 7.6 Hz, 2H), 0.61 (q, J = 8.0 Hz, 2H), 0.53 (m, 2H); [M+H] ⁺ 771.5.

Step 2: Preparation of 5-amino-7-(5-methyl-2-oxo-[1,3]dioxol-4-ylmethoxy)-3-β-D-ribofuranosyl -Thiazolo[4,5-d]pyrimidin-2-one (87)

In a manner similar to Step 3 in Example 32, the title compound was prepared from 86 as a white solid in 89% yield: ¹ H NMR (400 MHz, d ₆ -DMSO) δ 7.03 (br s, 2H), 5.90 (d , J=5.2Hz, 1H), 5.33(s, 2H), 5.02(d, J=4.8Hz, 1H), 4.83(q, J=5.6Hz, 1H), 4.71(t, J=6.0Hz, 1H ), 4.14(q, J=5.2Hz, 1H), 3.80(q, J=4.8Hz, 1H), 3.62(m, 1H), 3.47(m, 1H), 2.27(s, 3H); [M+ H] ⁺ 429.2.

Example 36: 5-Amino-3-β-D-ribofuranosyl-3H-thiazolo-[4,5-d]pyrimidin-2-one (90)

Step 1: Preparation of 5-amino-7-thioxo-3-(2',3',5'-tri-O-acetyl-β-D-nucleofuranosyl)-thiazolo[4,5-d ]pyrimidin-2-one(88)

To a solution of 75 (1 g, 2.26 mmol) in pyridine (50 mL) was added P ₂ S ₅ (2.13 g, 4.79 mmol) at room temperature. The solution was gently refluxed (bath temperature 130-140° C.) for 29 hours. The reaction mixture was evaporated to dryness in vacuo. Excess _P2S5 was decomposed by adding _H2O (40 mL) at ₆₀ °C. After stirring the mixture at 60°C for 1 hour, it was cooled to room temperature. The mixture was extracted with _CHCl3 (3 x 40 mL). The organic layer was evaporated to dryness (MgSO ₄ ) to give a syrup, which was purified by flash column chromatography (silica, acetone/CHCl ₃ =15%) to give 0.93 g (90%) of 88: ¹ H (400 MHz) as a yellow solid , d ₆ -DMSO). 12.50 (s, 1H), 7.35 (br s, 2H), 5.89 (m, 2H), 5.51 (t, J=6.4Hz, 1H), 4.36 (dd, J=12.0, 4.0 Hz, 1H), 4.24(m, 1H), 4.10(q, J=6.0Hz, 1H), 2.07(s, 3H), 2.06(s, 3H), 2.01(s, 3H); MS(+)- ES [M+H] ⁺ m/z 459.3.

Step 2: Preparation of 5-amino-3-(2',3',5'-tri-O-acetyl-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidine- 2-keto(89)

A suspension of ^Raney_2800 nickel (3 large spatulas, previously washed with _H2O , MeOH and acetone) in acetone (50 mL) was stirred at reflux for 1 h. The triacetate salt of 88 (0.93 g, 2.03 mmol) was then added to the above suspension at reflux. The mixture was stirred for 5 minutes and cooled to room temperature after 30 minutes. H ₂ S (g) was passed into the mixture for 2h to terminate the reaction. The resulting mixture was filtered through a ^Celite® short plug and washed with EtOH. The filtrate was concentrated and purified by flash column chromatography (silica, MeOH/CHCl ₃ =1-2%) to give 0.52 g of (60%) 89 as a white solid: mp 121-123°C; ¹ H (400 MHz, d ₆ - DMSO) 8.38(s, 1H), 6.93(s, 2H), 6.03(d, J=3.6Hz, 1H), 5.93(dd, J=6.4, 3.6Hz, 1H), 5.58(t, J=6.0Hz , 1H), 4.38(dd, J=11.6, 3.6Hz, 1H), 4.26(m, 1H), 4.11(q, J=6.0Hz, 1H), 2.08(s, 3H), 2.07(s, 3H) , 2.00(s, 3H); MS(+)-ES[M+H] ⁺ m/z 427.2. Calculated for elemental analysis of _{C 16} H ₁₈ N ₄ O ₈ S: 0.5CH ₃ OH·0.25H ₂ O: C, 44.34; H, 4.62; N, 12.54; S, 7.17. Found: C, 44.54; H, 4.88; N, 12.16;

Step 3: Preparation of 5-amino-3-β-D-ribofuranosyl-3H-thiazolo[4,5-d]pyrimidin-2-one (90)

To _a solution of 89 (0.52 g, 1.22 mmol) in MeOH (20 mL) was added _K2CO3 (25 mg, 0.18 mmol). The reaction was stirred overnight at room temperature, then neutralized with AcOH (21 μL, 0.36 mmol). The resulting mixture was further stirred at room temperature for 30 min, concentrated and triturated with H ₂ O (2 mL) to give 0.33 g of compound 90 (89%) as a white solid: mp 220°C (Dec); ¹ H (400 MHz, d ₆ -DMSO).8.34(s, 1H), 6.85(s, 2H), 5.90(d, J=4.8Hz, 1H), 5.31(d, J=5.6Hz, 1H), 4.98(d, J=5.6Hz , 1H), 4.81(q, J=5.2Hz, 1H), 4.67(t, J=6.0Hz, 1H), 4.11(q, J=5.2Hz, 1H), 3.77(dd, J=10.8, 4.8Hz , 1H), 3.58 (m, 1H), 3.44 (m, 1H); MS(+)-ES[M+H] ⁺ m/z 301.1. Elemental analysis calculated for C ₁₀ H ₁₂ N ₄ O ₅ S: 0.3H ₂ O: C, 39.29; H, 4.15; N, 18.33; S 10.49. Found: C, 39.51; H, 4.18; N, 17.95; S, 10.27.

