ZA200601632B - Administration of TLR7 ligands and prodrugs thereof for treatment of infection by hepatitis C virus - Google Patents

Description

WO» 2005/025583 PCT/US2004/028236

ADMINISTRATION OF TLR7 LIGANDS AND PRODBRUGS THEREOF

FOR TREATMENT OF INFECTION BY HEPATITIS C VIRUS

[0001] This application claims the benefit of U.S. Provisional Application No. 60/500,339, filed September 5, 2003, U.S. Provisional Applicatiom No. 60/518,996, filed

November 10, 2003, and U.S, Provisional Application No. 60/518,997, filed November 10, 2003. 1. FIELD OF THE INVENTION

[0002] This invention relates to methods for treating or preventing hepatitis C virus infections in mammals using Toll-Like Receptor (TLR)7 ligands &and prodrugs thereof.

More particularly, this invention relates to methods of orally administering a therapeutically effective amount of one or more prodrugs of TLR7 ligands for thes treatment or prevention of hepatitis C viral infection. Oral administration of these TLR7 L mmunomodulating ligands and prodrugs thereof to a mammal provides therapeutically effective amounts and reduced undesirable side effects. 2. BACKGROUND OF THE INVENTION

[0003] Immunomodulation by small molecules can be ach _ieved by identifying compounds that bind and activate Toll-Like Receptors (TLRs). T_LRs play an important role in innate immune responses in mammals and are often the firsst line of defense against pathogens such as bacteria and viruses. The various TLRs vary imm their abundance in different mammalian cell types and also vary regarding the molecwular structures that bind the TLR and activate signaling pathways. These signaling pathwamys lead to the range of : responses associates with innate immunity.

[0004] TLRs detect PAMPs (pathogen-associated molecular patterns) and stimulate immune cells via the MyD88-dependent interleukin 1 receptor (IL--1R)-TLR signaling _pathway, which leads to activation of the transcription factor NF-1<B2. Ten functional “family members of TLRs (TLR! to TLR 10) have been identified in humans. Akira S. et al,

Nature Immunol., 2, 675-680 (2001). TLR2, TLR4, and TLRS are crucial for the recognition of peptidoglycan, lipopolysacharide, and flagellin. Hemyashi, F. ef al., Nature, “410, 1099-1103 (2001). TLR6 associates with TLR2 and recognizes lipoproteins from mycoplasma. Ozinsky, A. et al., Proc. Natl. Acad. Sci US4., 97, “13766-13771 (2000). “TLR detects bacterial DNA containing unmethylated CpG motifss and TLR3 activates

Immune cells in response to double-stranded RNA. Hemmi, H. ef al., Nature, 408, 740-745 «(2000). 0005] A number of compounds, including guanosine analaogs, substituted w)rimidines, and imidazoquinolines have been reported as ligands for TLR7. See, e.g,

Hemmi et al., Nature Immunol., 3, 196-200 (2002) (imiquimod and R-8438 (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 gwianosine analogs loxoribine, 7-thia-8-oxoguanosine (isatoribine), and 7-deazaguanosines, and the imidazoquinolines imiquimod and R-848 (resiquimod) selectively activate TILR7).

[6006] Prior to being linked as potential TLR 7 ligands, guanosine analogs and other

D- and L-purine nucleosides have been the subject of considerable research the past two decades. See, e.g., Reitz et al., J. Med. Chem., 37, 3561-78 (1994); Michael et al., J. Med.

Chem., 36, 3431-36 (1993) (immuraomodulatory guanosine analogs having substituents at the 7-and/or 8-positions); Patent No. 5,821,236 to Krenitsky et al. (disclosing 6-alkoxy derivatives of arabinofuranosyl pur-ine derivatives that are useful for tumor therapy); U.S.

Patent No. 5,041,426 to Robins et cal. (certain pyrimido[4,5-d]pyridimine nucleosides are disclosed in as being effective in treatment against 1.1210 in BDF1 mice); Revankar ef al.,

J. Med. Chem., 27, 1489-96 (1984) (3-Deazaguanine nucleosides and nucleotides demonstrating significant broad spectrum antiviral activity against certain DNA and RNA viruses);

[0007] A number of compownds known to be immunostimulants have recently been identified in the literature as TLR7 Yigands, see, e.g., Heil et al., Eur. J. Immunol., 33(11), 2987-97 (2003), Lore et al., J. Immaunol., 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, etal., Am. J. Pathol., 163(2), 711-2 1 (2003), Doxsee et al, J. Immunol., 171(3), 1156-63 (2003), Bottcher et al., Neurosci. Lett., 344(1), 17-20 (2003), Kaisho ef al., Curr. Mol.

Med., 3(4), 373-85 (2003), Okada ez 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), Y amamoto 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)3 Hornung et al., J. Immunol., 168(9), 4531-7 (2002),

Hemmi et al., Nat. Immunol., 3(2), T.96-200 (2002); Bruno et al., Eur. J. Immunol., 31(11), 3403-12 (2001); Jarrossay et al., Eur. J. Immunol., 31(11), 3388-93 (2001); Miettinen 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).

} [0008] These TLR7 ligands are known to stimulate immune responses in vitro and in animal spe=cies, and this has led to testing of the uses of these compounds for several therapeutic u-ses, including antiviral and cancer therapies. These compowunds have been characterized as analogs or derivatives of a) guanosine, b) imidazoquino line, and ¢) pyrimidine. See Akira, Current Opinion, 15, 5-11 (2003). One member (imiquimod) of the imidazogquinoline chemical class has been found effective for treating togpical genital infections by~ papilloma virus. A second member of the imidazoquinolirme class, resiquimod, has been testesd for the treatment of HCV, but this compound failed to shrow anti-HCV effect at tolerated oral doses. Pockros et al., Gastroenterology, 124 (Suppl 1), A-766 (2003).

[0009] Thus, while there has been some limited use of TLR7 ligeands for the treatment of ®mmunological disease and viral infections; see, e.g., U.S. P®atent Nos. 5,041,426 and 4,880,784 to Robins et al. (3-B-D-ribofuranosylthiazolo[4-,5-d}pyridimines demonstrating significant immunoactivity, including murine spleen cell goroliferation and in vivo activity against Semliki Forest virus); United States Patent Applica@tion Publication No.

US 2003/019 9461 and WO 03/045968 to Averett er al. (3-B-D-ribofuraneosylthiazolo[4,5- dlpyrimidine nucleosides demonstrating activity against acute and chron-ic infections of both RNA an-d DNA viruses); to date ligands have proved ineffective for the treatment or prevention of” Hepatitis C virus.

[0010] It is also known that the oral administration of many purire nucleoside analogs are subject to difficulties arising from poor absorption, poor solubility, or degradation ir the digestive tract as a result of acidic or alkaline conditions or the action of enzymes, and/or combinations of these phenomena. Thus there remains =a need for purine nucleoside an alogs with improved oral availability and administration that are used to modulate aspescts of the immune system.

[0011] Moreover, immunomodulatory nucleosides have relatively. poor oral tolerability winen compared to that of the intravenous route. Also, the gasstrointestinal tract presents a particular tolerability barrier to immunologic agents by virtue of the large amount of immune tis sue associated with the intestinal wall (i.e., gut). Although -this is an important biologic mechanism for preventing invasion of the body by gut= flora, the immune tissue also ma-y become preferentially affected after oral administration of immunomodu latory compounds because of the high local concentrations «of the administered compound in the gut. This leads to undesirable side effects, for example in the case of immurae activating agents there is observed gastroenteritis and loc-alized hemorrhagic e=ffects.

[0012] Au solution to the problem of effective oral delivery of immunomodulators is not evident in thee literature. Available evidence indicates that systermic levels of administered drumgs in this class have been limited by gastrointestinazl toxicities arising after low oral doses. “Therefore there remains a need for immunomodulat-ing TLR7 ligands that have improved coral availability and reduced gastrointestinal irritancy. 3. SUMMARY OF THE INVENTION 3.1 TLRT7 Ligands

[0013]) This invention encompasses novel methods for the tresatment or prevention of hepatitis C viral infection, and novel pharmaceutical compositions which utilize TLR? ligands or pharmaceutically acceptable salts, hydrates, metabolites cwr stereoisomers thereof.

[0014] I one embodiment, the invention encompasses a memthod of treating or preventing a hepmatitis C virus infection in a patient in need thereof c-omprising administering to the patient a therapeutically or prophylactically effective amount «of a TLR7 ligand or a \ pharmaceuticall®y acceptable salt, hydrate, metabolite or sterecisome=r thereof or a pharmaceuticall=y acceptable salt or hydrate of said stereoisomer.

[0015] Ir another embodiment, the invention encompasses a method of treating or preventing a hepeatitis C virus infection in a patient in need thereof ceomprising administering to the patient a therapeutically or prophylactically effective amount eofa TLR7 ligand selected from an alogs and derivatives of a) guanosine b) imidazoqu-inoline ¢) adenine, and d) pyrimidine.

[0016] Im another embodiment, the invention encompasses a method of treating or preventing a hep atitis C virus infection in a patient in need thereof ceomprising administering to the patient a tExerapeutically or prophylactically effective amount fa TLR7 ligand selected from ir HN AA NH; ess al a3 fo) R' ~~) > HO / > ’ ws on RS oH R

Ia oh Ie 0 Rt S " pu " NH, an ( KANT) oY OH

EN or] rw ‘a

We on HS on s 1d Ie If

NH, a a y A HN NTN - KE me]

HO oH °

Ig Ih wherein: each R} is H, or a substituted or unsubstituted alkyl, alkenyl, or alkynyl, which may be interrupted by one or more O, S, or N heteroatoms, or a substituted or unsubstituted aryl or . heteroaryl;

R? is H, OH, SH, halo, or a substituted or unsubstituted alkyl, alkenyl, or alkyny}, which may be interrupted by one or more O, S, or N heteroatoms, or a substituted or unsubstituted -0O-(alkyl), -O-(aryl), -O-(heteroaryl), -S-(alky~1), -S-(aryl), -S-(heteroaryl), aryl, or heteroaryl;

R? is H, OH, or SH, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, -O-(alkyl), -O-(aryl), -O-(heteroarsyl), -S-(alkyl), -S-(aryl), -S-(heteroaryl), “NH{(alky), -NH(aryl), -NHheteroaryl), -NHCR (alkyl), -NH(R*)(aryD), or

NH(R*)(heteroaryl), wherein R* is a substituted or unsubstituted alkyl;

XisOorS;

Y is H, halo, OH, OR*, SH, SR, or a substitut-ed or unsubstituted alkyl or aryl,

Z is H, halo, OH, OR? SH, or SRY,

ora pharmaceutically acceptable salt, hydrate, metabolite or stereoisomer thereof or a pharmaceutically acceptable salt or hydrate of said stercoisomer.

[0017] In another embockiment, the invention encompasses a method of treating or preventing a hepatitis C virus infection in a patient in need thereof comprising administering to the patient a therapeutically ox prophylactically effective amount of a TLR7 ligand selected from Formula Ia, Ib, Ic, Id, Ie, If, Ig, and Th, wherein R! is H or a substituted or unsubstituted alkyl, alkenyl, or alkynyl; R2 is H, OH, halo, or a substituted or unsubstituted alkyl, alkenyl, or alkynyl, or —-CEL-O<(alkyl); R? is H, OH, or SH, or a substituted or unsubstituted -O-(alkyl), -S-(alk-yl), or -NH(alkyl); X is O or S; Y is H, halo, OH, OR*, SH, or SRY; and Z is H, halo, OH, OR’, SH, or SR*.

[6018] In another embodiment, the invention encompasses a method of treating or preventing a hepatitis C virus in-fection in a patient in need thereof comprising administering to the patient a therapeutically ox prophylactically effective amount of a TLR7 ligand selected from

J

0 9 lo} yoo JIS SIL § vs

SN N HaN =n N NN N

Ho oH, Ho oH | Ho OH

NH,

NH2 Qs

NZ

PY I H—oH 2 eS he x lo} <n ©

NH, ‘ NH,

Ne Ne N

I N | [ CH,0CH,CH

S | { 2 2a — , and OH . or a pharmaceutically acceptable salt, hydrate, metabolite or stereoisomer thereof.

[0019] In one aspect, thes invention encompasses a method for treating or preventing hepatitis C virus infection in a mammal in need thereof, preferably in a human in need thereof.

0020} Jn: an alternative embodiment, the invention encompasses a method for treating or prevemting hepatitis C virus infection in a patient in need thereof, comprising administering to the patient a therapeutically or prophylactically effective amount ofa

TLR7 ligand andl a pharmaceutically acceptable excipient, carrier, or vehicle.

[0021] Ine an alternative embodiment, the invention encompasses a method for treating or prevemting hepatitis C virus infection in a patient in need thereof, compmrising administering to the patient a therapeutically or prophylactically effective amount ofa

TLR7 ligand ora ly, mucosally, topically or transdermally.

[0022] Im a preferred embodiment, the invention encompasses a method fox treating or preventing hepatitis C virus infection in a patient in need thereof, comprising administering to the patient a therapeutically or prophylactically effective amount ofa

TLR7 ligand par-enterally.

[0023] Imm a separate embodiment, the invention encompasses a method for treating or preventing hepatitis C virus infection in a patient in need thereof, comprising administering to the patient a therapeutically or prophylactically effective amount «fa

TLR?7 ligand andl an additional therapeutic agent, preferably an additional antiviral or immunomodulateory agent.

[0024] The invention also encompasses pharmaceutical compositions suita ble for parenteral admin_istration to a patient comprising a therapeutically or pharmaceutically acceptable amount of a TLR7 ligand of the invention in a sterile form; pharmaceutical compositions sui table for oral administration to a patient comprising a therapeutically or pharmaceutically acceptable amount of a TLR7 ligand of the invention, wherein siach compositions ares formulated to reduce exposure of the subepithelial immune anatosmy to the

TLR7 ligand while improving systemic absorption of the TLR7 ligand; pharmaceutical compositions suitable for mucosal administration to a patient comprising a therape-utically or pharmaceutically acceptable amount of a TLR7 ligand of the invention, whereim such compositions are= formulated to reduce exposure of the subepithelial immune anatosmy to the

TLR? ligand while improving systemic absorption of the TLR7 ligand; and pharmaceutical compositions suitable for topical administration to a patient comprising a therapeutically or pharmaceutically acceptable amount of a TLR? ligand of the invention, wherein sumch compositions are formulated to reduce exposure of the subepithelial immune anato my to the

TLR? ligand while improving systemic absorption of the TLR7 ligand. Dependingz on the specific tissue to be treated, additional components, such as penetration enhancers, may be used prior to, in conjunction with, or subsequent to treatment with active ingredients of the invention. In a preferred embodiment, each of these compositions is in single unit dosage form and comprising an amount of active ingredient sufficient to treat or prevent htaman infection by he=patitis C virus. 0025) Tn a specific embodiment, the invention encompasses a pharmaceutical composition comprising a TLR7 ligand selected from analogs and derivatives of a) guanosine, b) &midazoguinoline, c) adenine, and d) pyrimidine.

[0026] In another specific embodiment, the invention encompasses 8 pharmaceutical composition ceomprising a TLR7 ligand selected from Formulas Ia, Ib, Ic, Id, Ie, If, Ig, and

Th, or a pharmaceutically acceptable salt, hydrate, metabolite or stereoisomer there=of or a pharmaceutically acceptable salt or hydrate of said stereoisomer. 32 TLR7 Ligand Prodrugs

[0027] This invention also encompasses novel methods for the treatment 0x prevention of “hepatitis C viral infection, and novel pharmaceutical compositions vwhich utilize TLR? Eigand prodrugs or pharmaceutically acceptable salts, hydrates, metabolites or stereoisomers thereof.

[0028] This invention also encompasses novel methods of treating diseasess responsive to immuno therapy with immunologic agents, comprising orally admin _istering a

TLR7 ligand gorodrug to a patient in need of immuno therapy, wherein the TLR7 prodrug achieves a the=rapeutically effective plasma concentration of the TLR7 ligand in th_e patient.

[0029] In one embodiment, the invention encompasses a method of treatin ga hepatitis C vixus infection in a patient comprising orally administering to the patiesnt a TLR7 ligand prodrug or a pharmaceutically acceptable salt, hydrate, or stereoisomer thereof, wherein the oral administration of the TLR7 ligand prodrug achieves a therapeutically effective plasmna concentration of the TLR7 ligand while reducing undesirable sid-e effects associated with TLR7 ligands. In a preferred embodiment, the TLR7 ligand prodmrug is a masked TLRZ ligand prodrug.

[0030] In another embodiment, the invention also encompasses a method «of treating diseases responsive to immuno therapy while reducing undesirable side effects as=sociated with immuno Jogic agents, comprising orally administering a TLR7 ligand prodrug to a patient in need of immuno therapy, wherein the TLR7 prodrug achieves a therapeutically effective plasama concentration of the TLR7 ligand in the patient. In a preferred embodiment, the TLR7 ligand prodrug is a masked TLR7 ligand prodrug.

[0031] In another embodiment, the oral administration of the TLR7 liganA prodrug improves the in vivo bioavailability of the TLR7 ligand. Ina preferred embodime=nt, the oral administacation of the TLR7 ligand prodrug achieves an in vivo effective plasrma concentration. of the TLR? ligand that is 10% to 500% of the effective in vivo exposure

VWW0 2005/025583 PCT/US2004/028236 obtained upon oral administration of the TLR7 ligand alone. In another preferred embodiment, the oral administration of the masked TLR7 ligand prodrug achieves an in vivo effective plasma concentration of the TLR7 ligand that is 50% wo 200% of the effective in vivo exposure obtained upon oral administration of the TLR7 ligand alone.