Example 37: 5-Amino-3-(2',3'-di-O-acetyl-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one (93)

Step 1: Preparation of 5-amino-3-(5'-O-tert-butyl-dimethylsilyl-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidine-2 -ketone(91)

To a solution of 90 (0.68 g, 2.28 mmol) in DMF (10 mL), imidazole (0.54 g, 7.93 mmol) and tert-butyldimethylsilyl chloride (0.68 g, 4.56 mmol) were added sequentially. After stirring the reaction mixture for 2 hours at room temperature, it was concentrated and purified by flash column chromatography (silica, MeOH/ _CHCl3 ; gradient = 5-20%) to give 0.49 g of a white solid (52%) 91: ¹ H(400MHz, d ₆ -DMSO).8.33(s, 1H), 6.87(s, 2H), 5.90(d, J=4.0Hz, 1H), 5.33(d, J=5.6Hz, 1H), 5.00( d, J=5.2Hz, 1H), 4.79(q, J=5.2Hz, 1H), 4.16(q, J=5.2Hz, 1H), 3.77(m, 2H), 3.64(dd, J=12.0, 7.2 Hz, 1H), 0.84(s, 9H), 0.00(s, 6H); MS(+)-ES[M+H] ⁺ m/z 415.4.

Step 2: Preparation of 5-amino-3-(2',3'-di-O-acetyl, 5'-O-tert-butyl-dimethylsilyl-β-D-ribofuranosyl)-3H -Thiazolo[4,5-d]pyrimidin-2-one (92)

To a solution of 91 (0.20 g, 0.48 mmol) in acetonitrile (5 mL) at 0°C, Et ₃ N (0.26 mL, 1.86 mmol) and Ac ₂ O (91 μL, 0.96 mmol) were added successively. After stirring the reaction mixture at room temperature for 24 hours, it was concentrated and purified by flash column chromatography (silica, acetone/ _CHCl3 : gradient = 5-10%) to give 0.22 g of a white solid (92%) 92: ¹ H(400MHz, d ₆ -DMSO).8.36(s, 1H), 6.90(s, 2H), 6.00(m, 2H), 5.57(t, J=6.0Hz, 1H), 4.07(q, J=5.2 Hz, 1H), 3.77(m, 2H), 2.07(s, 3H), 2.06(s, 3H), 0.83(s, 9H), 0.00(d, J=2.4Hz, 6H); MS(+)- ES [M+H] ⁺ m/z 499.5.

Step 3: Preparation of 5-amino-3-(2',3'-di-O-acetyl-β-D-ribofuranosyl)-3H-thiazolo[4,5-d]pyrimidin-2-one (93)

To a solution of 92 (0.22 g, 0.44 mmol) in THF (5 mL) was added HF/pyridine (0.70 mL) in a plastic test tube. The reaction was stirred for 2 h, concentrated and purified by flash column chromatography (silica, MeOH/ _CHCl3 : gradient = 5-10%) to afford 0.17 g (100%) of the title compound as a white solid: mp 109-111 ℃; ¹ H(400MHz, d ₆ -DMSO).8.37(s, 1H), 6.91(s, 2H), 6.00(m, 2H), 5.48(t, J=6.0Hz, 1H), 4.91(t, J=6.0Hz, 1H), 4.04(dd, J=10.4, 6.0Hz, 1H), 3.64(m, 1H), 3.52(m, 1H), 2.08(s, 3H), 2.05(s, 3H); Elemental analysis for MS(+)-ES[M+H] ⁺ m/z 385.3. Calcd. for C ₁₄ H ₁₆ N ₄ O ₇ S 0.5CH ₃ OH 0.2: C, 41.61; H, 4.32; N , 13.21; S 7.56: Found: C, 41.73; H, 4.29; N, 12.86; S, 7.33.

Example 38: [2-Ethoxymethyl-1-(2-hydroxy-2-methyl-propyl)-1H-imidazo[4,5-c]quinolin-4-yl]-carbamic acid Ethyl ester(39)

Step 1: Preparation of [2-ethoxymethyl-1-(2-hydroxy-2-methyl-propyl)-1H-imidazo[4,5-

In a manner similar to step 1 in Example 8, except using MeOH as solvent, from 1-(4-amino-2-ethoxymethyl-imidazo[4,5-c]quinolin-1-yl )-2-Methyl-propan-2-ol (38) (prepared according to the method described in International Publication No. WO 94/17043) and diethylpyrocarbonate to prepare the title compound as an oil in 39% yield: ¹ H NMR (400 MHz , CDCl ₃ ) δ8.36(d, J=8.0Hz, 1H), 8.05(d, J=8.0Hz, 1H), 7.70(t, J=7.2Hz, 1H), 7.61(t, J=8.0Hz , 1H), 4.96(br s, 2H), 4.80(s, 2H), 4.39(q, J=7.2Hz, 2H), 3.62(q, J=7.2Hz, 2H), 1.40(t, J=7.2 Hz, 3H), 1.36 (br s, 6H), 1.24 (t, J=6.8Hz, 3H); MS(+)-ES[M+H] ⁺ 387.4 m/z.

6.4 Masking effect of TLR7 ligand prodrugs

A typical experiment will use human peripheral blood mononuclear cells (PBMC) isolated from healthy donors and placed in duplicate cell culture wells; typically, 2 x ¹⁰⁶ to 5 x ¹⁰⁶ cells are seeded per well. At 37°C in a humidified environment containing 5% CO ₂ , PBMC were cultured in the absence of test compounds for 24 hours to stabilize the culture conditions, and then 100 μM of exatoribine, TLR7 ligand and the corresponding TLR7 ligand prodrugs were added to wells containing PBMC from the same donor; untreated controls were included. Concentrations of TLR7 ligand and TLR7 ligand prodrugs can be varied to suit a particular experiment, and PBMC cultures are then incubated at 37°C in a humidified environment with 5% _CO2 for 2-48 hours. Cell culture supernatants were sampled during the culture period. ELISA. Analysis of cytokine production. LC-MS method was used to analyze the amount of residual TLR7 ligand and TLR7 ligand prodrug at the end of incubation. Cytokine production was calculated relative to that in the exatoribine control and then subtracted from the untreated control. The results for cytokines are compared to determine the extent to which the TLR7 ligand is more active than the corresponding TLR7 ligand prodrug.

Therefore, a TLR7 ligand prodrug is considered to be "masking" if it produces a higher dose of interferon alpha (an easily measured cytokine) than the corresponding TLR7 ligand prodrug after similar exposure times and concentrations TLR7 Ligand Prodrugs. The reduction in cytokine production constituting the "masking" can be as little as 25% lower than that of the parental TLR, so that for a given level of tolerance the dose can be increased accordingly.

The data presented in Tables 9-14 show that various chemical classes of TLR7 ligands can be masked. The examples shown show comparable masking relative to the parent TLR7 ligand. The chemical substituents shown are exemplary and in no way limiting of the invention, therefore other chemical substituents may also exhibit masking effects and are included within the scope of the invention. Substituents can be introduced at multiple positions of the TLR7 ligand to achieve masking, and as shown in the table, many chemical bonds can be included. It is understood that preferred substituents and linkages may vary for different parent TLR7 ligands.

Table 9: Masking of Exatoribine Prodrugs Parent molecule and its prodrug Compound number Relative to the amount of INFa induced by 100 μM Exatoribine, % Parent Molecule: Exatoribine Prodrug: Amino Acid Ester Prodrug: Deoxy Prodrug: 6-Ethoxy Prodrug: 6-Methoxy Prodrug: Aminal Prodrug: Aminal Prodrug: Metadi Oxolone 2124937779848285 1001000000

The masking properties of the exatoribine prodrug can be studied using a PBMC assay. The results of the PBMC assay (Table 9) show the percentage of INFa released after 8 hours (valine-exatoribine, 24) or 24 hours (other prodrugs) of the parent compound and its prodrugs after exposure to an initial concentration of 100 μM. quantity. At 100 μM, the amount of INFa released was calibrated to the amount induced by 100 μM exatoribine in the same blood sample and at the same exposure time.

Table 10: Masking of loxoribine prodrugs Parent molecule and its prodrug Compound number Relative to the amount of INFa induced by 100 μM Exatoribine, % Parent Molecule: Loxoribine Prodrug: 6-Ethoxy Prodrug: Deoxy Prodrug: Valinate 17454368 50000

The masking properties of the loxoribine prodrug can be studied using a PBMC assay. The results of the PBMC assay (Table 10) show the amount of INFa released after 24 hours of exposure to the parent compound and its prodrug at an initial concentration of 100 [mu]M. The amount of INFa released was calibrated to the amount induced by 100 μM exatoribine at 100 μM in the same blood sample for the same exposure time.

Table 11: Masking of Imiquimod Prodrugs Parent molecule and its prodrug Compound number Relative to the amount of INFa induced by 100 μM Exatoribine, % Parent Molecule: Imiquimod Prodrug: Amyl Carbamate Prodrug: Ethyl Carbamate 313450 60-76 ^* 00

^* Results of two experiments performed on three different donors

The masking properties of imiquimod prodrugs can be studied using a PBMC assay. The results of the PBMC assay (Table 11) show the amount of INFa released after 24 hours of exposure to the parent compound and its prodrug at an initial concentration of 100 [mu]M. The amount of INFa released was calibrated to the amount induced by 100 μM exatoribine at 100 μM in the same blood sample for the same exposure time.