[0032] In another embodiment, the oral administration of the TLR7 ligand prodrug reduces adverse side effects. In a preferred embodiment, the side effect comprises gastrointestinal irritancy, wherein gastrointestinal irritancy comprises hemorrhage, lesions, and emesis.

[0033] In another embodiment, the TLR? ligand prodrug improves oral availability by at least 25% and reduces gastrointestinal irritancy by at least 50%% in a patient relative to the oral administration of the TLR7 ligand alone. In another embodiment, the TLR7 ligand prodrug improves oral availability by at least 50% and reduces gastrointestinal irritancy by such that other toxicities become limiting in a patient relative to the oral administration of the TLR7 ligand alone.

[0034] In a preferred embodiment, the TLR7 ligand prodrugs achieves a } therapeutically effective plasma concentration that is 25% to 200% of the effective in vivo concentration of the TLR7 ligand in a patient after oral administrati on, with minimal gastrointestinal irritancy.

[0035] In one embodiment, the methods of the invention en«<compass administering to a patient in need thereof a therapeutically or prophylactically effective amount of a prodrug of a TLR7 ligand selected from analogs and derivatives of a) guanosine, b) imidazoquinoline, c) adenine, and d) pyrimidine.

[0036] In another embodiment, the methods of the inventior encompass administering to a patient in need thereof a therapeutically or prophylactically effective amount of a prodrug of a TLR? ligand selected from analogs and derivatives of a) guanosine, b) imidazoquinoline, c) adenine, and d) pyrimidine, wherein the prodrug is an (a) amide, carbamate, or amidine moiety after conversion of a TLR7 ligand amine substituent, (b) ester, carbonate, carbamate, ether, imidate, acetal, aminal, or ketal moiety after conversion of a TLR7 ligand alcohol substituent, (c) acetal or ketal moiety after conversion ofa TLR7 ligand keto substituent, (d) imidate moiety after conversion of a TLR7 ligand carbonyl of an amido substituent, (¢) deoxygenated moiety after comversion of a TLR7 ligand oxo substituent of pyrimidine or guanosine, or (f) amine.

[0037] In another embodiment, the methods of the invention encompass administering to a patient in need thereof a therapeutically or prophylactically effective amount of a prodrug of a TLR7 ligand selected from og Rg gw a ,

HSN N HSH Vas Ru

N= N, 1 om] oe | hau

R¢ OR? RS ORS js

Rr?

Ia , 3113 , Ie ; [o] R' [o}

HN N HN N

] Y Y S—y

HoN A { HN A =

Rg 0) RS <0 Le) hs N 55 pb 65 BRS OT

RG OR RSG OR R NTR nd , Me , If ,

R® 7 | »=0 .

Rw HoN Asn N .

Ye ro o : RO. OR?

Og and Ih , wherein: each R! is H, or a substituted or unsubstituted alkyl, alkenyl, or alkynyl, which may be interrupted by one or more O, S, or N heteroatoms, or a substitusted or unsubstituted aryl or heteroaryl;

R? is H, OH, SH, halo, or a substituted or unsubstituted alkyl, allkenyl, or alkynyl, which may be interrupted by one or more O, S, or N heteroatoms, or a substituted or unsubstituted -O-(alkyl), -O-(aryl), -O-(heteroaryl), -S-(alkyl), -S~(aryl), -S-(h eteroaryl), aryl, or heteroaryl;

R? is H, OH, or SH, or a substituted or unsubstituted alkyl, alkerayl, alkynyl, aryl, heteroaryl, -O-(alkyl), -O-(aryl), -O-(heteroaryl), -S-(alkyl), -S-Caryl), -S-(heteroaryl), -

NH(alkyl), -NH(ary?), -NHheteroaryl), -NH®R*)(atkyl), NHR *)(aryl), or

NH(R*)(heteroaryl);

R* is a substituted or unsubstituted alkyl;

R? is independently H, ~C(O)(Cy-1salkyl), or a racemic, L-, or Dw»- amino acid group

~C(O)CHNH.R’;

RS is H, OR, or NR");

R’ is independently H or a substituted or unsubstituted —C(O)(Cy-1zalkyl) or —C(0XA(Ci- 1salkyl);

R® is H, —OH, -O-(alky!), ~OCO2(Ci-15alkyD), —OQC(O)(C;-15alkyl), or a racemic, L-, or D- amino acid group ~OC(O)CHNH;RY;

R? is H, or a substituted or unsubstituted alkyl, C(O)CH(C:1s alkyl)NH, or -C(O)CH(CH>- ary)NHz;

R'is independently H, Cy alkyl, Cs alkenyl, C37 alkynyl, «(CRZR*?)(C4-Co aryD), -(CR'ZR'*)(C5-Cy cycloalkyl), (CR*R"*)(Ca-Cio heterocyclic), «(CR R®), 0H, {CRYR™),0C0;C1.1 alkyl, and -(CRR")oo NR')COC118 alkyl, and SO,(aryl), wherein t is an integer from 0 to 6 unless otherwise inckicated, and wherein the alkyl, alkenyl, alkynyl, aryl, cycloalkyl, and heterocyclic moieties of the foregoing groups are optionally substituted with substituents independently selected from halo, cyano, nitro, trifluoromethyl, triflucromethoxy, Ci-Ce alkyl, C2-Cs alkenyl, C2-Cs alkynyl, hydroxy, C;-

Cs alkoxy, -NHa, -NH-alky}, -N(alkyl);, -NH-aryl, N(alkyl)(aryl), -N(aryl), -NHCHO, -NHC(O)alkyl, -NHC(O)aryl, -N(alkyh)C(O)H, N(alkyl)C(O)alkyl, -N(ary)C(O)H, -N(aryl)C(O)alkyl, -NHCO:alkyl, -N(alkyl)COzalkyl, -NHC(O)NHj, -N(alkyl)C(O)NHz, “NHC(O)NH-alky!, -NHC(O) N(alkyl), N(akkyl)C(O)NH-alkyl, Nalky)C(O) N(alkyl)z, 2NHSO,-alkyl, -N(alky)SO-alkyl, -C(O)alky 1, -C(O)aryl, -0C(0)alkyl, -OC(O)aryl, -CO2- alkyl, -CO,-aryl, -COzH, ~C(O)NHz, -C(O)NE3-alkyl, -C(O)N(alkyl), -C(O)NH-aryl, -C(O)N(aryl),, -C(O)N(alkyl)(aryl), -S(O)atks/], -S(O)aryl, -SOzalkyl, -SOqaryl, -SO;NH;, -

SO,NH-alkyl, and -SO;N(alkyl)z;

R! is independently H, Cy alkyl, C3-Cio cycRoalkyl, or together with nitrogen forms a 5- or 6-membered heterocyclic ring;

RZ and Rare independently H, C1 alkyl, Cas alkenyl, or Cy.¢ alkynyl;

RM is H, Cy alkyl, or -CHa-aryl;

XisOorS;

Y is H, halo, OH, OR’, SH, SR*, or a substituted or unsubstituted alky! or aryl; and

Z is H, halo, OH, OR’, SH, or SR; or a pharmaceutically acceptable salt, hydrate, metabolite or sterecisomer thereof or a pharmaceutically acceptable salt or hydrate of said stereoisomer.

[0038] In another embodiment, the invention encompasses a method of treating or preventing a hepatitis C virus infection in a patient in need thereof comprising administering to the patient a therapeutically or prophylactically effective amount of a TLR7 ligand selected from Formula La, Lib, Lic, Tid, Ile, IIf, [Kg, and Ith, wherein R! is H or a substituted or unsubstituted alkyl, alkenyl, or alkynyl; R? is ¥, OH, halo, or a substituted or unsubstituted atkyl, alkenyl, or alkynyl, or -CH2—O-(alkyl); R®isH, OH, or SH, ora substituted or unsubstituted -O-(alkyl), -S-(alkylD), or -NH(alkyl); R® is independently H, —C(O)(Cs-1galkyl), or a racemic, L-, or D- amino acid group _C(O)CHNH,R’, wherein R’ is an unsubstituted alkyl; R® is H or OR'®, wherein R'” is independently Cis alkyl, Cs alkenyl, Cs; alkynyl, -(CR'?R"*){(Cs-C1o aryl), -&CRR'*){C4-Cyo heterocyclic), and (CR RP)oNR")CO,C118 alkyl, wherein t is aan integer from 0 to 4 unless otherwise indicated, and wherein the alkyl, alkenyl, aryl, and heterocyclic moieties of the foregoing groups are optionally substituted with 110 3 subsstituents independently selected from halo, cyano, nitro, trifluoromethyl, trifluoromethoxy, €i-Cs alkyl, C,-Cs alkenyl, C;-Cg alkynyl, hydroxy, C;-Cs alkoxy, -CO2-alkyl, -CO»-aryl, —OC(O)alkyl, and -OC(O)aryl, and wherein

R!? and RP" are independently H, C1.6 alkyl, or Cy alkenyl; and R'is H, -CHj, or —CH,CHs; R is independently H or a substitute d or unsubstituted ~C(0)(Cy-1salkyl) or ~C(O),(Ci-1salicyt); R® is H, —OH, -O-(alkyl), —€0CO,(Cy-15alkyl), or a racemic, L-, or D- amino acid group —-OC(O)CEANH:R}; XisOorS;Y is H, halo, OH, ORY,

SH, or SR% and Z is H, halo, OH, OR®, SH, or SR".

[0039] In specific embodiment, the inve=ntion encompasses a method of treating or preventing a hepatitis C virus infection in a patient in need thereof comprising administering to the patient a therapeutically or prophylactically effective amount of a prodrug of a TLR7 ligand selected from

J

CHaCH,0

NZ SN Yr

LI Sy ha ~~ ~~

HO oH , wo on R

0

Ns (NG ! AX 0 (o] as No = I =o Yo

Sw N Sy N nS Va fo)

Ho “on > wo om R nd on )

NHz NH, oy py

NN" N0CH,CH, X

Br Br ,

Q lo) NH,

NP ON Soke

XN x

[6] (o) r o 0 ~~ ~~

N ’d N ~ N > : LY \ 1

NHy NH» NH,

Oh Yoo Yoo CH 2Y3 ia 7 9 Ia q

OCH3 ~ . OCH 9 borg O 0

NHz 0 NH, o J

Yd I Soca Zo” NH 0 OCH. ~ ) C © N

Hs , CH , ~ ,

NH2 0, NH, fo) NH2 Ys

Yd Ys Hq CY CeHya

I i Yo i Sd

Ha , OCH, ,OCHs 2

NH 0 NH, 3 ~ 2 N, \ OCrHis NT” N ] jess Yr 7 0” ON N oN" N

C A ‘. ’

NHz pS — NH, 3 ~ geal Id lo / ae > ’ A he

CHa ’ CHa bd

NIH; \ NH, fo)

N Zz N (o] jes alli evo

J Na N 0 SN N cl

OCH, > C 3 lo) fo) CH3CH,0

Le Le Xe Ie en Ay NN N nS N Sh

AcO AcO HO HO

Ad OAC, ald onc , wo on, HO OH

Q

No

I 0 Po

HN S, NZ S, NZ S

CLI gos

SN EN N Sh N

Oo. 0

Pas & I Ss 5 5 YL

HO on, HO oH , HO OH a

[o}

CHL0 or

Cr X= PL

Han SN N NN N EN N

HO HO HO

Ho oH, al OAc , Hoe on and 0

Yo 0

NZ NS jeer

HN" ON : ~~)

A al Ac or a pharmaceutically acceptable salt, hydrate, or stereoisomer thereof or a pharmaceutically acceptable salt or hydrate of said stereoisomer.

[0040] In another preferred embodiment of the invention, the TLR7 ligand prodrug is an amin-o acid ester prodrug of the TLR? ligand. In another preferred embodim ent, the amino acid ester prodrug of the TLR7 ligand is a valyl ester.

[0041] In one embodiment of the invention, R’ is not a racemic, L-, or D- zamino acid group» ~C(O)CHNH,R®. In another embodiment, R® is not a racemic, L-, or ID- amino acid group» —C(O)CHNH,R® when the TLR7 ligand prodrug is selected from a compound of

Formula 1h.

[0042] In another alternative embodiment, the invention encompasses a method for treating or- preventing hepatitis C virus infection ina patient in need thereof, comporising administer-ing to the patient a therapeutically or prophylactically effective amount: of a prodrug oX a TLR7 ligand and a pharmaceutically acceptable excipient, carrier, or vehicle.

[0043] In a separate embodiment, the invention encompasses a method fom treating or preventing hepatitis C virus infection in a patient in need thereof, comprising administewing to the patient a therapeutically or prophylactically effective amount= of a prodrug of a TLR7 ligand and an additional therapeutic agent, preferably an additional antiviral oor immunomodulatory agent.

[0044] The invention also encompasses pharmaceutical compositions suitable for parenteral administration to a patient comprising a therapeutically or pharmaceutically acceptable amount a prodrug of a TLR7 ligand of the invention im a sterile form; pharmaceutical compositions suitable for parenteral administration to a patient comprising a thmerapentically or pharmaceutically acceptable amount of a prodrug of a TLR7 ligand of the iravention; pharmaceutical compositions suitable for mucosal adnministration to a patient comprising a therapeutically or pharmaceutically acceptable amowunt of a prodrug of a TLR7 1i_gand of the invention; and pharmaceutical compositions suitable for topical administration to a patient comprising a therapeutically or pharmaceutically acceptable amount ofa prodrug of a TLR7 ligand of the invention. Depending on the specific tissue to be treated, additional components, such as penetration enhancers, may be us-ed prior to, in conjunction with, or subsequent to treatment with active ingredients of the inwention. In a preferred embodiment, each of these compositions is in single unit dosage form and comprising an a_mount of active ingredient sufficient to treat or prevent human infection by hepatitis C wirus.

[40045] In a specific embodiment, the invention encompasses a pharmaceutical composition comprising a prodrug of a TLR7 ligand selected fromm Formula Ila, IIb, IIc, Hd,

I Xe, XIf, Ig, and Ith, or a pharmaceutically acceptable salt, hydrate or stereoisomer thereof or a pharmaceutically acceptable salt or hydrate of said stereoisome=r. [ 0046] In another embodiment of the invention, and depending on the specific tissue t-o be treated, additional components including, but not limited tow penetration enhancers, molecules which target the area of the infection and molecules w=hich reduce the in vivo toxicity of the prodrug of a TLR7 ligand may be used prior to, inc conjunction with, or subsequent to treatment with one or more prodrugs of TLR7 ligaends of the invention.

[0047] The TLR7 ligand prodrugs are useful as immune system enhancers and have

Certain immune system properties including modulation, mitogemicity, augmentation, and/or potentiation or they are intermediates for compounds that have these properties. The compounds are expected to express effects after administration t« a mammal on at least one ofthe cell populations characterized as the natural killer cells, m_acrophages, dendritic cells, and lymphocyte cells of the immune system of a host. Because ofthese properties they are wiseful as an anti-infective including, but not limited to antiviral agents, and as antitumor agents or as intermediates for the same. They can be used to treat an affected host by sserving as the active ingredients of suitable pharmaceutical comypositions.

RF 0048] In one aspect of the invention, TLR7 ligand prodarugs are utilized to treat the ull range of viral diseases in mammals by administering to the rmammal a therapeutically effective amount of the compounds. Viral diseases contemplated to be treated with TLR7

Rigand prodrugs include acute and chronic infections caused by both RNA and DNA viruses. Without limiting in any way the range of viral infections that may be treated, TLR7 ligand prodrugs are particularly useful in the treatment of infections «caused by adenovirus, cytomegalovirus, hepatitis A virus (HAV), hepatitis B virus (HBV), ~flaviviruses including

Yellow Fever virus, hepacivirus including hepatitis C virus (HCV), Inerpes simplex type 1 and 2, herpes zoster, human herpesvirus 6, human immunodeficiency virus (HIV), human papilloma virus (HPV), influenza A virus, influenza B virus, measless, parainfluenza virus, pestivirus, poliovirus, poxvirus (including smallpox and monkeypox— virus), rhinovirus, coronavirus, respiratory syncytial virus (RSV), multiple families of wyiruses that cause hemorrhagic fevers, including the Arenaviruses (LCM, Junin virus, MMachupo virus,

Guanarito virus, and Lassa Fever), the Bunyaviruses (Hanta viruses .and Rift Valley Fever) and Filoviruses ( Ebola and Marburg virus), a range of viral encephaalitides including West

Nile virus, LaCrosse virus, California Encephalitis virus, Venezuelamn Equine Encephalitis virus, Eastern Equine Encephalitis virus, Western Equine Encephalistis virus, Japanese

Encephalitis virus, Kysanur Forest virus, and tickborne viruses such as Crimean-Congo

Hemorrhagic fever virus.

[0049] In another aspect of the invention, TLR7 ligand prodsrugs are utilized to treat bacterial, fungal, and protozoal infections in mammals by administering to the mammal a therapeutically effective amount of the prodrugs. The full range of pathogenic microorganisms is contemplated to be treatable by the TLR7 ligand prodrugs of the present invention, including without limitation those organisms that are resistant to antibiotics. The ability of TLR7 ligand prodrugs to activate multiple components of ~the immune system bypasses resistance mechanisms commonly found to reduce susceptibility to antibiotics, and thus treatment of infections in a mammal caused by such resistant microorganisms by TLR7 ligand prodrugs is a particular utility of the present invention.