Table 12: Masking of resiquimod prodrugs

The masking properties of the resiquimod prodrug can be studied using a PBMC assay. The results of the PBMC assay (Table 12) show the amount of INFa released after 24 hours of exposure to the parent compound and its prodrug at an initial concentration of 1 or 100 [mu]M. The amount of INFa released was calibrated to the amount induced by 100 μM exatoribine at 100 μM in the same blood sample for the same exposure time.

Table 13: Masking of Bropirimine Prodrugs Parent molecule and its prodrug Compound number Relative to the amount of INFa induced by 100 μM Exatoribine, % Parent Molecule: Bropirimine Prodrug: Deoxy Prodrug: Ethoxy Prodrug: Ethyl Carbamate Prodrug: Amyl Carbamate 3548373649 220000

The masking properties of the bropiriramine prodrug can be studied using a PBMC assay. The results of the PBMC assay (Table 13) show the amount of INFa released after 24 hours of exposure to the parent compound and its prodrug at an initial concentration of 100 [mu]M. The amount of INFa released was calibrated to the amount induced by 100 μM exatoribine at 100 μM in the same blood sample for the same exposure time.

Table 14: Masking of adenine prodrugs Parent molecule and its prodrug Compound number Relative to the amount of INFa induced by 100 μM Exatoribine, % Parent Molecule Prodrug: Methoxy Prodrug: Ethoxy Prodrug: Deoxy Prodrug: Ethyl Carbonate Prodrug: Amyl Carbonate 296564625154 1280.1μM0100μM010μM00.1μM1832μM1510μM

The masking properties of adenine prodrugs can be studied using a PBMC assay. The results of the PBMC assay (Table 14) show the amount of INFa released after 24 hours of exposure to different starting concentrations of the parent compound and its prodrug. The amount of INFa released was calibrated to the amount induced by 100 μM exatoribine at 100 μM in the same blood sample for the same exposure time.

The conversion of TLR7 ligand prodrugs to active parent TLR7 ligand can also be assessed in vitro. This can be determined by incubating the prodrugs in cell culture of blood, plasma or hepatocytes. Samples were taken at selected time intervals to determine residual prodrug and TLR7 ligand production. Assays are readily performed using analytical tools known in the art, such as LC-MS. The extent to which masked TLR7 ligand prodrugs were converted to parent TLR7 ligand was determined for interpretation of the data, where masking was evident with shorter incubation times and lost with longer incubation times in the PBMC assay. The rate of conversion of the prodrug to the TLR7 ligand was measured to ensure that the significant increase in cytokine outcome was due to exposure to the prodrug and not due to exposure to the TLR7 ligand produced by the rapid conversion of the prodrug under the conditions tested.

6.5 Biological tests of TLR7 ligand prodrugs

Increased oral availability and reduced side effects

Oral availability

The improved bioavailability of TLR7 ligand prodrugs was evaluated by in vivo assays. In this experiment, the candidate prodrug is orally administered to mice, rats, monkeys and/or dogs, and blood samples are taken at selected time intervals. Blood samples were analyzed for prodrugs and desired TLR7 ligands. Other blood or liver samples can be analyzed for the presence of interferons and other cytokines indicative of functional activation of the TLR7 pathway in vivo. Desired candidate compounds will exhibit blood contact prodrugs and exhibit blood exposure of the desired TLR7 ligand in the range of 10% to 99% of the applied dose based on a molar assay.

A representative example is the results obtained with the TLR7 ligand prodrug valine-exatoribine (24), which produced significant amounts of the parent TLR7 ligand exatoribine in the blood of mice and dogs as described below (twenty one). See, US Patent Application No. 10/305,061 (incorporated herein by reference).

Concentration of interferon alpha (Mu-IFN-α) in mice

Normal mice provide a useful system for evaluating the extent to which substances of the invention improve oral delivery of 21 (exatoribine). Not only can the plasma concentration of exatoribine be determined after oral administration of the prodrug, but extensive immunological studies in mice provide a suitable method for the determination of interferon alfa, a protein that reflects the desired biology of exatoribine. active one of the cytokines of interest) levels of reagents.

Using the murine system in a series of experiments, it was shown that 24, 21 (valine-exatoribine) 5'-valine ester induced an interferon response substantially greater than that given by exatoribine itself.

Table 15 records the test results of murine interferon α in mouse plasma after oral administration of 50 mg/kg of exatoribine in bicarbonate form twice. Results showed that interferon was undetectable, even after repeated dosing 4 hours later.

Table 15: Interferon α (Mu-IFN-α) plasma concentration (picogram/ml) in mice after two oral administrations of 50 mg/kg dose of exatoribine after 4 hours interval

BQL ⁿ - below the upper limit of measurement < n pg/mL

Table 16 records the experimental results of murine interferon α in mouse plasma by firstly administering bicarbonate and then orally administering exatoribine in the form of bicarbonate at a concentration of 50 mg/kg 4 hours later. Interferon was present in the plasma of four mice, including two given the sodium bicarbonate vehicle. All values recorded in this experiment were low, and the recorded interferon levels were not consistent with all three mice recorded at each time point, suggesting that this information is an error due to approaching the lower limit of analysis.

Table 16: Interferon-alpha (Mu-IFN-α) plasma concentrations (picograms/ml) in mice after one vehicle dose and one 50 mg/kg dose of exatoribine 4 hours later

BQL ⁿ - below the upper limit of measurement < n pg/mL.

NR - not detected

Table 17 records the concentration of murine interferon alfa in mouse plasma after oral administration of valine-exatoribine, dissolved in bicarbonate, at a molar-based dose equivalent to 50 mg/kg of exatoribine test results. Clearly, interferon was readily detectable at 1.0 hour, 1.5 hour and 2.0 hours after administration. Interferon was detected in all mice at a given time point, suggesting the reliability of the effect after administration of valine-exatoribine. Therefore, a single dose of valine-exatoribine is superior to single or multiple doses of exatoribine.

Table 17: Plasma concentrations of interferon alpha (Mu-IFN-α) in mice after a single administration of 73.0 mg/kg dose of valine-exatoribine (pg/ml)

BQL-below the measurement limit <12.5pg/mL

BQL ⁿ - below the upper limit of measurement < n pg/mL

NR - not detected

The data presented in Tables 15, 16 and 17 can also be considered in terms of the incidence of measurable interferon levels. In the test of Exatoribine, only 4 of the 114 mice could detect interferon in the plasma, but given valine-Exatoribine, there were 10 of the 30 mice in the plasma. Interferon can be detected. Thus, the prodrug increased the proportion of mice showing an interferon response from 4% to 30%, and increased both the mean and peak response two-fold (100%).

In other embodiments, exatoribine is administered intravenously, the plasma levels of exatoribine and interferon alpha in mice are determined, and these plasma levels are compared to those after oral administration of valine-exatoribine. The levels of satoribine and interferon alpha were compared. These data are summarized in Figure 1. As shown, oral administration of valine-exatoribine ("val-isator") (50 mg/kg exectoribine molar equivalent) induced levels of interferon-alpha similar to intravenous administration of 25 mg/kg Levels of Isatoribine ("isator") in kilograms. Thus, oral valine-exatoribine provided approximately 50% of the levels of exatoribine and interferon that were observed after intravenous administration of exatoribine itself.

beagle dog

The effect of systemic levels of the prodrug (valine-exatoribine, 24) exatoribine (21) after oral administration to beagle dogs was studied. Exatoribine was prepared in sodium bicarbonate solution. Valine-exatoribine and exatoribine were prepared in the following formulations, which were selected to ensure solubility:

Formulation 1: Exatoribine in sodium bicarbonate solution, 1 and 4 mg/ml.

Formulation 2: Valine-Exatoribine in Phosphate Buffered Saline, 1.62 and 6.48 mg/ml, on a molar basis, equivalent to 1 and 4 mg/ml Exatoribine.

Four male and four female adult beagle dogs weighing 15-27 kg and approximately 1-2 years of age were used at the outset of the study. The animals were divided into 2 groups, 2 males and 2 females in each group. The test substances were administered by gavage on days 1 and 8, with a washout period of 7 days between the two administrations. Blood samples (2 mL) were collected from each animal at predose, 15 minutes, 30 minutes, 1, 2, 3, 4, 6, 8 and 10 hours after each dose into lithium heparin tubes. Plasma was frozen at -70°C until analysis. Analysis of exatoribine in plasma by HPLC-MS/MS.

The pharmacokinetic parameters of Exatoribine from Exatoribine or Valine-Exatoribine in each dog are summarized in Tables 18 and 19. The ratios of the total key pharmacokinetic parameters calculated by the maximum concentration (Cmax) and the area under the time-concentration curve (AUC) of the prodrug and the 50 mg/kg dose of bicarbonate solution are summarized in Table 20. For prodrug 24, the Cmax ratio was 2.98±0.695 and the AUC ratio was 2.38±0.485. The results suggest that the prodrug valine-exatoribine has significantly higher Cmax and greater bioavailability than the bicarbonate solution of exatoribine at a dose of 50 mg/kg.

The Cmax and AUC ratios of the prodrug to the 10 mg/kg dose in bicarbonate solution are summarized in Table 21. For the prodrug, the Cmax ratio was 2.24±0.249 and the AUC ratio was 1.82±0.529. These results suggest that the prodrug valine-exatoribine has a higher Cmax and greater bioavailability than the bicarbonate solution of exatoribine at a dose of 10 mg/kg.

Thus, at doses of 10 and 50 mg/kg, the maximum concentration of exatoribine following oral administration of the prodrug valine-exatoribine was at least doubled compared to exatoribine itself, and exatoribine Systemic levels of satoribine were increased approximately 2-fold.