[0050] In another aspect of the invention, TLR? ligand prodmrugs are utilized to treat tumors in mammals by administering to the mammal a therapeutical ly effective amount of the prodrugs. Tumors or cancers contemplated to be treated include= both those arising from aberrations in normal cellular processes as well as those caused by wirus, and the effect may involve inhibiting the spread of cancerous cells, accelerating the kill-ing of cancerous cells, inhibiting transformation of virus-infected cells to a neoplastic state, inhibiting the spread of viruses from transformed cells to other normal cells, and/or arresting the growth of virus- transformed cells. The prodrugs of TLR7 ligands are expected to bes useful against a broad spectrum of tumors including but not limited to carcinomas, sarcom-as, and leukemias.

Included in such a class are mammary, colon, bladder, lung, prostatez, stomach, and pancreas carcinomas and lymphoblastic and myeloid leukemias,

[0051] In another aspect of the invention, a method of treating a mammal comprises administering a therapeutically and/or prophylactically effective amount ofa pharmaceutical containing a TLR7 ligand prodrug of the invention. In this aspect the effect may relate to modulation of some portion of “the mammal’s immune system, especially modulation of cytokine activities of Thi and Th2, including but not restricted to the interleukin family, e.g., IL-1 through IL-12, zand other cytokines such as TNF alpha, and interferons including interferon alpha, interfesron beta, and interferon gamma, and their downsteam effectors. Where modulation of “Th1 and Th2 cytokines occurs, itis contemplated that the modulation may inclucle stimulation of both Th1 and Th2, suppression of both Thi and Th2, stimulatiomn of either Thl or Th2 and suppression of the other, or a bimodal modulation in which one: effect on Th1/Th2 levels (such as generalized suppression) occurs at a high concentration, “while another effect (such as stimulation of either Th1 or Th2 and suppression of the oth_er) occurs at a lower concentration.

[0052] In another aspect of this invertion, pharmaceutical compositions containing a prodrug of a TLR? ligand are administered im therapeutically effective doses to a mammal that is receiving immunomodulatory drugs not included in this invention. Ina preferred aspect, the doses of the immunomodulatory «drug are reduced below their customary effective dose, to reduce adverse effects. In a second preferred aspect, the immunomodulatory drug is used at its custoxmary dose, but with an improved therapeutic effect when a prodrug of a TLR7 ligand is allso administered.

[0053] In another aspect of the invermtion, pharmaceutical compositions containing a prodrug of a TLR7 ligand are administered in a therapeutically effective dose to a mammal that is receiving anti-infective drugs not included in this invention. Ina preferred aspect of this invention, the pharmaceutical compositi_ons containing a prodrug of a TLR7 ligand are administered in a therapeutically effective dese with anti-infective drug(s) that act directly upon the infectious agent to inhibit the grow~th of or kill the infectious agent. 4, BRIEF DESCRIPTION OF THE IDRAWINGS

[0054] Figure 1 is a graphical depict ion of plasma levels of isatoribine and interferon alpha in mice.

[0055] Figure 2 is a graphical depict jon of Viral Load Changes in HCV infected

Patients receiving isatoribine. 5. DETAILED DESCRIPTION OF WHE INVENTION 5.1 DEFINITIONS

[0056] Where the following terms are used in this specification, they are used as defined below: 10057) The terms “comprising” and “including” are used herein in their open, non- limiting sense. [00s8) The term “nucleoside” refers to a compound composed of any pentose or modified pentose moiety attached to a specific pos ition of a heterocycle or to the natural position of a purine (9-position) or pyrimidine (1-position) or to the equivalent position in an analog.

[0059] The term “purine” refers to nitrogerious bicyclic heterocycles.

[0060] The term “D-nucleosides” refers to the nucleoside compounds that have a D- ribose sugar moiety (e.g., Adenosine).

[0061] The term “L-nucleosides” refers to the nucleoside compounds that have a L- ribose sugar moiety.

[0062] The term “immunomodulator” refe 1s to natural or synthetic products capable of modifying the normal or aberrant immune system through stimulation or suppression.

[0063] The term “NOAEL?” is the No Observed Adverse Event Level, which is a toxicology term for the dose of drug that results ira no significant toxicity under the specified conditions of dose level, frequency, duration in a selected species.

[0064] “Ligand” means a low molecular weight molecule capable of binding to a biologic receptor. A ligand may be either an agomist or an antagonist, or may have no effect.

[0065] An “agonist” is a ligand that, upon_ binding, stimulates the receptor to exert a biologic response that is consistent with the normal biologic activity of the receptor.

[0066] An “antagonist” is a ligand that, upon binding, causes the receptor to not exert the normal biologic activity of the receptor.

[0067] The term “mammal” includes bot animals and humans.

[0068] The term "preventing" refers to the ability of a compound or composition of the invention to prevent a disease identified hereian in mammals diagnosed as having the disease or who are at risk of developing such disevase. The term also encompasses preventing further progression of the disease in mammals who are already suffering from or have symptoms of such disease.

[0069] The term "treating" refers to: 0) preventing a disease, disorder, or condition from occurring in a mammal that may be predisposed to the disease, «disorder and/or condition, but has not yet been diagnosed as having it;

(ii) inhibiting the disease, disorder, or condition, i.e., arresting its development; and (iii) relieving the disease, disorder, or condition, i.e., causing regression of the disease, disorder, and/or condition.

[0070] The terms “0 and “B” indicate the specific stereochemical configuration ofa substituent at an assymmetric carbon atom in a chemical structure as drawn.

[0071] The terms “patient” or “subject” mean an animal (e.g., COW, horsez, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit, guinea pig, etc.) or a mannmal, including chimeric 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, most preferably a Jhuman. In a specific embodiment the patient or subject is infe«cted by or exposed to the hepatitis C virus. In certain embodiments, the patient is a human infant (age 0-2), child (age 2- 17), adolescent (age 12-17), adult (age 18 and up) or geriatric age 70 and up) patient. In adcdition, the patient includes immunocompromised patients such as HIV positive patients, Cancer patients, patients undergoing immunotherapy or chemotherapy. In a particular embocliment, the patient is a healthy individual, i.e., not displaying symptoms of other viral infections.

[0072] The term a “therapeutically effective amount” refers to an amount of the

TLR?7 ligand or prodrug of a TLR7 ligand of the invention sufficient to provide & benefit in the treatment or parevention of viral disease, to delay or minimize symptoms associated with viral infection or wiral-induced disease, or to cure or ameliorate the disease or infection or cause thereof. In =particular, a therapeutically effective amount means an amount sufficient to provide a therapeutic benefit in vivo. Used in connection with an amount of & compound of the invention, the term preferably encompasses a non-toxic amount that improves overall therapy, reduces car avoids symptoms or causes of disease, or enhances the therapeutic efficacy of or synesrgies with another therapeutic agent. {0073] Thee term a “prophylactically effective amount” refers to an amount of a compound of the invention or other active ingredient sufficient to result in the prevention of infection, recurrerace or spread of viral infection. A prophylactically effective armount may refer to an amount sufficient to prevent initial infection or the recurrence or spre ad of the infection or a dise=ase associated with the infection. Used in connection with an amount ofa compound of the invention, the term preferably encompasses a non-toxic amoumt that improves overall gorophylaxis or enhances the prophylactic efficacy of or synergizes with another prophylacstic or therapeutic agent.

[0074] The tersm “in combination” refers to the use of more than one prophaylactic and/or therapeutic agents simultaneously or sequentially and in a manner that theim respective effects are additive or synergistic.

[0075] The term “pharmaceutically acceptable salts” refer to salts preparecd from pharmaceutically accesptable non- toxic acids or bases including inorganic acids ard bases and organic acids and bases. If the inventive TLR7 ligand prodrug is a base, the Hesired pharmaceutically acceptable salt may be prepared by any suitable method available in the art, for example, treatmment of the free base with an inorganic acid, such as hydroc-hloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, or ~with an organic acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumar-ic acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidy1 acid, such as glucuronic acid or galacturonic acid, an alpha-hydroxy acid, such as citric acid_ or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, sucha as benzoic acid or cinnammic acid, a sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid, or the like. If the inventive TLR7 ligand prodrug is an acid, the desired pharmaceutically acceptable salt may be prepared by any suitable method, for example, treatment of the free acid with an inorganic or organic base, such as an amine (pr “mary, secondary or tertiary)», an alkali metal hydroxide or alkaline earth metal hydroxid e, or the like. Illustrative exarmples of suitable salts include organic salts derived from am ino acids, such as glycine and arginine, ammonia, primary, secondary, and tertiary amines, and cyclic amines, such as piper-idine, morpholine and piperazine, and inorganic salts derive=d from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, alumintam and lithium.

[0076] The teerm "prodrug" is intended to mean any chemical entity that aafier administration is con-verted via metabolic actions or solvolysis to a different chermical entity that retains biological activity.

[0077] The tesrm "TLR7 ligand prodrug" is intended to mean any chemical entity that after administrat-ion is converted via metabolic actions or solvolysis to a diffserent chemical entity that retains biological activity and that is a ligand for TLR7. A TLR7 ligand prodrug may itself bes a ligand for TLR7, or it may be "masked" in that it does not function efficiently as a TLR7 ligand.

[6078] The tesrm "masked TLR7 ligand prodrug" is intended to mean any~ chemical entity that after admi nistration is converted via metabolic actions or solvolysis to a different chemical entity that retains biological activity and that is a ligand for TLR7, and where the administered chemical entity is a less efficient ligand for TLR7 than the chemical entity aris=ing from metabolic conversion or solvolysis.

[00-79] The term “a pharmaceutically active metabolite” &is intended to mean a phasrmacologically active product produced through metabolism in the body of a specified compound or salt thereof.

After entry into the body, most drugs are substrates for chemical reactions that may change their physical properties and biologic effects.

These metabolic comversions, which usually affect the polarity of the TLR7 ligand, alter the way in which dru _gs are distributed in and excreted from the body.

However, im some cases, metabolism of a drug is required for therapeutic effect.

For example, many anticancer drugs of the anti- metabolite class must be converted to their active forms after thesy have been transported into a cancer cell. [00»80] As used herein, unless otherwise specified, the team “alkyl” means a satwurated straight chain or branched non-cyclic hydrocarbon hav-ing from 1 to 20 carbon ato ms, preferably 1-10 carbon atoms and most preferably 1-4 carbon atoms.

Representative satwrated straight chain alkyls include -methyl, -ethyl, -n-propyl,, -n-butyl, -n-pentyl, -n- hexyl, -n-heptyl, -n-octyl, -n-nonyl and -n-decyl; while saturatecl branched alkyls include - isopropyl, -sec-butyl, -isobutyl, tert-butyl, -isopentyl, 2-methylBsutyl, 3-methylbutyl, 2- mesthylpentyl, 3-methylpentyl, 4-methylpentyl, 2-methylhexyl, 3-methylhexyl, 4- me=thylhexyl, 5-methylhexyl, 2,3-dimethylbutyl, 2,3-dimethylpemtyl, 2,4-dimethylpentyl, 2,3 -dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-d imethylpentyl, 2,2- dimnethylhexyl, 3,3-dimtheylpentyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylpentyl, 3- eth-ylpentyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3- eth ylpentyl, 2-methyl-4-ethylpentyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2- me=thyl-4-ethylhexyl, 2,2-diethylpentyl, 3,3-diethylhexyl, 2,2-diethylhexyl, 3,3-diethylhexyl and the like.

An alkyl group can be unsubstituted or substituted [Om81] Ass used herein, unless otherwise specified the term “aryl” means a carbocyclic aromatic ring containing from 5 to 14 ring atoms.

T he ring atoms of a carbocyclic aryl group are all carbon atoms.

Aryl ring structures include compounds having one or more ring structures such as mono-, bi-, or tricyclic compounds as well as benzo- fus ed carbocyclic moieties such as 5,6,7,8-tetrahydronaphthyl ard the like.

Preferably, the aryl group is a monocyclic ring or bicyclic ring.

Representative aryl groups include phenyl, tol], anthracenyl, fluorenyl, indenyl, azulenyl, phenanthrenyl ard naphthyl.

A carbocyclic ary~1 group can be unsubstituted or substituted. [om82] The term “substituted” means that the specified gzroup or moiety bears one or monre substituents.

The term "unsubstituted" means that the specified group bears no substituents. A “substituted alkyl” or “substituted aryl” is substitsited by one or more substituents including halogen (F, Cl, Br, or I), lower alkyl (C16) “OH, -NO3, -CN, -CO.H, -O-lowe: alkyl, -aryl, -aryl-lower alkyl, -CO.CH, -CONH,, -OCM];CONH,, -NH;, -

SO,NH_=, haloalkyl (e.g, -CFs3, -CH2CF3), -O-haloalkyl (e.g, -OCF;, -OCHF?,), and the like.

[0083] As used herein and unless otherwise indicated, the= term “optically pure” or “stereonmerically pure” means a composition that comprises one sstereoisomer of a compound and is substantially free of other stereoisomers of that compound. For example, a stereommerically pure compound having one chiral center will bes substantially free of the opposites enantiomer of the compound. A typical stereomerically~ pure compound comprises greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, more preferably greater than about 90% by- weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, 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 stereoissomers of the compound, and most preferably greater thara about 97% by weight of one ster-eoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound. Since many of the compounds of the invention comprise sacchar-ides which can exist in either the D or L forms, the inven tion encompasses either or both D and L sugars. As such, for example, a stereomerically puare D sugar will be substarmmtially free of the L form. In an alternative embodiment, the use of L forms of a

TLR?7 1 igand will be substantially free of the D form. Thus, the “methods and compositions disclosed herein include in an alternative embodiment the use of such levorotatory sugars or polyme=rs made therefrom.

[0084] The compounds of the invention may exhibit the phenomenon of tautomeerism. While Formulas I and II cannot expressly depict all possible tautomeric forms, itis to be understood that Formula 1 is intended to represesnt any tautomeric form of the depwicted compound and are not to be limited merely to a spe=cific compound form depicte=d by the formula drawings. For example, it is understood that regardless of whether or not the substituents are shown in their enol or their keto form, they represent the same compound (as shown in the Formula Ila example below). o) OH

HN z

IS UR ES IPN

AS HO oH AN we om

5.2 Identification of TLR7 Ligands

[0085] Known TLR? ligands include, but are not limited to (1) guanosine analogs, such as 7-deazaguanosine and related compounds, inclvading but not limited to those described in Townsend, J. Heterocyclic Chem, 13, 1363 (1976), and Seela, et al, Chem.

Ber., 114(10), 3395-3402 (1981); 7-ally}, 8-ox0-guanossine (loxorabine) and related compounds, including but not limited to those described in Reitz, et al., J. Med. Chem, 37, 3561-3578 (1994); 7-methyl, 9-deazaguanosine and re] ated compounds including, but not limited to, those described in Girgis et al., J. Med. Chewn., 33, 2750-2755 (1990); 8- bromoguanosine and other 8-halogen substituted purines compounds including, but not limited to, those described in United States Patent No. 4,643,992; 6-amino-9-benzyl-2- butoxy-9H-purin-8-ol, and other 2, 6, 8, 9 -substituted gourines including, but not limited to, those described in 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 ef al., J. Org. Chem., 64 (22), 8411-8412 (19°99), Barrio et al; J. Org. Chem., 61, 6084-6085 (1996), United States Patent No. 4,539,205, United States Patent No. 5,011,828,

United States Patent No. 5,041,426, United States Patent No. 4,880,784, and International

Patent Application Publication Nos. WO 94/07904; (23 imidazoquinolines, including but not limited to 1-(4-amino-2-ethoxymethyl-imidazo[4,5-c]quinolin-1-y])-2-methyl-propan-2-ol (imiquimoid), as described in International Patent App lication Publication No. WO 94/17043; 1-isobutyl-1H-imidazo[4,5-c]quinolin-4-ylasmine (resiquimoid) as described in

International Patent Application Publication No. WO 94/17043 and United States Patent

Application Nos. 10/357,777 (United States Patent Application Publication No. US 2003/0195209), 10/357,733 (United States Patent App lication Publication No. US 2003/0186949), 10/358,017(United States Patent Appkication Publication No. US 2003/0176458), 10/357,995(United States Patent AppRication Publication No. US 2003/0162806), 10/165,222 (United States Patent Appelication Publication No. US 2003/0100764), 10/011,921(United States Patent AppRication Publication No. US 2003/0065005) and 10/013,059 (United States Patent Application Publication No. US 2002/0173655); United States Patent No. 5,395,937; Imternational Patent Application

Publication No. WO 98/17279; and (3) pyrimidine derivatives, including but not limited to 2-amino-6-bromo-5-phenyl-3 H-pyrimidin-4-one (bropirimine), and similar substituted pyrimidines including, but not limited to, those described in Wierenga et al., 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, ef al., J. Med. Chem., 23, 237-239 (1980), and

Fujiwara ef al., Bioorg. Med. Chem. Lett., 10(12) 13177-1320 (2000). The entireties of each of the patents, patent publications and publications identified herein are incorporated herein by reference.

[0086] In addition to the above TLR7 ligands, additional TLR7 ligands can be readily identified by known screening methods. See, e.g., Hirota ef al., J. Med. Chem., 45, 5419-5422 (2002); and Akira S. et al., Immunology Letters, 85, 85-95 (2003). Using a variant of one of these known screening methods (as described in Section 6.1), analogs and derivatives of adenine were also identified as TLR 7 ligands. Adenine derivatives known in the art are described in European Patent Application Publication Nos. EP 1035 123,EP 1 043 021, and EP 0 882 727; United States Patent No. 6,376,501; United States Patent No. 6,329,381; United States Patent No0.6,028,076, and United States Patent Application

Publication No. US 2003/0162806.