Table 18: Pharmacokinetic Parameters of Exatoribine in Dogs at 50 mg/kg

Table 19: Pharmacokinetic Parameters of Exatoribine in Dogs at 10 mg/kg

Table 20: Ratio of Pharmacokinetic Parameters of Exatoribine in Dogs at the 50 mg/kg Dose

Table 21: Ratio of Exatoribine Pharmacokinetic Parameters in Dogs at 10 mg/kg

The prodrug valine-exatoribine is preferred for the following reasons. First, the prodrug is readily prepared to provide a high ratio of active agent. This allows the preparation of small capsules for a given dose, which is advantageous for oral medicines. Second, at the doses tested, valine-exatoribine provided plasma concentrations of exatoribine well within the range of desired biological effects after oral administration, whereas for exatoribine Libin itself was not like that.

macaque

2-4 male or female macaques were used in this animal experiment. Test compounds are formulated in a vehicle suitable for oral or intravenous administration to animals. The vehicles used are aqueous buffers or solutions containing polyoxyethylene castor oil. Each animal was dosed by oral gavage or intravenous bolus. Blood samples (approximately 0.5 mL) were collected at predetermined time points (typically, predose, 15 minutes, 30 minutes, 45 minutes, 1, 1.5, 2, 2.5, 3, 4, 8, and 24 hours post-dose), and the blood samples Place in a test tube containing disodium EDTA. After sampling the samples were placed on an ice bath and the plasma was separated as quickly as possible. Plasma samples were dispensed into single test tubes and stored frozen at approximately -20°C until shipped on dry ice to the originator. Approximately 4 hours after dosing, the animals were dosed with chow and water.

Plasma samples were analyzed for sampling and parent compounds using well-known LCMS/MS quantitative techniques by a triple quadrupole device such as the Sciex API3000. Area under the curve (AUC) values from time zero to 24 hours (PO AUC0-24h) were calculated using the quantitative results of oral administration of the parent compound itself or oral administration of its prodrug to deliver the parent compound. The relative oral availability of the prodrugs was calculated by comparing the AUC values of the parent compound delivered into the systemic circulation with the AUC values of the prodrugs. The results are shown in Table 22-26. Absolute oral bioavailability was calculated by dividing the PO AUC (0-24h) of the prodrug by the IV AUC (0-24h) of the parent molecule when the parent compound was given intravenously administered AUC data (AUCIV) for the parent compound itself Spend.

Table 22: Oral bioavailability of exatoribine and its prodrugs in monkeys

Toribine Prodrug: Amino acid ester Prodrug: Deoxy Prodrug: 6-Ethoxy Prodrug: 6-Methoxy Prodrug: Aminal Prodrug: Aminal Prodrug: Dioxolone 24937779848285 7-9 ^* 80282114417

^* Average of multiple experiments at different doses

Table 23: Oral bioavailability of loxoribine and its prodrugs in monkeys

Loxoribine Prodrug: 6-Ethoxy Prodrug: Deoxy 4543 913

Table 24: Oral bioavailability of imiquimod and its prodrugs in monkeys Parent molecule and its prodrug Compound number Oral bioavailability in macaques, % Parent Molecule: Imiquimod Prodrug: Amyl Carbamate Prodrug: Ethyl Carbamate 313450 100AUC(0-24h)＝9.0555AUC(0-24h)＝50234AUC(0-24h)＝21.1

Table 25: Oral bioavailability of bropirimine and its prodrugs in monkeys

^* Oral bioavailability greater than 100% may be related to sex differences, as the parent compound was studied in male monkeys and the prodrug was studied in female monkeys.

Table 26: Oral bioavailability of adenine prodrugs in monkeys

Reduced gastrointestinal irritation

The TLR7 ligand prodrugs of the present invention also exhibit greatly reduced unexpected toxic effects, especially reduced GI irritation.

The gastrointestinal tract ("GI") is lined with numerous immune tissues (eg, Peyer&apos;s plaques, etc.). As the agent passes through the lymphoid tissues in the gastrointestinal tract, TLR7 ligand prodrugs can mask the active structure, minimizing activation of lymphoid tissues and reducing GI irritation.

Robins et al. showed that the activity was lost by removing the 5'-hydroxyl of the esartoribine nucleoside. See Robins et al., Adv. Enzyme Regul., 29, 97-121 (1989). Without being bound by any particular theory, it is hypothesized that blocking the hydroxyl site with an ester substituent would similarly eliminate activity but allow transport in the systemic circulation, where valine ester would be cleared to expose exatoribine.

The above hypothesis was found to be confirmed. Toxicity studies of intravenous exatoribine and oral exatoribine and valine-exatoribine were performed in beagle dogs. Toxicity results of orally administered exatoribine are from a study conducted by the ICN/Nucleic Acid Research Institute.

The oral toxicity of 21 and 24 and the intravenous toxicity of 21 were compared in dogs. It was observed that the toxicity of oral 24 was closer to that of intravenous 21 than oral 21. Specifically, the dose-limiting toxicity of oral 3 was qualitatively similar to that of intravenous 21, occurring when blood concentrations were similar to those observed after intravenous administration of 21. In contrast, oral 21 had a different limiting toxicity (gastrointestinal injury) that was observed at lower toxic doses than intravenous 21 or oral 24. Also, emesis was observed in dogs treated with Oral 21 at doses lower than that of Oral 24 that caused emesis. See Table 27. Other systems for evaluating emesis are also known, such as ferrets, to compare oral and intravenous administration of compounds. See, eg, Strominger N. et al., Brain Res. Bull, 5, 445-451 (2001).

Each animal is administered the compound in solution, either by gavage or by intravenous infusion. Various parameters were evaluated in routine and toxicity studies. LC/MS was used to evaluate the ability to speak and read Exatoribine in the study to enhance the higher potential Exatoribine levels. Scores for observed GI phenomena are listed in Table 27.

Table 27: Effect of Exatoribine (21) or Valine-Exatoribine (24) on GI Tolerance in Dogs in Toxicity Study, as Systemic Levels of Exatoribine (AUC) score

For oral administration of exatoribine, the main findings were related to GI tolerance, as measured by GI irritation. The clinical presentation shown in Table 27 is vomiting and/or loose stools. These clinical signs were more common at 10 mg/kg, and one animal also had bloody stools at this dose. Gross histopathological evaluation of the gastrointestinal tract revealed numerous scattered erythema on the intestinal mucosa in 4 of 8 dogs at 10 mg/kg, and microscopic evaluation revealed cellular congestion and hemorrhage, suggesting local inflammation. The effect of GI was determined with a NOAEL of 5 mg/kg.

Intravenous administration of exatoribine resulted in vomiting and/or diarrhea commonly seen in dogs; this effect occurred at much higher applied doses than oral administration of exatoribine. Neither histopsy nor histopathological evaluation revealed gastrointestinal injury. GI toxicity does not respond to the NOAEL, which has been determined to be 12.5 mg/kg based on other findings.

Oral administration of valine-exatoribine showed a toxicity profile similar to intravenous administration of exatoribine. At higher applied doses, vomiting and diarrhea were observed. No GI lesions were found, although this was the focus of the evaluation in this study. For intravenous exatoribine, the NOAEL was determined based on other findings. The observed toxicity and systemic levels of exatoribine were of concern in this study; thresholds of exatoribine AUC for vomiting and diarrhea were observed similar to IV exatoribine and oral valamine Acid-Exatoribine (Table 27).

The data in Table 27 suggest that oral administration of valine-exatoribine provides an improved toxicity profile over oral administration of exatoribine, and is consistent with the hypothesis that chemical substitution by hydroxyl at the 5'-position of the nucleoside Esters enable chemical masking of exatoribine activity. As shown in Tables 9-14, any TLR7 ligand can be chemically masked using a variety of substituents. Allowing the aforementioned substituents to be cleared in vivo provides useful systemic levels of compound activity without the limitations of GI toxicity imposed by the anatomy of the gastrointestinal tract. As shown in Tables 22-25, chemical substituents that can be cleared after administration can be designed on masked TLR7 ligands. In this way, masked ligand prodrugs can be used with any TLR7 ligand. This allows the administration of much higher molar doses than are acceptable, resulting in greater potency and reduced side effects, compared to administering only the parent "unmasked" compound.

6.6 Oral group

Table 28 shows batches of formulations and single dose unit formulations containing 100 mg of valine-exatoribine

Table 28: 100 mg Tablets Material weight percentage Quantity (mg/tablet) Quantity (kg/batch) Valine-Exatoribine 40% 100.00 20.00 Microcrystalline Cellulose, NF 53.5% 133.75 26.75 Pluronic F-68 Surfactant 4.0% 10.00 2.00 Croscarmellose Sodium Type A, NF 2.0% 5.00 1.00 Magnesium Stearate, NF 0.5% 1.25 0.25 Total 100.0% 250.00mg 50.00kg

The microcrystalline cellulose, croscarmellose sodium, and valine-exatoribine components were passed through a #30 mesh screen (approximately 430μ-655μ). Pluronic F-68_ (manufactured by JRH Biosciences, Inc., Lenexa, KS) surfactant was passed through a #20 mesh screen (about 457μ-1041μ). Pluronic F-68_surfactant and 0.5 kg croscarmellose sodium were charged to a 16qt. twin shell tumble blender and mixed for about 5 minutes. Any Transfer the mixture to a 3 ft3 double shell tumbling mixer, add the microcrystalline cellulose and mix for about 5 minutes. Thalidomide was added and mixed for an additional 25 minutes. This premix is passed through a tumbler press with a hammer mill at the outlet and back into a tumble mixer. Add the remaining croscarmellose sodium and magnesium stearate to the tumbling mixer and mix for about 3 minutes. The final blend was compressed on a rotary tablet press, 250 tablets per batch (200,000 tablets per batch).

6.7 Mucosa composition

A concentrate was prepared by mixing Exatoribine and 12.6 kg of trichloromonofluoromethane in a sealed stainless steel vessel with a high shear mixer. Mix for about 20 minutes. Then, the concentrate is mixed with a certain amount of propellant in the batch product tank in a sealed container, the temperature is controlled at 21-27° C., and the pressure is controlled at 2.8-4.0 to prepare a large amount of suspension. The 17 ml aerosol container with metering valve is designed to allow 100 inhalations of the composition of the invention. Each container has the following ingredients:

Valine-Exatoribine 0.0120g

Trichlorofluoromethane 1.6960g

Dichlorodifluoromethane 3.7028g

Dichlorotetrafluoroethane 1.5766g

Total amount 7.0000g

6.8 Intravenous Composition

Intravenous formulations are prepared by reconstituting a compound of the invention in a suitable liquid medium, such as water for injection (WFI) or 5% dextrose solution. An appropriate amount of a compound of this invention is reconstituted with an appropriate volume of liquid medium to obtain the desired concentration for intravenous formulation. Intravenous formulations at desired concentrations provide to a patient, preferably a mammal, more preferably a human, in need of an intravenous pharmaceutical formulation, a therapeutically effective amount of a compound of the invention and maintain a therapeutically preferred level of the compound of the invention in the patient. A therapeutically effective dose will depend upon the rate at which the intravenous formulation is delivered to the patient and the concentration of the intravenous formulation.