[0087] The TLR7 ligands of Formulas Ia-Ih can be synthesized using methods known to one of skill in the art, particularly in lighat of the references and patents listed above. 53 Preparation of TLR7 Ligand Prosdrugs

[0088] The TLR7 ligand prodrugs of the iravention are prepared by making an (2) amide, carbamate, or amidine moiety after converssion of a TLR7 ligand amine substituent, (b) ester, carbonate, carbamate, ether, imidate, acetal, or ketal moiety after conversion of a

TLR?7 ligand alcohol substituent, (c) acetal or ketal moiety after conversion of a TLR7 ligand amine substituent, (d) imidate moiety after conversion of a TLR7 ligand carbonyl of an amido substituent, (¢) deoxygenated moiety after conversion of a TLR7 ligand oxo substituent of pyrimidine or guanosine, or (f) amine. For example, TLR7 ligand prodrugs are prepared by either (1) converting an hydroxyl (OH) substituents of the TLR7 ligand into an amino acid ester, or (2) making an amine subst ituent of the TLR7 ligand into an amide or carbamate. The process for preparing prodrugs is well known in the art and is described by

Burger's Medicinal Chemistry and Drug Chemistay, 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 , 765-767; Bagshawe, Drug Dev. Res., 34, 220-2300 (1995); Bodor, Advances in Drug Res., 13, 224-331 (1984); Bundgaard, Design of Prodrzigs (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).

[0089] Schemes 1-18 show a general procsedure to prepare representative compounds of Formula II.

[0090] Schemes 1-6 describe how 5'-amiro acid esters can be synthesized from analogs and derivatives of guanosine.

Scheme 1 0 fo e wT HN HN \ HN TY ge i na SN No, BAS Np Sy Nog HNTNT TN

HO o HO 0 mo No © HN 0)

Sx sz [lo] = EES 2 a>

HO OH S 2 ~N 65d AN nd oH 1 2 3 4

Townsend, JHC, 13, 1876. 1363

Seela, et al, Chem. Ber, 114, 10, 1981, 3395-3402 a) 2,2-dimethoxypropane, acetone, DMSO, MeSO3H, 0 °c b) BOC-NHCHR'COZH, EDC, DMAP, PhMe, 0 °C - 1t c) anh. HCI, PrOAc, PrOH

Scheme 2 [o} CH, [0] CH, o CH, 0 CH, yt HN N HN N HN pe

IL, =o Ye Lo LIL

ASN No HSN Nob aS c ha N

RH 0 HCG

HO HO LEN LN

WO OH 6,0 0A 8.0 AN Ho oH 6 7 8

Retz, et a), JMC, 37, 1984, 3561-3578 a) 2,2-dimethoxypropane, acetone, DMSO, MeSOgH, 0 °c b) BOC-NHCHR'COZH, EDC, DMAP, PhMe, 0 °C - rt ¢) anh. HCI, PrOAc, iPrOH

Scheme 3 o] CH, 0 Hs 0 CH, a CH,

SQ ® SQ yw; sv

HAZ { a Ha { HN" SN c HNN

H —T 2HCl go

Ho N° HOP moo AL 0 wo 0

ST Ean o_AL St AN S %

HO OH a. .0 0.0 HO OH

Pal X 9 10 11 12 , Girgls, et al, JMC, 33, 1980, 276€-2755 a) 2,2-dimethoxypropane, acetone, DMSO, MeSO3H, 0 °C b) BOC-NHCHR'!CO2H, EDC, DMAP, PhMe, 0 °C - rt c) anh. HC, PrOAG, /PrOH :

Scheme 4 fo a 0 0

Ae HAS Te OY POY

HNTSNTN HNN b HN SN N © HNTSNTN - - HO TT Hol g

H 0 Hom © ot HN Jo 0 won 'y AN AN wd on

Pal Pa 13 14 15 16 8&-bromoguanosine [303136-79-0] commercially available a) 2,2-dimethoxypropane, acetome, DMSO, MeSO3H, 0 °C b) BOG-NHCHR'CO,H, EDC, DMAP, PhMe, 0 °C - rt ©) anh. HCI, IPrOAc, ProH

[0091] In a typical synthetic route, the 2',3'-hydroxyl groups of the B-D-ribose moiety of Formulaa Ia, Ib, Id, le, or Th can first protected, preferably with an acetonide as shown for 2, 6, 10, or 14. The free 5'-hydroxyl can then be subjected to a variety of esterification methods with a N-protected amino acid to form 3, 7, 11, or 15. The nitrogen of the amino acid ester an d the 2',3'-hydroxyls of the ribose unit can then be subjected to various deprotection condlitions, preferably concurrently, followed by salt formation of the free amine of the amino a«<id ester as illustrated for 4, 8, 12, or 16. ’

Scheme S

PRY, YET my wo ay wy

I »=o0 =o fo

VN a a NSN Lo b HAS No HSN N —_— —_— H — 2HCI ~~) oe] ot Jon I ne oH 4% AS S5_o AN ud on < x 17 18 19 20

Reitz, et al, JMC, 37, 1894, 3561-3578 : a) 2,2-dimethoxypropane, acetone, DMF, M@SO3H, 0 °c b) BOC-NHCHR'CO,H, EDC, DMAP, PhMe, 0 °C - 1t ¢) AcCl, CH3OH

Scheme 6 [o} rhe wr 2 yes

J 0 © ] 0 [ >=o0 han ey a HNN ! Vey b Han 's c HNN ¥ = — H © 2H ~~) o~) word Son vo Joo 0

He oH i: A do AN wd om x Pat 2 2 23; 24

Kinl, et al, JMC, 34, 1981, 3006-3010 a) 2,2-dimethoxypropane, acetone, DMSO. MeSOH, 0 °C b) BOC-NHCHR'CO2H, EDC, OMAP, Phivte, 0 °C - rt ¢) anh. HCI, IPrOAc, iPrOH

[0092] In the synthetic routes shown in Schemes 5 and 6, the 2',3'-hydroxyl groups of the B-D-ribose moiety of compound 17 and 21 were first protected with an acetonide to form 18 and 22 respectively. The free 5'-hydroxyl was then subjected to esterification with a N-tert-butoxycarbonyl valine to ¥orm 19 and 23 respectively. The nitrogen of the amino acid ester and the 2',3"-hydroxyls of the ribose were concurrently deprotected forming the hydrochloride salts as illustrated for 20 and 24.

[0093] Schemes 7 and 8 describe how carbamates and carbonates can be synthesizecd from analogs and derivatives of aclenine.

Scheme 7

NH2 0

H

I oH BuO. Tom oy N + hf : Ee

AN

S cs NO2

Hirota, et al, JMC, 45, 26 Gangwar, etal, JOC, 62, 2002, 5419-5422 1997, 1366-1382

H 9 fo} 0 0

Boy oo " oli o—o tm

OH I[ HoH ra a b Ea N° 27 28 (Example of IIf) . a) HOBT, DMF,CHClp, 0 °C b) TFA, CHaClg, 0 °C - rt

[0098] In a typical synthetic route, the amino group of Forneula If can be subjected to a variety of conditions with carbonates or chloroformates to forn carbamates. In the case of 27 , the N-terminal protected amine of the resulting amino acid esster can be subjected to deprotection conditions to form salts such as 28.

Scheme 8

NH, NH; } 8 NZ OCgHy3 [Yon Qa

SN N 2, ia N i's oS 5 Iv 29 30

Kurimota, et al, Bioorg. Med. Chem., 12, 2004, p.1091-1099 a) CgH130C(O)CY, (iPr) NEL, CHyCly, MeOH, DMAP, 0-35 °C [0095S] In Scheme 8 the hydroxy! group of adenine derivative 29 was esterified with n-hex=yl chlorofomate to give carbonate 30. [0096s] Schemes 9 and 10 describes how carbamates and car be synthesized from imidemzoquinoline analogs.

Scheme 9

NHz o 0 lo] lo]

WA AL, I Ao x N + _— Z N ~ HMPA, rt NTT =""N

NO; ~~ 31 Nicolaides et al, 32 Alexander, et al, JMC, 31, 33 (Example of Tic)

WO 94/17043 1988, 318-322

[00977] In a typical synthetic route, the amino group of analogss of Formula Ic can be subjected to a variety of conditions with carbonates, pyrocarbonates om chloroformates to . form carbamates.

Scheme 10 fo)

NH ~~oP

NZ NPN a) [CsH110C(O)]20, NEt3, CHCl3, 40 °C

[0098] In Scheme 10 imidazoquinoline 31 was treated with n—pentyl pyrocarbonate to give pentyl carbamate 34.

[009] Schemes 11-12 described how to synthesize carbamatess and imidates of pyrimmidines of formula Ig.

Scheme 11 lo}

NH; IN

EN NAN

No a Yo

Br Br 36 (Example of IXg)

Wierenga et al, JMC, 23, Fan et al, JHC, 30, 1980, 239-240 1993, 1273-1276 a) [EtO(CO)1,0, NEt3, DMF, 65 °C [00:00] In a typical synthesis of carbamates, the amino group «of 35 was subjected to ethy/1 pyrocarbonate under conditions shown above to form carbamatee 36.

Scherme 12

NH NH

NH NN

Np a ] "“OCH,CHz r 37 (Example of Ig)

Wierenga et al, JMC, 23, 1980, 239-240 a) polymer supported PPhy, EXOH, DEAD, THF, rt

[00101] In a typical synthesis of imidates, the amino group of 35 was subjected to ethyl alcohol under Mitsunobu type condition s shown above to form ethoxy derivative 37.

[00102] Scheme 13 describes how carbramates and can be synthesized from imidazoquinoline analogs. :

Scheme 13 x

Et0” “NH 1 N o— NN o/

NU EtO,CCl NTT >

NN —_— x pyr, toluene, — 0°C- st bn

OH OH

38 as al, 39 (Example of Ic)

[00103] In a typical synthetic route, the amino group of a derivative of Formula Ic can be subjected to a variety of conditions wi th carbonates, pyrocarbonates or chlaroformates to form carbamates.

[00104] Scheme 14 shows a general procedure for preparing 7-allyl-2-Amino-9-8-D- ribofuranosyl-7,9-dihydro-purin-8-one.

Scheme 14 o oJ a J

N

Yes HN Ne N“ ] Se 0 0 8 0

HSN Ne a HSN Ps b nS N —— ———eeefe o oC ~ ~~)

HO OH AO DdAc AO OAc 17 40 41

J J

NY eo AY =o c HNN N d HNN N eC) ~~

AD BAC HS on ry) 43 a) Ac,0, DMAP, NEt;, CH;CN b) POC, 75 °C ©) Zn-Cu, AcOH, 70 °C d) KzCO3, CH3OR, 1t

[00105]) In a typical synthetic routes, 7-allyl-2-amino-9-B-D-ribofuranosyl-7,9- dihydro-1H-purine-6,8-dione 17, was protected at the 2',3',5’-hydroxyl groups of the B-D- ribose, preferably with acyl groups as shown for 40, can be subjected to a variety of conditions to convert the carbonyl at the C-6 position to various groups, including but not limited to halogen, as shown for 41, that are susceptible to reduction. Following reduction under hetero- or homogeneous reaction conditioms, the 2',3',5 >-hydroxyls of the ribose unit are then subjected to appropriate deprotection conditions, to produce 43. Compound 43 can further be appropriately modified if so desired.

[00106] Scheme 15 shows a general procesdure for preparing 7-allyl-2-Amino-6- ethoxy-9-p-D-ribofuranosyl-7,9-dihydro-purin-&-one.

Scheme 15 lo] A CHaCHy A CH3CH,0 A eo AL hee

HN ‘a a HAS a b NSN N 0 ~) oC) oC)

AG OAc AG OAc HG OH 40 44 45 a) polymer supported PPh, EtOH, DEAD, THF, tt b) K,CO3, GHyOH, it

[00107] In a typical synthetic route, 40 can be subjected to a variety of conditions to . convert the carbonyl at the C-6 position to various imido-ethers, including but not limited to ethyl, as shown for 44. The 2',3',5’-hydroxyls of the ribose unit are then subjected to appropriate deprotection conditions, to produce 45. Compound 45 can further be appropriately modified if so dessired.

[00108] Scheme 16 describes how ethers can be synthesized from analogs and : derivatives of adenine.

Scheme 16

NH, NH; NH, oY Oe YY -emc ia \ J “Sw a b oN 0 oS 5 Io! ig o 62 63 64

Kurimota, et al, Bioorg. Med. Cher., 12, 2004, p. 1091-1089 a) Bra, CHCl, b) NaOEt, EtOH

[00109] In a typical synthetic route, the adenine derivative can be halogenated at C-8.

The halogen can then be displaced with an appropriate alkoxide to form derivatives such as 64.

[00110] Scheme 17 shows a general procedure for preparing 7-allyl-2-Amino-6- substituted alkoxy-9-B-D-ribofuranosyl-7,9-dihydro-purin-8-ones.

Scheme 17 i A i A or ya. 0 | o

SN a a HSN se b

HG BH EtSIO BsiEL 17 69 0 0 yg 3 a > 0 J LJ 0 _— « IL =o

HAS N HSN N

EtsSiO HO

EtsSi0 DSE HG OH 70 mn a) Et3SiCl, imidazole, DMF, it

Ao b) Bon polymer supported PPhy, DEAD, THF, rt ©) HF-NEty, CHyOH, it

[00111] In a typical synthetic route, the hydroxyl groups on ribose of 17 can "be protected as silyl ethers. The carbonyl at the C-6 position of 69 can be subjected to =a variety of conditions to comvert the carbonyl to various imido-ethers, including but not limited to the ether of 4-hydreoxymethyl-5-methyl-[1,3]dioxol-2-one, as shown for 70. The 2',3'5°- hydroxyls of the riboose unit are then subjected to appropriate deprotection conditioras, to produce 71.

[00112] Schesme 18 shows a general procedure for preparing 7-allyl-2-Aminc-6- substituted alkoxy-—9-B-D-ribofuranosyl-7,9-dihydro-purin-8-ones.

Scheme 18

/ x J x J ~ . - £0 pe ~ Eo” N iy wind WAL AT uso ~°) sor) oC)

ElSI6 bsiety Et,Si0 sie, HG oH 69 ” 73 8) HOGH,N(CHy)CO,EY, polymer supported PPhs, DEAD, THF, it b) HF-NEta, HOH, rt [0®113] The carbonyl at the C-6 position of 69 can be subjected to a variety of conditions to convert the carbonyl to various imido-ethers, including but: not limited to the ether of N-methyl-N-(hydroxymethyl)urethane, as shown for 72. The 2',3"5°-hydroxyls of thee ribose unit are then subjected to appropriate deprotection conditions, to produce 73.

Compound 73 can further be appropriately modified if so desired. 54 METHODS OF TREATMENT AND PREVENTION OF

HEPATITIS C VIRAL INFECTIONS

[0-0114] The present invention provides methods for treating or preventing a hepatitis

C virus infection in a patient in need thereof. [0»0115] The present invention further provides methods for introcducing a th_erapeutically effective amount of a TLR7 ligand or a prodrug thereof, or combination of such ligands and prodrugs into the blood stream of a patient in the treatranent and/or prevention of hepatitis C viral infections.

[0116] The magnitude of a prophylactic or therapeutic dose of 2 TLR7 ligand or

TLR7 ligand prodrug of the invention or a pharmaceutically acceptable salt, solvate, hwdrate, or stereoisomer thereof in the acute or chronic treatment or prevention of an imfection will vary, however, with the nature and severity of the infecticen, and the route by which the active ingredient is administered. The dose, and in some casess the dose frequency, will also vary according to the infection to be treated, the agee, body weight, and response of the individual patient. Suitable dosing regimens can be reaclily selected by thyose skilled in the art with due consideration of such factors.

[00117] The methods of the present invention are particularly we=l] suited for human patients. In particular, the methods and doses of the present invention csan be useful for irmmunocompromised patients including, but not limited to cancer patie=nts, HIV infected patients, and patients with an immunodegenerative disease. Furthermowre, the methods can bee useful for immunocompromised patients currently in a state of remisssion. The methods amd doses of the present invention are also useful for patients undergoirag other antiviral

: treatments. The prevention methods of the present invention are particularly useful for patients at risk of viral infection. These patients include, but are not limited to health care workers, e.g., doctors, nurses, hospice care givers; military personnel; teachers; childcare workers; patients traveling to, or living in, foreign locales, in particular third world locales including social aid workers, missionaries, and foreign diplomats. Finally, the methods and compositions include the treatment of refractory patients or patients resistant to treatment such as resistance to viral polymerase inhibitors, preotease inhibitors, etc.

Doses

[00118] Toxicity and efficacy of the compounds of the invention can be determined by standard pharmaceutical procedures in cell cultwares or experimental animals, e.g., for determining the LDs (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LDso/EDso.

N [00119] The data obtained from the cell cultwire assays and animal studies can be used in formulating a range of dosage of the compoundss for use in humans. The dosage of such compounds lie preferably within a range of circula®ing concentrations that include the EDso with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration util ized. For any compound used in the method of the invention, the therapeutically effecti ve dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the ICso Cie., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture; alternatively, the dose of the TLR7 ligand prodrug may be formulated in animal models to achieve a circulating plasma concentrati on range of the TLR7 ligand that corresponds to the concentration required to achiewe a fixed magnitude of response. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[00120] The protocols and compositions of the invention are preferably tested in vitro, and then in vivo, for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays which can be used to determine whether administration of a specific therapeutic protocol iss indicated, include in vitro cell culture assays in which cells that are responsive to the effects of the TLR?7 ligands are exposed to the ligand and the magnitude of response is measured by an appropriate technique. The assessment of the TLR7 ligand potency is then evaluated with respect to the TLR7 ligand prodrug potency, and the degree of conversion of the TLR7 ligand prodrug. Compounds for use in methods of the invention can be tested in suitable animal model systems prior to testing in humans, including but not limited to ir rats, mice, chicken, cows, monkeys, rabbits, hamsters, etc. The compounds can then “be used in the appropriate clinical trials.