For example, a vial containing the composition (eg, 50 mg of a compound of the invention/vial) is reconstituted with 5% dextrose solution (14 ml of 5% dextrose solution/vial) to yield a total of 25 mL of solution. The reconstituted solution was added to the glucose solution in the infusion bag and quantified to 50 ml to obtain a solution containing 1 mg/ml of the compound of the present invention suitable for intravenous infusion. For liquid media, the preferred concentration of the compound of the present invention in the infusion bag is about 0.001-3 mg/ml, preferably about 0.75-1 mg/ml.

The foregoing has set forth the relevant and important features of the invention. Many changes and variations of this invention can be made without departing from the spirit and scope of the invention, as will be apparent to those skilled in the art. The specific embodiments described herein are exemplary only, and the invention is to be limited only by the claims appended hereto and the scope of equivalents to which such claims are entitled.

The contents of all references cited herein are hereby incorporated by reference, as is the contents of each individual publication or patent or patent application which is specifically and individually indicated to be incorporated by reference.

Claims (29)

1.TLR7 the purposes of part in the medicine of preparation treatment infection with hepatitis C virus, wherein, described TLR7 part is selected from following:

2. purposes as claimed in claim 1 is characterized in that described medicine is used for the people.

3. purposes as claimed in claim 1, described medicine also comprises pharmaceutically acceptable excipient, carrier or vehicle.

4. purposes as claimed in claim 1, described medicine also comprises other therapeutic agent.

5. purposes as claimed in claim 4 is characterized in that, described other therapeutic agent is an antiviral agent.

6. purposes as claimed in claim 1 is characterized in that, the treatment of described TLR7 part or prevention effective dose are 0.001-100 mg/kg every day.

7. purposes as claimed in claim 1 is characterized in that, the treatment of described TLR7 part or prevention effective dose are 0.01-50 mg/kg every day.

8. purposes as claimed in claim 1 is characterized in that, the treatment of described TLR7 part or prevention effective dose are 0.1-20 mg/kg every day.

9. purposes as claimed in claim 1 is characterized in that parenteral route gives described medicine.

10. purposes as claimed in claim 1 is characterized in that intravenous gives described medicine.

11. purposes as claimed in claim 1 is characterized in that, the described medicine of orally give.

12. purposes as claimed in claim 1 is characterized in that, through mucous membrane gives described medicine.

13. treatment or pharmaceutically the TLR7 part prodrug of sheltering of effective dose or the purposes of its pharmaceutically acceptable salt in the medicine of preparation treatment infection with hepatitis C virus, the described TLR7 part prodrug of sheltering is selected from:

14. purposes as claimed in claim 13 is characterized in that, by effective plasma level concentration in the body of the described medicine acquisition of orally give TLR7 part, its 10%-500% for contacting in effective body that only orally give TLR7 part obtains.

15. purposes as claimed in claim 13 is characterized in that, by effective plasma level concentration in the body of the described medicine acquisition of orally give TLR7 part, its 50%-200% for contacting in effective body that only orally give TLR7 part obtains.

16. purposes as claimed in claim 13 is characterized in that, obtains the treatment effective plasma level concentration of corresponding TLR7 part and does not produce GI irritation by the described medicine of orally give.

17. purposes as claimed in claim 13 is characterized in that, compares with the side effect of only orally give TLR7 part generation, the described medicine of orally give reduces the adverse side effect among the patient.

18. purposes as claimed in claim 13 is characterized in that, compares 50% adverse side effect among the described medicine reduction of the orally give patient with the side effect of only orally give TLR7 part generation.

19. purposes as claimed in claim 18 is characterized in that, described side effect is a GI irritation.

20. purposes as claimed in claim 18 is characterized in that, described stimulation is hemorrhage.

21. purposes as claimed in claim 18 is characterized in that, described stimulation is damage.

22. purposes as claimed in claim 18 is characterized in that, described stimulation is vomiting.

23. purposes as claimed in claim 13 is characterized in that, described medicine is used for the people.

24. purposes as claimed in claim 13, described medicine also comprises pharmaceutically acceptable excipient, carrier and vehicle.

25. purposes as claimed in claim 13, described medicine also comprises other therapeutic agent.

26. purposes as claimed in claim 25 is characterized in that, described other therapeutic agent is an antiviral agent.

27. purposes as claimed in claim 13 is characterized in that, described treatment or prevention effective dose are 0.001-100 mg/kg every day.

28. purposes as claimed in claim 13 is characterized in that, described treatment or prevention effective dose are 0.1-25 mg/kg every day.

29. purposes as claimed in claim 13 is characterized in that, described treatment or prevention effective dose are 0.1-20 mg/kg every day.

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