[00121] The magnitude of a prophylactic cr therapeutic dose of a prodrug of a TLR7 ligand of the invention or a pharmaceutically acceptable salt, solvate, hydrate, or stereoisomer thereof in the acute or chronic treatment or prevention of an infection or condition will vary with the nature and severity of the infection, and the route by which the active ingredient is administered. The dose, and. perhaps the dose frequency, will also vary according to the infection to be treated, the age, body weight, and response of the individual patient. Suitable dosing regimens can be readily selected by those skilled in the art with due consideration of such factors. In one embodime=nt, the dose administered depends upon the specific compound to be used, and the weight and condition of the patient. Also, the dose may differ for various particular TLR7 ligands prodrugs; suitable doses can be predicted on the basis of the aforementioned in vitro measurements, in particular by use of such measurements of the TLR7 ligand to which the “TLR7 ligand prodrug is related, and on the basis of animal studies, such that smaller doses “will be suitable for those TLR7 ligand prodrugs that show effectiveness at lower conce=ntrations than other TLR7 ligand prodrugs when measured in the systems described or refesrenced herein. In general, the dose per day is in the range of from about 0.001 to 100 mg/kg, preferably about 1 to 25 mg/kg, more preferably about 5 to 15 mg/kg. For treatment of humans infected by hepatitis C viruses, about 0.1 mg to about 15 g per day is administe=red in about one to four divisions a day, preferably 100 mg to 12 g per day, more preferably from 100 mg to 8000 mg per day. Ina preferred embodiment for compounds such as prodrugs of 3-B-D-ribofuranosylthiazolo[4,5- d]pyrimidines from 200 mg to 8000 mg per day is administered in about one to four divisions a day. Additionally, the recommended daily dose ran can be administered in cycles as single agents or in combination with Other therapeutic agents. In one embodiment, the daily dose is administered in a single dose Or in equally divided doses. In a related embodiment, the recommended daily dose can be administered once time per week, two times per week, three times per week, four times per week or five times per week.

[00122] In a preferred embodiment, the compounds of the invention are administered to provide systemic distribution of the compound within the patient. In a related embodiment, the compounds of the invention awre administered to produce a systemic effect in the body.

[00123] In another embodiment the compounds of the invention are administered via oral, mucosal (including sublingual, buccal, rectal, nasal, or vaginal), parenteral (including subcutaneeous, intramuscular, bolus injection, intraarterial, or= intravenous), transdermal, or topical administration. In a specific embodiment the compo=unds of the invention are administesred via mucosal (including sublingual, buccal, rect al, nasal, or vaginal), parenteral (includingz subcutaneous, intramuscular, bolus injection, intr-aarterial, or intravenous), transdermal, or topical administration. In a further specific embodiment, the compounds of the invent-ion are administered via oral administration. In a “further specific embodiment, the compounds of the invention are not administered via oral adaministration.

[00124] Different therapeutically effective amounts ray be applicable for different infectionsz, as will be readily known by those of ordinary skill in the art. Similarly, amounts sufficient to treat or prevent such infections, but insufficient= to cause, or sufficient to reduce, acdverse effects associated with conventional therapi_es are also encompassed by the above described dosage amounts and dose frequency schedules.

Combination Therapy

[00125] Specific methods of the invention further corprise the administration of an additional therapeutic agent (i.e., a therapeutic agent other t"han a compound of the invention). In certain embodiments of the present inventior, the compounds of the invention. can be used in combination with at least one othemr therapeutic agent. Therapeutic agents include, but are not limited to antibiotics, antiemetic agents, antidepressants, and antifunga_l agents, anti-inflammatory agents, antiviral agentss, anticancer agents, immunonmodulatory agents, B-interferons, alkylating agents_, hormones or cytokines. Ina preferred embodiment the invention encompasses the administration of an additional therapeut-ic agent that is HCV specific or demonstrates anti—HCYV activity.

[00126] The TLR7 ligands prodrugs of the invention can be administered or formulate=d in combination with antibiotics. For example, t#hey can be formulated with a macrolide (e.g., tobramycin (Tobi®)), a cephalosporin (e.g. , cephalexin (Keflex®), cephradirae (Velosef®), cefuroxime (Ceftin®), cefprozil (Ceefzil®), cefaclor (Ceclor®), cefixime (Suprax®) or cefadroxil (Duricef®)), a clarithrom_ycin (e.g, clarithromycin (Biaxin®»)), an erythromycin (e.g., erythromycin (EMycin®)), a penicillin (e.g., penicillin V (V-Cillin K® or Pen Vee K®)) or a quinolone (e.g., ofloxacin (Floxin®), ciprofloxacin (Cipro®) or norfloxacin (Noroxin®)),aminoglycoside antibwiotics (e.g., apramycin, arbekacira, bambermycins, butirosin, dibekacin, neomycin, mneomycin, undecylenate, netilmicim, paromomycin, ribostamycin, sisomicin, and spe-ctinomycin), amphenicol antibioticss (e.g., azidamfenicol, chloramphenicol, florfeniccol, and thiamphenicol), ansamycin antibiatics (e.g., rifamide and rifampin), carbace=phems (e.g., loracarbef), carbapeneems (e.g., biapenem and imipenem), cephalosporirs (e.g., cefaclor, cefadroxil,

cefamandole, cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide, and cefpirome), cephamycins (e.g., cefbuperazone, cefmetazole, and cefminox), monobactams (e.g., aztreonam, carumonam, and tigemonzam), oxacephems (e.g., flomoxef, and moxalactam), penicillins (e.g., amdinocillira, amdinocillin pivoxil, amoxicillin, bacampicillin, benzylpenicillinic acid, benzzylpenicillin sodium, epicillin, fenbenicillin, floxacillin, penamccillin, penethamate hydmriodide, penicillin o-benethamine, penicillin 0, penicillin V, penicillin V benzathine, penic:iliin V hydrabamine, penimepicycline, and phencihicillin potassium), lincosamides (e._g., clindamycin, and lincomycin), amphomycin, bacitracin, capreomycin, colistin, enduraciclin, enviomyecin, tetracyclines (e.g. apicycline, chlortetracycline, clomocycline, and demeclocycline), 2,4-diaminopyrimidines (e.g., brodimoprim), nitrofurans (e.g., furaltadon. €, and furazolium chloride), quinolones and analogs thereof (e.g., cinoxacin,, clinafloxaxcin, flumequine, and grepagloxacin), sulfonamides (e.g., acetyl sulfamethoxypymrazine, benzylsulfamide, noprylsulfamide, phthalylsulfacetamide, sulfachrysoidine, ard sulfacytine), sulfones (e.g., diathymosulfone, glucosulfone sodium, and solasulfone), cycloserine, mupirocin and tuberin.

[00127] The TLR7 ligand prodrugs «of the invention can also be administered or formulated in combination with an antiemetic agent. Suitable antiemetic agents include, buat are not limited to, metoclopromide, dompesridone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondamsetron, granisetron, hydroxyzine, acethylleucine monoethanolamine, alizapride, azasetron, bbenzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dimenhydwinate, diphenidol, dolasetron, meclizine, methallatal, metopimazine, nabilone, oxyp-emdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiethylperazine, th_ioproperazine, tropisetron, and mixtures thereof.

[00128] The TLR? ligand prodrugs «of the invention can be administered or formulated in combination with an antidep ressant. Suitable antidepressants include, but are * not limited to, binedaline, caroxazone, cita lopram, dimethazan, fencamine, indalpine, indeloxazine hydrocholoride, nefopam, nommifensine, oxitriptan, oxypertine, paroxetine, sertraline, thiazesim, trazodone, benmoxinee, iproclozide, iproniazid, isocarboxazid, : nialamide, octamoxin, phenelzine, cotinines, rolicyprine, rolipram, maprotiline, metralindoles, mianserin, mirtazepine, adinazolam, amitriptyline, amitriptylinoxide, amoxapine, butriptyline, clomipramine, demexiptiline, desipramine, dibenzepin, dimetacrine, dothiepine, doxepin, fluacizine, imipramine, imipramime N-oxide, iprindole, lofepramine, melitracen, metapramine, nortriptyline, noxiptilin, opigpramol, pizotyline, propizepine, protriptyline, quinupramine, tianeptine, trimipramine, adirafinil, benactyzine, bupropion, butacetin, dioxadrol, duloxetine, etoperidone, febarbaamate, femoxetine, fenpentadiol, fluoxetine,

fluvoxamine, hemactoporphyrin, hypericin, levophacetoperane, medifoxamine, milnaciperan, - minaprine, moclobemide, nefazodone, oxaflozane, piberaline, prolintane, pyrisuccidearol, ritanserin, roxindol e, rubidium chloride, sulpiride, tandospirone, thozalinone, tofenacin_, toloxatone, tranylczypromine, L-tryptophan, venlafaxine, viloxazine, and zimeldine.

[00129] The TLR? ligands or TLR7 ligand prodrugs of the invention can be administered or formulated in combination with an antifungal agent. Suitable antifung al agents include but are not limited to amphotericin B, itraconazole, ketoconazole, fluconazole, intrathecal, flucytosine, miconazole, butoconazole, clotrimazole, nystatin, terconazole, tiocommazole, ciclopirox, econazole, haloprogrin, naftifine, terbinafine, undecylenate, and griseofuldin.

[00130] The= TLR7 ligands or TLR7 ligand prodrugs of the invention can be administered or formulated in combination with an anti-inflammatory agent. Useful axati- inflammatory agerts include, but are not limited to, non-steroidal anti-inflammatory drugs such as salicylic acid, acetylsalicylic acid, methyl salicylate, diflunisal, salsalate, olsal=zine, sulfasalazine, acetaminophen, indomethacin, sulindac, etodolac, mefenamic acid, meclofenamate sodium, tolmetin, ketorolac, dichlofenac, ibuprofen, naproxen, naproxeen sodium, fenoprofe n, ketoprofen, flurbinprofen, oxaprozin, piroxicam, meloxicam, ampiroxicam, droxicam, pivoxicam, tenoxicam, nabumetome, phenylbutazone, oxyphenbutazone, antipyrine, aminopyrine, apazone and nimesulide; leukotriene antagonists including, but not limited to, zileuton, aurothioglucose, gold sodium thionmalate and auranofin; steroids including, but not limited to, alclometasone diproprionate, amcinonide, beclomethasone dipropionate, betametasone, betamethasone benzoate, betamethasone diproprionate, betamethasone sodium phosphate, betamethasone valerate, clobetasol propriomate, clocortolone pivalate, hydrocortisone, hydrocortisone derivativses, desonide, desoximmatasone, dexamethasone, flunisolide, flucoxinolide, flurandrenolide halcinocide, medrwsone, methylprednisolone, methprednisolone acetate, methylprednisolorae sodium succinate, mometasone furoate, paramethasone acetate, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebuaatate, prednisone, triamcinolone, triamcinolone acetonide, triamcinolone diacetate, and triamcinolone hex acetonide; and other anti-inflammatory agents including, but not limited to, methotrexate, colchicine, allopurinol, probenecid, sulfinpyrazone and benzbromarcone. 100131] The TLR7 ligands or TLR7 ligand prodrugs of the invention can be administered or formulated in combination with another antiviral agent. Useful antiviral agents include, but are not limited to, protease inhibitors, nucleoside reverse transcriptase inhibitors, non-nuccleoside reverse transcriptase inhibitors and nucleoside analogs. Th.e antiviral agents include but are not limited to zidovudine, acyclovir, gan _gcyclovir, vidarabine=, idoxuridine, trifluridine, levovirin, viramidine and ribavirin, as well as foscarnet, amantadine, rimantadine, saquinavir, indinavir, amprenavir, leopinavir, ritonavir, the alpha--interferons, beta-interferons, gamma-interferons, adefovir, cle=vudine, entecavir, and plecomaril.

[00132] The TLR? ligands or TLR? ligand prodrugs of the invermtion can be administered or formulated in combination with an immunomodulatory= agent.

Immunomaodulatory agents include, but are not limited to, methothrexa-te, leflunomide, cyclophosphamide, cyclosporine A, mycophenolate mofetil, rapamycim (sirolimus), mizoribire, deoxyspergualin, brequinar, malononitriloamindes (e.g., le—flunamide), T cell receptor mmodulators, and cytokine receptor modulators, peptide mimetIics, and antibodies (e.g., hurman, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFv—s, Fab or F(ab)2 fragments or epitope binding fragments), nucleic acid molecules (e.g., antisense nucleic acid molecules and triple helices), small molecules, organic compounds, amd inorganic compounds. Examples of T cell receptor modulators include, but are rot limited to, anti-T cell receptor antibodies (e.g., anti-CD4 antibodies (e.g., cM-T412 (Boeeringer), IDEC-

CE9.1® (IDEC and SKB), mAB 4162W94, Orthoclonc and OKTcdr4-a (J anssen-Cilag)), anti-CD3 antibodies (e.g., Nuvion (Product Design Labs), OKT3 (Joh-nson & Johnson), or

Rituxan (IDEC)), anti-CD5 antibodies (e.g., an anti-CD5 ricin-linked immunoconjugate), anti-CD 7 antibodies (e.g., CHH-380 (Novartis)), anti-CD8 antibodies, anti-CD40 ligand monoclonal antibodies (e.g., IDEC-131 (IDEC)), anti-CD52 antibodies (e.g., CAMPATH 1H (Ilex<)), anti-CD2 antibodies, anti-CD11a antibodies (e.g., Xanelinm (Genentech)), and anti-B7 antibodies (e.g., IDEC-114 (IDEC)) and CTLA4-immunoglo®bulin. Examples of cytokine receptor modulators include, but are not limited to, soluble cytokine receptors (e.g., th_e extracellular domain of a TNF-a receptor or a fragment thereof, the extracellular domain_ of an IL-1 receptor or a fragment thereof, and the extracellu_lar domain of an IL-6 receptor or a fragment 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 -g, interferon (IFN)-a,

IFN-B, IFN-y, and GM-CSF), anti-cytokine receptor antibodies (e.g., anti-IFN receptor antibodies, anti-IL-2 receptor antibodies (e.g., Zenapax (Protein Design Labs)), anti-IL-4 receptor antibodies, anti-IL-6 receptor antibodies, anti-IL-10 receptor antibodies, and anti-

IL-12 receptor antibodies), anti-cytokine antibodies (¢.g., anti-IFN amtibodies, anti-TNF-o antiboclies, anti-IL-1p antibodies, anti-IL-6 antibodies, anti-IL-8 anti bodies (e.g., ABX-IL-8 (Abgenix)), and anti-IL-12 antibodies).

[00 133] The TLR7 ligands or TLR ligand prodrugs of the inventior can be administered or formulated in combination with an agent which inhibits v=iral enzymes, including but not limited to inhibitors of HCV protease, such as BILN 20«61 and inhibitors of INS5b polymerase such as NM107 and its prodrug NM283 (Idenix Pharmaceuticals, Inc.,

Cambridge, MA).

[00134] The TLR7 ligands or TLR7 ligand prodrugs of the inventi on can be administered or formulated in combination with an agent which inhibits BEICV polymerase such as those described in Wu, Curr Drug Targets Infect Disord. 2003;3(3):207-19 or in : coambination with compounds that inhibit the helicase function of the virmus such as those described in Bretner M, et al Nucleosides Nucleotides Nucleic Acids. 2003 122(5-8):1531, or with inhibitors of other HCV specific targets such as those described £n Zhang X. IDrugs. 20802;5(2):154-8.

[00135] The TLR? ligands or TLR7 ligand prodrugs of the invention can be administered or formulated in combination with an agent that inhibits vimral replication. [0 0136] The TLR7 ligands or TLR ligand prodrugs of the invention can be administered or formulated in combination with cytokines. Examples off cytokines include, buat are not limited to, interleukin-2 (IL-2), interleukin-3 (IL-3), interleuk<in-4 (IL-4), in-terleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleuki n-9 (IL-9), in terleukin-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-

C SF), granulocyte colony stimulating factor (G-CSF), macrophage colo. ny stimulating factor (M-CSF), prolactin, and interferon (IFN), e.g., IFN-alpha, and I[FIN-gamma). [®0137] The TLR7 ligands or TLR?7 ligand prodrugs of the invention can be aciministered or formulated in combination with hormones. Examples of hormones include, but are not limited to, luteinizing hormone releasing hormone (LHRH), growth hormone (CGH), growth hormone releasing hormone, ACTH, somatostatin, somat=otropin, somatomedin, parathyroid hormone, hypothalamic releasing factors, insulin, glucagon, emkephalins, vasopressin, calcitonin, heparin, low molecular weight hegoarins, heparinoids, synthetic and natural opioids, insulin thyroid stimulating hormones, anc endorphins. [©0138] The TLR7 ligands or TLR7 ligand prodrugs of the invermtion can be administered or formulated in combination with B-interferons which include, but are not limited to, interferon beta-1a, interferon beta-1b. [©0139] The TLR7 ligands or TLR7 ligand prodrugs of the invermtion can be administered or formulated in combination with a-interferons which include, but are not limited to, interferon alpha-1, interferon alpha-2a (x-oferon), interferon alpha-2b, intron, Peg-

Intron, Pegasys, consensus interferon (infergen) ana albuferon.

[00140] The TLR7 ligands or TLR7 ligand prodrugs of the invention can be administered or formulated in combination with an_ absorption enhancer, particularly those which target the lymphatic system, including, but rot limited to sodium glycocholate; sodium caprate; N-lauryly-D-maltopyranoside; EIDTA; mixed micelle; and those reported in Muranishi Crit. Rev. Ther. Drug Carrier Syst., 77-1-33, which is hereby incorporated by reference in its entirety. Other known absorption enhancers can also be used. Thus, the invention also encompasses a pharmaceutical com_position comprising one or more TLR7 ligand prodrugs of the invention and one or more absorption enhancers.

[00141] The TLR7 ligands or TLR7 ligand prodrugs of the invention can be administered or formulated in combination with am alkylating agent. Examples of - alkylating agents include, but are not limited to nitrogen mustards, ethylenimines, methylmelamines, alkyl sulfonates, nitrosoureas, ®riazenes, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, hexamethylmelaine, thiotepa, busulfan, carmustine, streptozocin, dacarbazine amd temozolomide.

[00142] The compounds of the invention amd the other therapeutics agent can act additively or, more preferably, synergistically. Imm a preferred embodiment, a composition comprising a compound of the invention is administered concurrently with the administration of another therapeutic agent, which can be part of the same composition or in a different composition from that comprising the compounds of the invention. In another embodiment, a compound of the invention is administered prior to or subsequent to administration of another therapeutic agent. In a separate embodiment, a compound of the invention is administered to a patient who has not previously undergone or is not currently undergoing treatment with another therapeutic ageent, particularly an antiviral agent.

[00143] In one embodiment, the methods of the invention comprise the administration of one or more TLR7 ligands or T"LR7 ligand prodrugs of the invention without an additional therapeutic agent.

PHARMACEUTICAL COMPOSITIONS AND DOSAGE FORMAS

[00144] Pharmaceutical compositions and single un-it dosage forms comprising a TLR7 ligand or prodrug of the invention, or a pharmaceutically acceptable salt, hydrate or stereoisomer thereof, are also encompassed by the= invention. Individual dosage forms of the invention may be suitable for oral, mucosal (#ncluding sublingual, buccal, rectal, nasal, or vaginal), parenteral (including subcutanecus, intramuscular, bolus injection, intraarterial, or intravenous), transdermal, or topical adruinistration.

Pharmaceutical compositions and dosage forms of the invention typically also comprise one or more pharmaceutically acceptable excipients. Sterile dosage forms are also contemplated. : [00145] In an alternative embodiment,a pharmaceutical composition encompassed by this embodiment includes a TLR? ligand or prodrug of the invention, or a pharmaceutically acceptable salt, hydrate or stereoisomer thereof, and at least one additional therapeutic agent. Examples of additional therapeutic agents include, but are= not limited to, those listed above in section 5.2.2.

[00146] The composition, shape, and type of dosage formss of the invention will typically vary depending on their use. For example, a dosage for-m used in the acute treatment of a disease or a related disease may contain larger amounts of one or more of the active ingredients it comprises than a dosage form used in the chmronic treatment of the same disease. Similarly, a parenteral dosage form may contain smallex" amounts of one or more of the active ingredients it comprises than an oral dosage form used to treat the same disease or disorder.

[00147] These and other ways in which specific dosage forms encompassed by this invention will vary from one another will be readily apparent to t-hose skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990).

Examples of dosage forms include, but are not limited to: tablets; caplets; capsules, such as soft elastic gelatin capsules; cachets; troches; lozenges; dispersiowns; suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; cr-eams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid doseage forms suitable for oral or mucosal administration to a patient, including suspensions (e.#2., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be rezconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.

[00148] Typical pharmaceutical compositions and dosag-e forms comprise one or more carriers, excipients or diluents. Suitable excipients are weell known to those skilled in the art of pharmacy, and non-limiting examples of suitable excTpients are provided herein.

Whether a particular excipient is suitable for incorporation inte a pharmaceutical composition or dosage form depends on a variety of factors we=1] known in the art including, but not limited to, the way in which the dosage form will be administered to a patient. For example, oral dosage forms such as tablets may contain excipi-ents not suited for use in parenteral dosage forms. The suitability of a particular excipient may also depend on the specific active ingredients in the dosage form.

[00149] This invention further encompasses anhydrous pharmaceutical compositions and dosage forms comprising active ingredients, since water c-an facilitate the degradation of some compounds. For example, the addition of water (e.g.» 5%) is widely accepted in the pharmaceutical arts as a means of simulating long-term storagse in order to determine characteristics such as shelf-life or the stability of formulations over time. See, e.g., Jens T.

Carstensen, Drug Stability: Principles & Practice, 2d. Ed., Marcel Dekker, NY, NY, 1995, pp. 379-80. In effect, water and heat accelerate the decomposition of some compounds.

Thus, the effect of water on a formulation can be of great sigrificance since moisture and/or humidity are commonly encountered during manufacture, haradling, packaging, storage, shipment, and use of formulations.

[00150] Anhydrous pharmaceutical compositions and cosage forms of the invention can be prepared using anhydrous or low moisture containing -ingredients and low moisture or low humidity conditions.

[00151] An anhydrous pharmaceutical composition sheould be prepared and stored such that its anhydrous nature is maintained. Accordingly, amhydrous compositions are preferably packaged using materials known to prevent exposrure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

[00152] The invention further encompasses pharmacewtical compositions and dosage forms that comprise one or more compounds that reduce the rate by which an active ingredient will decompose. Such compounds, which are referred to herein as “stabilizers,” include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.

[00153] Like the amounts and types of excipients, the amounts and specific types of active ingredients in a dosage form may differ depending on factors such as, but not limited to, the route by which it is to be administered to patients. Heowever, typical dosage forms of the invention comprise TLR7 ligand prodrugs of the invention, or a pharmaceutically acceptable salt, hydrate, or stereoisomers thereof comprise 0.1 mg to 1 500 mg per unit to prowide doses of about 0.01 to 200 mg/kg per day.

Ora] Dosage Forms [00M 54] Pharmaceutical compositions of the invention that are suitable for oral administration can be presented as discrete dosage forms, such as, but are not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flasvored syrups). Such dos age forms contain predetermined amounts of active ingredients, ard may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington’s

Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (199 0). [00»155] Typical oral dosage forms of the invention are prepare=d by combining the active ingredient(s) in an intimate admixture with at least one excipie=nt according to corventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administra_tion. For example, excipients suitable for use in oral liquid or aerosol dosage forms incliade, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents.

Examples of excipients suitable for use in solid oral dosage forms (eg, powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugarss, micro-crystalline ceXMulose, diluents, granulating agents, lubricants, binders, and disinteegrating agents.

[000156] Because of their ease of administration, tablets and caapsules represent the mest advantageous oral dosage unit forms, in which case solid excip -ients are employed. If desired, tablets can be coated by standard aqueous or nonaqueous techniques. Such dosage forms can be prepared by any of the methods of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intima ately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary.

[0157] For example, a tablet can be prepared by compressiomn or molding.

Compressed tablets can be prepared by compressing in a suitable maachine the active imgredients in a free-flowing form such as powder or granules, optionally mixed with an excipient. Molded tablets can be made by molding in a suitable maechine a mixture of the powdered compound moistened with an inert liquid diluent.

[00158] Examples of excipients that can be used in oral dosagze forms of the invention ' include, but are not limited to, binders, fillers, disintegrants, and luboricants. Binders suitable for use in'pharmaceutical compositions and dosage forms imaclude, but are not limited to, corn starch, potato starch, or other starches, gelatin, natu-ral and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetat=e, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrroliclone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, (e.g. , Nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.

[00159] Examples of fillers suitable for use in the pharmaceutic al compositions and dosage forms disclosed herein include, but are not limited to, tale, calcium carbonate (e.g. granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof. The binder or filler in pharmaceutical compositions of the invention is typ ically present in from about 50 to about 99 weight percent of the pharmaceutical composition or dosage form.

[00160] Suitable forms of microcrystalline cellulose include, but are not limited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICEL RC-581, AVICEL-PH- 105 (available from FMC Corporation, American Viscose Division, AAvicel Sales, Marcus

Hook, PA), and mixtures thereof. A specific binder is a mixture of microcrystalline . cellulose and sodium carboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or low moisture excipients or additives include AVICEL-"PH-103™ and Starch 1500 LM.

[00161] Disintegrants are used in the compositions of the invention to provide tablets that disintegrate when exposed to an aqueous environment. Tablets that contain too much disintegrant may disintegrate in storage, while those that contain toc little may not disintegrate at a desired rate or under the desired conditions. Thus, =a sufficient amount of disintegrant that is neither too much nor too little to detrimentally akter the release of the active ingredients should be used to form solid oral dosage forms off the invention. The amount of disintegrant used varies based upon the type of formulati_on, and is readily discernible to those of ordinary skill in the art. Typical pharmaceut:ical compositions comprise from about 0.5 to about 15 weight percent of disintegrant,, specifically from about 1 to about 5 weight percent of disintegrant.

[00162] Disintegrants that can be used in pharmaceutical cormpositions and dosage forms of the invention include, but are not limited to, agar-agar, algzinic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crosgpovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof.

[00163] Lubricants that can be used in pharmaceutical compositions and dosage forms of the invention include, but are not limited to, calcium stearate, magnesium stearate,

mineral oil, light ruineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, otZher glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (€.8.. peanut oil, cottonseed oil, sumflower oil, sesame oil, olive oil, com oil, and soybean oil), zin_c stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof. Additional lubricants inc=1lude, for example, a syloidl silica gel (AEROSIL 200, manufactured by W.R. Grace Co. of Baltimore,

MD), a coagulate=d aerosol of synthetic silica (marketed by Degussa Co. of Plano, TX),

CAB-O-SIL (a pyrogenic silicon dioxide product sold by Cabot Co. of Boston, MA), and mixtures thereof. If used at all, lubricants are typically used in an amount of les-s than about 1 weight percent of the pharmaceutical compositions or dosage forms into which they are incorporated.

Delayed Release Dosage Forms

[00164] A _ctive ingredients of the invention can be administered by contreoiled release means or by deli-very devices that are well known to those of ordinary skill in thae art.

Examples include, but are not limited to, those described in U.S. Patent Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; and 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,566, each of which is in_corporated ! herein by refererace. Such dosage forms can be used to provide slow or control led-release of one or more active ingredients using, for example, hydropropylmethyl cellul ose, other polymer matricess, gels, permeable membranes, osmotic systems, multilayer cozatings, microparticles, Liposomes, microspheres, or a combination thereof to provide tte desired release profile ir varying proportions. Suitable controlled-release formulationss 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 invention. The invention thus encompasses single unit dosage forms suitable for oral administration such as, but not limited to, tamblets, capsules, gelcapys, and caplets that are adapted for controlled-release.

[00165] All controlled-release pharmaceutical products have a common goal of improving drug: therapy over that achieved by their non-controlled counterparts, Ideally, the use of an optimally designed controlled-release preparation in medical treatme-nt is characterized by a minimum of drug substance being employed to cure or cont=rol the condition in a minimum amount of time. Advantages of controlled-release for-mulations include extendesd activity of the drug, reduced dosage frequency, and increasec patient compliance. In. addition, controlled-release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the drug, and can thus affect the occurrence of side (e.g., adverse) effects.

100166] Most controlled-release formulations are designed to initially rele=ase an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this lev el of therapeutic or perophylactic effect over an extended period of time. In order to nnaintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replaces the amount of drug being metabolized and excreted from the body.

Controlled-rele=ase of an active ingredient can be stimulated by various conditions including, but not limited to, pH, temperature, enzymes, water, or other physiological conditions or compounds.

Parenteral Dos age Forms }

[00167] Parenteral dosage forms can be administered to patients by various routes including, but ot limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Because their administration typically bypasses patients’ natural defensess against contaminants, parenteral dosage forms are preferably sterile or capable of beimg sterilized prior to administration to a patient. Examples of pamenteral dosage forms &nclude, but are not limited to, solutions ready for injection, dry and/or lyophylized products ready to be dissolved or suspended in a pharmaceutically- acceptable vehicle for injection (reconstitutable powders), suspensions ready for injection , and emulsions.

[00168] Suitable vehicles that can be used to provide 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, Rinzger’s Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated IRinger’s Injection; water-miscible vehicles such as, but not limit-ed to, ethyl alcohol, polyesthylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limitead to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, ana benzyl benzoate.

[00169] Compounds that increase the solubility of one or more of the active ingredients di_sclosed herein can also be incorporated into the parenteral dosagse forms of the invention.

Transdermal Dosage Forms

[00170] Transdermal dosages forms include “reservoir type” or “matrix type” patche:s, which can be applied to the skin amd worn for a specific period of time to permit the penetration of a desired amount of active ingredients.

[00171] Suitable excipients «(e.g., carriers and diluents) and other materials that can be used to provide transdermal andl topical dosage forms encompassed by this invention are well known to those skilled in the pharmaceutical arts, and depend on the particular tissue 10 which a given pharmaceutical composition or dosage form will be applied. With that fact in mind, typical excipients include, bsut are not limited to, water, acetone, ethanol, ethylene glycol, propylene glycol, butane-1 ,3-diol, isopropyl myristate, isopropyl palmitate, mineraal oil, and mixtures thereof.

[00172] Depending on the specific tissue to be treated, additional components may be used prior to, in conjunction with, or subsequent to treatment with active ingredients of thme invention. For example, penetrati_on enhancers can be used to assist in delivering the acti-ve ingredients to the tissue. Suitable penetration enhancers include, but are not limited to: acetone; various alcohols such as ethanol, oleyl, and tetrahydrofuryl; alkyl sulfoxides sucsh as dimethyl sulfoxide; dimethyl a«cetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such as polyvinylpymrolidone; Kollidon grades (Povidone, Polyvidone); urea; and various water-soluble or inso Juble sugar esters such as Tween 80 (polysorbate 80) amd

Span 60 (sorbitan monostearate).

[00173] The pH of a pharmaceutical composition or dosage form, or of the tissue &o which the pharmaceutical composition or dosage form is applied, may 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 svach 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 resgard, stearates can serve as a lipid vehicle for the formulation, as an emulsifying aggent or surfactant, and as a delivery-enhancing or penetration-enhancing agent. Di ferent salts, hydrates or solvates of the active ingredierats can be used to further adjust the properties of the resulting composition.

Topical Dosage Forms

[00174] Topical dosage foyrms of the invention include, but are not limited to, crezams, lotions, ointments, gels, solution_s, emulsions, suspensions, or other forms known to one- of skill in the art. See, e.g., Remington's Pharmaceutical Sciences, 18th eds., Mack

Publishing, Easton PA (1990); and Introduction to Pharmaceutical Dosage Forms, 4th ed.,

Lea & Febiger, Philadelphia (1 985).

[00175] Suitable excipients (e.g-, carriers and diluents) and other materials that can be used to provide transdermal and topical dosage forms encompassed by this invention are well known to those skilled in the pharmaceutical arts, and depend on the particular tissue to» which a given pharmaceutical composition or dosage form will be applied. With that fact im mind, typical excipients inclucie, but are not limited to, water, acetone, ethanol, ethylene glycol, propylene glycol, butasne-1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof.

[00176] Depending on the specific tissue to be treated, additional components may be used prior to, in conjunction with, or subsequent to treatment with active ingredients of the invention. For example, penetration enhancers can be used to assist in delivering the actives ingredients to the tissue. Suitable penetration enhancers include, but are not limited to: acetone; various alcohols such as ethanol, oleyl, and tetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethyl acetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such as polyvimylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; and various water-soluble or insoluble sugar estcrs such as Tween 80 (polysorbate 80) and

Span 60 (sorbitan monosteamate).

Mucosal Dosage Forms

[00177] Mucosal dos age forms of the invention include, but are not limited to, ophthalmic solutions, spray=s and aerosols, or other forms known to one of skill in the art.

See, e.g., Remington’s Pharmaceutical Sciences, 18th eds., Mack Publishing, Easton PA (1990); and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger,

Philadelphia (1985). Dosage forms suitable for treating mucosal tissues within the oral cavity can be formulated as mouthwashes or as oral gels. In one embodiment, the aeroso-] comprises a carrier. In anowther embodiment, the aerosol is carrier free.

[00178] The TLR7 Ligands or TLR? ligand prodrugs of the invention may also be administered directly to the lung by inhalation. For administration by inhalation, a TLR7 ligand can be conveniently delivered to the lung by a number of different devices. For example, a Metered Dose Inhaler (“MDI”) which utilizes canisters that contain a suitable low boiling propellant, e.g=., dichlorodiflucromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas can be used to deliver a

TLR7 ligand directly to thhe lung. MDI devices are available from a number of suppliers such as 3M Corporation, Aventis, Boehringer Ingleheim, Forest Laboratories, Gilaxo-

Wellcome, Schering Plough and Vectura.

fod179) Alternatively, a Dry Powder Inha®er (DPI) device can be used to administer a

TLR7 ligand to the lung (see, e.g., Raleigh ef al. , Proc. Amer. Assoc. Cancer Research -

Annual Meeting, 1999, 40, 397, which is herein incorporated by reference). DPI devices typically use a mechanism such as a burst of gass to create a cloud of dry powder inside a container, which can then be inhaled by the patisent. DPI devices are also well known in the art and can be purchased from a number of venclors which include, for example, Fisons,

Glaxo-Wellcome, Inhale Therapeutic Systems, IML Laboratories, Qdose and Vectura. A popular variation is the multiple dose DPI (“MIDDPI”) system, which allows for the delivery of more than one therapeutic dose. MIDDPI devices are available from companies such as AstraZeneca, GlaxoWellcome, IVAX, Schering Plough, SkyePharma and Vectura.

For example, capsules and cartridges of gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch for these systems.

[00180] Another type of device that can “be used to deliver a TLR7 ligand or TLR7 ligand prodrug to the lung is a liquid spray dev -ice supplied, for example, by Aradigm

Corporation. Liquid spray systems use extremely small nozzle holes to aerosolize liquid drug formulations that can then be directly inhaaled into the lung.

[00181] In a preferred embodiment, a ne=bulizer device is used to deliver TLR7 ligands or TLR ligand prodrugs to the lung. Nebulizers create aerosols from liquid drug formulations by using, for example, ultrasonic energy to form fine particles that can be readily inhaled (See e.g., Verschoyle et al., British J. Cancer, 1999, 80, Suppl 2, 96, which is herein incorporated by reference). Example=s of nebulizers include devices supplied by

Sheffield/Systemic Pulmonary Delivery Ltd, Aventis, and Batelle Pulmonary Therapeutics.

See U.S. Pat. Nos. 5,954,047; 5,950,619; 5,97=0,974, which are herein incorporated by reference.

[00182] In a particularly preferred embeodiment, an electrohydrodynamic (“EHD”) aerosol device is used to deliver TLR7 ligandss and TLR7 ligand prodrugs to the lung. EHD aerosol devices use electrical energy to acrosoolize liquid drug solutions or suspensions (see, e.g., Noakes et al., U.S. Pat. No. 4,765,539; Coffee, U.S. Pat. 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, which are herein incorporated by reference). The electrochemical properties of the TLR7 ligancds and TLR7 ligand prodrugs formulation may be important parameters to optimize when deJivering this drug to the lung with an EHD aerosol device and such optimization is routiraely performed by one of skill in the art. EHD aerosol devices may more efficiently delivery driags to the Jung than existing pulmonary delivery technologies. Other methods of intra-ptalmonary delivery of TLR7 ligand and

TLR7 ligand prodrugs will be known to the skill ed artisan and are within the scope of the invention. (00183) Liquid drug formulations suitable for use with nebulizers and liquid spray devices and EHD aerosol devices will typically include a TLR7 ligand or TLR? ligand prodrug with a pharmaceutically acceptable carxcier. Preferably, the pharmaceutically acceptable carrier is a liquid such as alcohol, water, polyethylene glycol or a perfluorocarbon. Optionally, another material ray be added to alter the aerosol properties of the solution or suspension of the TLR7 ligan_d or prodrug of a TLR7 ligand. Preferably, this material is liquid such as an alcohol, glycol, polyglycol or a fatty acid. Other methods of formulating liquid drug solutions or suspension suitable for use in aerosol devices are known to those of skill in the art (see, e.g., Bie=salski, U.S. Pat. Nos. 5,112,598; Biesalski, 5,556,611, which are herein incorporated by re=ference) A TLR?7 ligand or prodrug of a

TLR7 ligand can also be formulated in rectal oer vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[00184] In addition to the formulations described previously, a TLR7 ligand or TLR7 ligand prodrug can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injectiorm. Thus, for example, the compounds can be formulated with suitable polymeric or hydrop=hobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[00185] Alternatively, other pharmacerutical delivery systems can be employed.

Liposomes and emulsions are well known ex_amples of delivery vehicles that can be used to deliver TLR7 ligands and TLR7 ligand prodrugs. Certain organic solvents such as dimethylsulfoxide can also be employed, altbaough usually at the cost of greater toxicity. A

TLR7 ligand or prodrug of a TLR7 ligand caan also be delivered in a controlled release system. In one embodiment, a pump can be used (Sefton, CRC Crit. Ref Biomed Eng., 1987, 14, 201; Buchwald et al., Surgery, 19830, 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, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); R_anger and Peppas, J. Macromol. Sci. Rev.

WF 0 2005/025583 PCT/US2004/028236

Macromol. Chem., 1983, 23, 61; see also Levy et al., Scienc=e, 1985, 228, 190; During et al.,

Ann. Neurol, 1989,25,351; Howard ef al., 1989, J. Neurosurg. 71, 105). In yet another embodiment, a controlled-release system can be placed in pmroximity of the target of the compounds of the invention, e.g., the lung, thus requiring orly a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controelled Release, supra, vol. 2, pp. 115 (1984)). Other controlled-release system can be used (ssee, e.g. Langer, Science, 1990, 249, 1527).

[00186] Suitable excipients (e.g., carriers and diluents) and other materials that can be used to provide mucosal dosage forms encompassed by ®his invention are well known to those skilled in the pharmaceutical arts, and depend on the goarticular site or method which a given pharmaceutical composition or dosage form will be asdministered. With that fact 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 mixtures thereof, which are non-toxic and pharmaceuticall®y acceptable, Examples of such additional ingredients are well known in the art. See, e.g., “Remington’s Pharmaceutical

Sciences, 18th eds., Mack Publishing, Easton PA (1990).

[00187] The pH of a pharmaceutical composition or dosage form, or of the tissue to which the pharmaceutical composition or dosage form is agpplied, 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 stearates can also be added to pharmaceutical compositi_ons or dosage forms to advantageously alter the hydrophilicity or lipophilicity of ne or more active ingredients so as to improve delivery. In this regard, stearates can serve aas a lipid vehicle for the formulation, as an emulsifying agent or surfactant, and as aa delivery-enhancing or penetration-enhancing agent. Different salts, hydrates or ssolvates of the active ingredients can be used to further adjust the properties of the resulting; composition.

KITS

[00188] The invention provides a pharmaceutical paack or kit comprising one or more containers comprising a TLR7 ligand prodrug useful for tie treatment or prevention of a

Hepatitis C virus infection. In other embodiments, the invwention provides a pharmaceutical pack or kit comprising one or more containers comprisingz a TLR7 ligand prodrug useful for the treatment or prevention of a Hepatitis C virus infection and one or more containers comprising an additional therapeutic agent, including but mot limited to those listed in section 5.2.2 above, in particular an antiviral agent, an inteerferon, an agent which inhibits viral enzymes, or an agent which inhibits viral replication, preferably the additional therapeutic agent is HCV specific or demonstrates anti—HCV activity.

[00189] 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 container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

ASSAYS

[00190] The TLR7 ligands, TLR ligand prodrugs, compositions and dosage forms of the invention can be tested in vitro or in vivo by a vari ety of methods known in the art to test activity. See, for example, the methods discussed below and used throughout the examples.

[00191] A range of assays for the purpose of ewaluating TLR7 activity are available, and are described in the following publications, each «of which is incorporated-by-reference:

Hirota et al., J. Med. Chem., 45, 5419-5422 (2002); amd Akira S. ef al., Immunology Letters, 85, 85-95 (2003). For example, one system useful for the assay of TLR7 ligands is where the gene for TLR7 is cloned by methods known to those of skill in the art and transfected into an appropriate cell type such that TLR7 is expressed and coupled to a NFkB-luciferase reporter plasmid. In this cell system, exposure to TLR7 ligand prodrugs in cell culture results in a measurable luminescence signal. See, ¢.&., Lee et al., Proc. Nat. Acad. USA, 100, 6646-6651 (2003); Hemmi et al, Nat. Immunol, 3, 196-200 (2002); and Jurk et al.,

Nat. Immunol. 3, 499 (2002) (wherein the Lee et al. , Hemmi et al., and Jurk er al. references are all incorporated herein by reference).

[00192] Another example of an in vitro method is to expose human peripheral blood mononuclear cells (PBMC) to the candidate TLR7 ligand prodrug for a predetermined interval (e.g., 2 hours to 24 hours), followed by mea surement of immunologic activity.

Such immunologic activity may include induction of the synthesis of cytokines, which can be measured in the culture supernate by ELISA assay of the cytokine protein, such as a

Type 1 interferon (interferon alpha, interferon beta) or Type 2 interferon (interferon gamma). Alternatively, the PBMC can be harvested after incubation with the candidate

TLR? ligand prodrug, the PBMC RNA extracted, and the level of induction of immune system genes determined by RNAse protection assays of the extracted RNA. Genes typically induced include 2°5’-OAS, or interferon geamma, but a range of cytokines can be measured. See, e.g., Hirota ef al., J. Med. Chem., 4&5, 5419-5422 (2002).

[00193] RNAse protection assays (RPA) are a method known in the art wherein RNA analytes are quantitated by first hybridizing them to a radioalabeled RNA sequence specific for the analyte RNA, followed by digestion with enzymes that selectively degrade single stranded RNA. The sample is then submitted to gel electrophoresis under conditions that resolve the hybridized, protected doumble stranded RNA. The gel is then autoradiographed to reveal the location and intensity of time bands. These can be quantitated by methods well known in the art. Multiple analyte R INA species can be simultaneously assayed, if the protected fragments are sufficiently clifferent in size to allow their separation by gel electrophoresis. Comparison of the L evels of an analyte RNA to control RNA species that is expressed at constant levels in cells provides an internal control that enable changes in levels of the analyte RNA species to be monitored even if the total amount of RNA varies,

Such RNAse protection assays can be performed as follows:

[00194] RNA is purified from PMBC pellets using the “RNAcasy” kit (Qiagen), according to the manufacturer’s instructions. Template sets may be obtained from

PharMingen (BD Biosciences); a useful set that is commercially available from this supplier contains materials allowing the assay of TNF-a, IL12 p35, IP10, IL-1a, [L-1b, IL-6,

Interferon gamma, and the control RMA species L32 and GAPDH. This template set contains DNA that is appropriate for synthesis of the proper RNA probe for each of the listed genes.

[00195] Probe synthesis is performed using the PharMingen in vitro transcription kit provided in the kit. The reaction contains RNase inhibitor; transcription buffer; 50 ng of the tempate set; 0.1375 mM of each of rGIP, rCTP, rATP; 0.003 mM rUTP;10 mM DTT, 0.010 mCi of [alpha 32P] UTP, and 20 Units of T7 RNA polymerase in a volume of 20 microliters. The reaction mixture is in cubated for one hour at 37 °C, and then stopped by addition of 2 units of RNAse free DNAse, with an additional incubation of 30 minutes at 37 °C. The RNA probes synthesized in this incubation are extracted once with 5.2 mM EDTA, pl of Tris Saturated phenol, 25 ul. of chloroform, and 4 pg of yeast tRNA, and then extracted with 50 pL of chloroform. T he RNA is precipitated by addition of 50 pL of 4M ammonium acetate and 250 uL of ice-cold 100% ethanol, and after incubation at —80 °C for minutes the preparation is centrifuged at high speed for 30 minutes. The pellet is washed in 100 % ethanol, and after removal of the ethanol the probe is resuspended and used in the

RNAse protection incubation.

[00196] The RNAse protection assay uses the probe material prepared above and

RNA extracted from PBMC. The PBMEC RNA is washed in 100% ethanol, quantitated by absorbance at 260 nm. The RPA is performed as described in the protocol provided in the

PharMingen RiboQuant kit. Eight pL of the PBMC RNA samples are mixed with the 2 pL of the probe set in a thin-walled PCR tube, mixed well, briefly centrifuged and then overlaid with mineral oil. The tube is then placed in a 90 °C PCR block, cooled to 56 °C, and incubated for 16 hours. The samples are then cooled to 37 °C and RNAse A and RNAse T1 are added. The mixture is incubated for 45 minutes at 30 °C, and the reaction stopped with a mix of protease K and yeast tRNA. The RNA is extracted with phenol-chloroform, and then precipitated frorm the phenol-chloroform with ammonium acetate-ethanol. The pellet is washed with ethanol and resuspended in buffer for electrophoresis. The samples are analysed by gel electrophoresis by methods well known in molecular biology.

[00197] A nurmber of assays may be employed in accordance with the present invention in order to determine the degree of anti-viral activity of a compound of the invention such as ceL1 culture, animal models, and administration to human subjects. The assays described herein may be used to assay viral growth over time to determine the growth characteristics of a virus in the presence of a compound of the invention.

[00198] In another embodiment, a virus and a compound of the invention are administered to animal subjects susceptible to infection with the virus. The incidence, severity, length, virus load, mortality rate of infection, etc. can be compared to the incidence, severity, Rength, virus load, mortality rate of infection, etc. observed when subjects are administered the virus alone (in the absence of a compound of the invention).

Anti-virus activity of the compound of the invention is demonstrated by a decrease in incidence, severity, Rength, virus load, mortality rate of infection, etc. in the presence of the compound of the invention. In a specific embodiment, the virus and the compound of the invention are administered to the animal subject at the same time. In another specific embodiment, the virus is administered to the animal subject before the compound of the invention. In another specific embodiment, the compound of the invention is administered to the animal subject before the virus.

[00199] In another embodiment, the growth rate of the virus can be tested by sampling biological fluids/clinical samples (e.g., nasal aspirate, throat swab, sputum, broncho-alveolar lawage, urine, saliva, blood, or serum) from human or animal subjects at multiple time points post-infection either in the presence or absence of a compound of the invention and measuring levels of virus. In specific embodiments, the growth rate of a virus is assayed by assessing the presence of virus ina sample after growth in cell culture, growth. on a permissible graswth medium, or growth in subject using any method well-known in the art, for example, but not limited to, immunoassay (e.g., ELISA; for discussion regarding

ELISAs see, e.g., Ausubel er al., eds, 1994, Current Protocols in Molecular Biology, Vol. I,

John Wiley & Sons, Inc., New York at 11.2.1), immunofluorescent staining, or immunoblot analysis using an antibody which immunospecifically recognizes the wirus to be assayed or detecztion of a virus-specific nucleic acid (e.g., by Southern blot or RT-PCR analysis, etc.). [602&0] In a specific embodiment, viral titers can be dettrminec by obtaining biolosgical fluids/clinical samples from infected cells or an infected sulidject, preparing a seria] dilution of the sample and infecting a monolayer of cells that are= susceptible to infec=tion with the virus (e.g. primary cells, transformed cell lines, pati ent tissue samples, etc) ata dilution of the virus that allows for the emergence of single pMaques. The plaques can then be counted and the viral titer expressed as plaque forming un its per milliliter of sample.

[0021] In one specific embodiment, the growth rate of a virus in a subject can be estimated by the titer of antibodies against the virus in the subject. Aratibody serum titer can be destermined by any method well-known in the art, for example, but not limited to, the amotant of antibody or antibody fragment in serum samples can be quantitated by, e.g.,

ELISA. [002@2] Additionally, in vivo activity of a TLR7 ligand or prodrug of a TLR7 ligand can bee determined by directly administering the compound to a test an=imal, collecting biolo-gical fluids (e.g., nasal aspirate, throat swab, sputum, broncho-alweolar lavage, urine, saliva, blood, or serum) and testing the fluid for anti-virus activity.

[00203] In embodiments where samples to be assayed for virus levels are biological fluidss/clinical samples (e.g., nasal aspirate, throat swab, sputum, broncho-alveolar lavage, urine, saliva, blood, or serum), the samples may or may not contain in tact cells. Samples from subjects containing intact cells can be directly processed, wherea_s isolates without intact cells may or may not be first cultured on a permissive cell line (e.g. primary cells, transformed cell lines, patient tissue samples, etc) or growth medium Ce.g., LB broth/agar,

YT baroth/agar, blood agar, etc.). Cell suspensions can be cleared by ceentrifugation at, e.g., 300x ag for 5 minutes at room temperature, followed by a PBS, pH 7.4 (Ca++ and Mg++ free) ~wash under the same conditions. Cell pellets can be resuspended. in a small volume of

PBS ffor analysis. Primary clinical isolates containing intact cells can tbe mixed with PBS and ceentrifuged at 300xg for 5 minutes at room temperature. Mucus iss removed from the interf2ace with a sterile pipette tip and cell pellets can be washed once rnore with PBS under the sazme conditions. Pellets can then be resuspended in a small volume of PBS for analysis.

[002004] In another embodiment, a compound of the invention is administered to a humamn subject infected with a virus. The incidence, severity, length, v iral load, mortality rate of infection, etc. can be compared to the incidence, severity, length, viral load,

mortality rate of infection, etc. observed in h-uman subjects infected with a virus in the absence of a compound of the invention or ify the presence of a placebo. Anti-viral activity of the compound of the invention is demonstrated by a decrease in incidence, severity, length, viral load, mortality rate of infection. etc. in the presence of the compound of the invention. Any method known in the art cara be used to determine anti-viral activity ina subject such as those described previously.

[00205] Additionally, in vivo activity ofa TLR7 ligand or prodrug of TLR7 ligand can be determined by directly administeringz the compound to an animal or human subject, collecting biological fluids/clinical samples (e.g., nasal aspirate, throat swab, sputum, broncho-alveolar lavage, urine, saliva, blood, or serum) and testing the biological fluids/clinical samples for anti-viral activity” (e.g., by addition to cells in culture in the presence of the virus).

[00206] The foregoing has demonstrated the pertinent and important features of the present invention. One of skill in the art will be appreciate that numerous modifications and embodiments may be devised. Therefore, itis intended that the appended claims cover all such modifications and embodiments. 6. EXAMPLES

[00207] The following examples are for the purpose of illustration only and are not intended as limiting the scope of the invent ion. 6.1 TLR7 Ligand Identification

[00208] There are three known chemical classes of TLR7 ligands: guanosines, imidazoquinolines, and pyrimidines (see Section 5.2). As described above, additional TLR7 ligands are readily identified by known screening methods. For example, adenine analogs and derivatives were identified as TLR ligands by using the following screening procedure.

See Tables 1 and 2.

A stable HEK293-hTLR7 cell line ~was obtained from Invivogen (San Diego,

California), transfected with pNiFty2-Luc, an NF-kB inducible luciferase reporter plasmid (Invivogen) and (dual) stable transfectants selected. The resultant dual (hTLR7/pNiFty2-

Luc) cell lines were functionally tested by response to loxoribine and isatoribine as measured by fold luciferase induction relative to a no drug control. The C23 line was chosen due to its satisfactory response and? sensitivity profile with these (and other) TLR7 agonists. The biological rationale which connects TLR7 engagement and NF-kB activation has met with widespread acceptance (for a review, see Akira S. et al., Immunol. Lett., 83, 85-95 (2003)

and as a consequence the HEK293-TLR-NF-kB inducible reporter system is accepted as a standard assay which has been consistently used to assay TLIR(7) agonists, in either transient or stable system format. See, e.g., 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).

[00209] For a typical C23 assay, cells are seeded at 6x=10* cells/well in 96 well plates and 4-24 hours later are treated with various concentrations of compound. After 2-48 hours exposure the cell monolayers are lyzed with passive lysis buffer (Promega) and the firefly luciferase assay carried out using the luciferase assay reagen.t (Promega) as specified by the manufacturer. Relative luciferase activities are expressed as folds of induction compared to the no drug control. A two-fold induction over background is considered a bona fide TLRT agonist, dependent on this being a statistically significant increase.

Table 1: Isatoribine Activates Human TLR7 in HEK293 Assay

Compound No. 21 3.1 34 62 109 (0.2) 0.1) 0.50 0.7

Table 2: Adenine Derivative Activates Human TI_R7 in HEK293 Assay

Compound No. 20.8 373 47.5 43.4 @&11) @0.5) *0.2D #04)

[00210] In Table 1, isatoribine, was added to C23 cel 1s for forty-eight hours, and the cells were then harvested and assayed for luciferase activity. Each time-point was assayed in triplicate. The data displayed is the mean fold induction ccompared to the no drug control, together with the standard deviation in parentheses. :

[00211] In Table 2, an adenine derivative 29 was addled to C23 cells for twenty-four hours, and the cells were then harvested and assayed for lucziferase activity. Each time-point was assayed in triplicate. The data displayed is the mean. fold induction compared to the no drug control, together with the standard deviation in parentheses. 6.2 TLR7 Ligands Tested as anti-HCV Agents

HCV Viral Load Reduction

[00212] Isatoribine investigational drug product was supplied as a 1 mg/mL solution in sterile normal saline contained in a 50 mL vial. Isator-ibine was administered in humans by intravenous infusion once daily for 7 days, at 200, 4040, 600 or 800 mg per dose. All doses were administered by constant rate infusion over a. 60-minute period, except the 800 mg dose was administered 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/mim for the 400 mg dose; 8.33 mL/min for the 500 mg dose; or 10.0 mL/min for the 600 mg and. 800 mg dose.

[00213] Four to twelve patients were enrolled in e=ach of the dose groups (200 mg, 400 mg, 600 mg and 800 mg per dose) and received once daily intravenous infusions for 7 days. Prior to dosing, a blood sample was drawn from ezach patient for assessment of the genotype of the HCV virus.

[00214] Plasma HCV RNA was dctcrmined at bas eline (an average of 2 pre-treatment measurements taken on Day —1 or pre-treatment and on Day 1) and once daily prior to the start of the first daily isatoribine intravenous infusion on Days 2 through 7 for these daily (x7 days) dosing groups. See Figure 2. The viral load w-as measured by the branched DNA method (Versant™ v3.0 bDNA assay, Bayer Diagnostics:). For plasma HCV RNA, the maximum change from the pre-treatment baseline was es timated using log-transformed values.

[00215] Plasma HCV RNA decreased over the course of isatoribine treatment, with the larger changes generally occurring in patients who received the higher daily doses (Figure 2). Eight of 12 patients who received isatoribine 800 mg QD x 7 days showed a plasma viral load decrease of more than 0.5 log10 units, vith a mean change in these 12 patients of -0.76 log10 units and range of —2.85 to +0.21 Mog10 units. This decrease in viral load was statistically significant for the 800 mg QD dose group (p=0.008). Plasma viral load declines generally reversed upon cessation of treatmeent.

HCV Replicon-Based Viral Bioassay

[00216] It has been demonstrated that HCV replicosns are highly sensitive to the inhibitory effects of interferon-a and interferon-y. Therefore the HCV replicon becomes a very useful system for measuring the amount of biologically active interferons in supernatants from human PBMCs stimulated with a TLR agonist. A quantitative assay was developed which is based on measuring the activity o»fthe luciferase reporter gene that was integrated Zinto an HCV replicon. By using this system, interferons from TLR7 agonist- treated PBMCs= were measured and their inhibitory activity was asssessed using the luciferase repomrter replicon. . : [00217] “Human PBMCs isolated from a healthy donor were placed in replicate cell culture wells (5 x 10° cells per well). The PBMCs are incubated im the absence of test compounds at 337 °C in a humidified atmosphere containing 5% C®O, for 24 hours to allow stabilization to the culture conditions, and then the TLR7 ligand o_r a drug free control is added to replicaate wells containing PBMCs from the same donor. The concentrations of

TLR7 ligand mmay be varied to suit the particular experiment, and the PBMC cultures are then incubated at 37 °C in a humidified atmosphere containing 5% CO; for eight hours.

Samples of cell culture supernatant are taken at the eight hour tim-€ point (or twenty four hour time poin®t in case of Loxoribine and its prodrugs) from TLR_7 ligand treated and control wells amnd are assayed for interferon-alpha production by EELISA. Supernatants from compound-treamted cells and a no-drug control were diluted at 1:10, 1:100, and 1:1000 in

RPMI media amd applied to a 96-well plate of Huh7 hepatocyte cells containing the luciferase reporter replicon. Cells were incubated for 48 hours at 37 °C in a tissue culture incubator.

[00218] _ After the incubation period, the 96-well plates are =washed 2X with PBS and are lysed with gpassive lysis buffer (Promega). Plates are shaken amt room temperature for 20 minutes and steandard luciferase assay reagent (Promega) is added to each well by injection and the plate is: read on an Lmax luminometer (Molecular Devicess). The raw relative light units are converted to a percent inhibition that is compared to the -no-drug control wells to determine the 1 evel of inhibition observed in the replicon assay. The estimated maximal concentration Of interferons required to inhibit HCV replicon repl-ication was determined to be at a 1:10 dil-ution of supernatant of PBMC-stimulated cells whitch fell within our test range of the dilution series. For all TLR7 agonists tested, 100% imhibition was observed on the luciferase r-eporter replicon system at the 1:10 dilution.

[00219] ‘The data presented in Tables 3-8 represents the inhibition of the HCV replicon systercn by the supernatant collected after exposure of PBIMC cells to the compound at an initial coracentration for the incubation time indicated and diluted as specified in the first column (“PBMC exposed fo compound”). A supernatant col lected from PBMC cells non-exposed tc the compound and diluted as specified in the first column was used as a control (“Blank supernatant”). The PBMC cells were isolated from a single blood donor as specified.

Table 3= Antiviral Effect of Isatoribine in the in vitro HCW Replicon Bioassay

No. 1

Incubation time: 8 hours

Initial concentration: 100 pM

Blood donor number: FL.72035 supernatant to compo—und

I ON I SRY SO” NA

No. 2

Incubation time: 8 hours

Initial concentration: 100 pM

Blood donor number: FL75287 1 HCV repliconinhibition, %a supernatant to compomund wo Fo 1 100 |] 100 [0 | 87 |]

No. 3

Incubation time: 24 hours

Initial concentration: 100 uM

Blood donor number: FL75864

T — HCVrepliconinhibition, %& supernatant to comporand wwe [0 7] 100 i200 23 Fee 0] [1100 J 0 — 1 — 64 — ]

Table 4: Antiviral Effect of Loxoribine irm the in vitro HCV Replicon Bioassay

No. 1

Incubation time : 24 hours

Initial concentration: 100 uM

Blood donor number: FL.75864 supernatant to compou_nd

IR 1 I I TE A I uriweo [23 17a]

Table S: Antiviral Effect of Imiquimod in the in vitro HCV Replicon Bioassay

No. 1

Incubation time: 8 hours

Initial concentration: 3.2 uM

Blood donor number: FL75287 supernatant to compound two 1 0 1 10 _mao00 [0 | 8

No. 2

Incubation time: 8 hours

Initial concentration: 3.2 pM

Blood edonor number: FL75287

Dilution of Blank PBMC sugpernatant to compound mo | 3 1 100 woo | 13 75

IN TT I NN RS

Mable 6: Antiviral Effect of Resiquimod in the in_ vitro HCV Replicon Bioas

No. 1

Incubaztion time: 8 hours

Initial concentration: 10 uM

Blood edonor number: F1,75287

Di lution of Blank PBMC suppernatant to compound wo Pw [wo

MEN © NN SEN BE 93m000 Jo | 21

No. 2

Incubation time: 8 hours

Initial concentration: 10 uM

Blood adonor number: FL75287

Di: lution of PBMC exposed supoernatant to compound wo 7 "3 100 — mae00 [3 [6

T able 7: Antiviral Effect of Bropirimine in the in —vifro HCV Replicon Bioassay

No. 1 .

Incubation time: 8 hours

Initial concentration: 100 pM

Blood cdonor number: FL.72035

HCV replicon inhibition, %

Dilution of PBMC exposed supernatant to compound rio [6 | 100 doo | 0 {95s [ @:000 | 0 [0

No. 2

Incubat=ion time: 8 hours

Initial cconcentration: 100 pM

Blood donor number: FL72036

Dilution of Blank PEBMC supernatant to compound nfo [oto too | 8 | 95 |] woo [0 | 3

Table 8: Antiviral Effect off Adenine Derivative in the in vitro HCV Replicon Bioass.a

No. 1

Incubation time: 8 hours

Initial concentration: 0.1 pM

Blood donor number: FL. 7641 8

Form of the compound: a TFA salt supernatant to compound tio [0 | 10 — _wwo | 3 | 7B wooo [3 | 0

No. 2 k

Incubation time: 8 hours

Initial concentration: 0.1 puMC

Blood donor number: FL764 18

Form of the compound: a TFA salt supernatant to compound tio [0 | 160 |] qae0 | 3 | "ea wooo | 3 [9 6.3 Preparation of” TLR7 Ligand Prodrugs

[00220] Compounds o-f the invention can be synthesized using the methodology described in Schemes 1-18 above. Unless otherwise indicated all temperatures are set —forth in degrees Celsius and all par-ts and percentages are by weight. Reagents are purchasecl from commercial suppliers such as Aldrich Chemical Company or Lancaster Synthesis Ltd. and are used without further gpurification unless otherwise indicated. Tetrahydrofuran (THF) and N,N-dimethylfora:mide (DMF) are purchased from Aldrich in Sure Seal bottles and used as received. Unless otherwise indicated, the following solvents and reagents are distilled under a blanket of dary nitrogen. THF and Et;0 are distilled from Na- benzophenone ketyl; CH2Clz (DCM), diisopropylamine, pyridine and Et;N are distilled from CaH,; MeCN is distilled first from POs, then from CaH,; MeOH is distilled fron Mg;

PhMe, EtOAc and i-PrOAc are distilled from CaHy; TFAA was purified via simple atmospheric distillation undesr dry argon.

The reactions set forth are done generally under a positive pressure of amrgon at an ambient temperature (unless otherwise stated) in anhydrous solvents, and the re=action flasks are fitted ~with rubber septa for the introduction of substrates and reagents via s¥yringe.

Glassware is oven dried and/or heat dried. The reactions are assayed by TLC a=nd terminated as judged by the consumption of starting material. Analytical thin L ayer chromato graphy (TLC) is performed on aluminum-backed silica gel 60 F254 0 .2 mm plates (EM Science), and visualized with UV light (254 nm) followed by heating wit commercial ethanolic phosphomolybdic acid. Preparative thin layer chromatography (TLC) is performed on aluminum-backed silica gel 60 F254 1.0 mm plates (EM Sciences) and visualized with UV light (254 nm). HPLC is performed on a Waters Micromass ZQ system consisting of a model 2525 binary gradient pump with an Alltech model 800 E_LSD detector and a Waters model 996 photodiode array detector.

[00221] Work-ups are typically done by doubling the reaction volume with the reaction Solvent or extraction solvent and then washing with the indicated aqueous solutions using 25%% by volume of the extraction volume unless otherwise indicated. Product solutions are dried over anhydrous Na,SO, and/or MgSO, prior to filtration ard evaporati_on of the solvents under reduced pressure on a rotary evaporator and moted as solvents sremoved in vacuo. Column chromatography is completed under positive pressure ~ using 230-400 mesh silica gel or 50-200 mesh neutral alumina. Hydrogenolys-isis done at the presseure indicated in the examples or at ambient pressure.

[00222] "H-NMR spectra is recorded on a Varian Mercury-VX400 instraument operating at 400 MHz and '*C-NMR spectra are recorded operating at 75 MHz. NMR spectra amre obtained as CDCl; solutions (reported in ppm), using chloroform ass the references standard (7.27 ppm and 77.00 ppm), CD3;OD (3.4 and 4.8 ppm and 4-9.3 ppm),

DMSO-d, or internally tetramethylsilane (0.00 ppm) when appropriate. Other= NMR solvents sare used as needed. When peak multiplicities are reported, the follow~ing abbreviatzions are used: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broadened), dd (doublet of doublets), dt (doublet of triplets). Coupling constants, when given, ares reported in Hertz (Hz).

[00223] Infrared (IR) spectra are recorded on a Thermo Nicolet Avatar 370 FT-IR as neat oils -or solids, and when given are reported in wave numbers (cm™). Masss spectra reported «+)-ES Thermo Finnegan LCQ LC/MS conducted by the Analytical Chemistry

Department at Anadys Pharmaceuticals, Inc. Elemental analyses are conducted by the

Atlantic Microlab, Inc. in Norcross, GA or by NuMega, in San Diego, CA. M-elting points (mp) are determined on an open capillary apparatus, and are uncorrected.

[200224] The described synthetic pathways and experimental procedures utilize many common chemical abbreviations, THF (tetrahydrofuran), DMF (N ~N-dimethylformamide),

EtOAc (ethyl acetate), DMSO (dimethyl sulfoxide), DMAP (4-dimaethylaminopyridine),

IDBU (1,8-diazacyclo[5.4.0Jundec-7-ene), DCM (4-(dicyanomethy~lene)-2-methyl-6-(4- dimethylamino-styryl)-4-H-pyran), MCPBA (3-chloroperoxybenz=oic acid), EDC (1-3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), HATUJ (O-(7-azabenzotriazol-

L-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), HOBT (1 -hydroxybenzotriazole hydrate), TFAA (trifluoroacetic anhydride), pyBOP (benzotriazol—1- yloxy)tripyrrolidinophosphonium hexafluorophosphate), DIEA (diisopropylethylamine), and the like.

Example 1: 7-Allyl-2-amino-9-B-D-ribofuranosyl-7,9-dihydro-gpurin-8-one 43)

J

NEN oo ~~) 6 on

Step 1: Preparation of 7-Allyl-2-amino-9-(2",3",5 -tri-O-acetyl- p-sD-ribofuranosyl)-7,9- ddihydro-1H-purine-6,8-dione (40)

[00225] A heterogeneous mixture of 7-allyl-2-amino-9-B-D—ribofuranosyl-7,9- lihydro-1H-purine-6,8-dione 17 (1.00 g, 2.95 mmol, prepared acceording to Reitz et al,

FMC, 37, 3561-3578 (1994)), DMAP (0.036 mg, 0.29 mmol) and ENEt3 (2.05 mL, 14.74 mmol) was stirred in dry acetonitrile (25 mL). Acetic anhydride (0.862 mL, 9.13 mmol) was added slowly to the suspension and the reaction mixture was stirre d at ambient temperature feor 16 h. The solvent was removed under vacuum and the residue clissolved in

Qichloromethane (DCM). The organic phase was then washed witla saturated aqueous s-odium bicarbonate (NaHCO), brine and thereafter dried with anbmydrous magnesium sulfate (MgSOs). The solvent was concentrated under vacuum and dried at room temperature under high vacuum to give 1.33 g of 40 (97%) as a pale yellow solid: 'H NMR (<400 MHz, CDCl3) § 6.12 (t, J = 6.0 Hz, 1H), 6.01 (d, J = 3.6 Hz, 11H), 5.89 (m, 1H), 5.82 (8, J =6.0 Hz, 1H), 5.39 (br s, 2H), 5.21 (m, 2H), 4.58 (br 5, 2H), 4=.51 (m, 1H), 4.32 (mm, 2H), 2.16 (s, 3H), 2.15 (s, 3H), 2.10 (s, 3H); MS (+)-ES [M+H]" 4-66.2 m/z.

Step 2: Preparation of 7-Allyl-2-amino-6-chloro-9-(2'3",5 -tri-O-aacetyl- -D- r@bofuranosyl)-7,9-dihydro-purin-8-one (41)

[00226] 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 mixtumre was concentrated under vacuum and the crude product dissolved in DCM. The mixture was then washed with NaHCO solution, brine, dried (MgSO4) and filtered. The filtrate vvas concentrated under vacuum and purified by flash chromatography using 10 to 50% gradient of ethyl acetate in hexanes.

Removal of the solvent afforded 280 mg (41%) of the desired product 41: "HNMR (400

MHz, CDCl) 5 6.04 (d, J= 4.0 Hz, 1H), 6.03 (t, J= 5.6 ¥iz, 1H), 5.87 (m, 1H), 5.86 (t, J = 5.6 Hz, 1H), 5.18 (m, 4H), 4.59 (d, J = 8.0 Hz, 2H), 4.45 (4, J=7.6 Hz, 1H), 4.31 (m, 2H), 2.10 (s, 3H), 2.08 (s, 3H), 2.04 (s, 3H); MS (+)-ES [M+HE]" 484.2 m/z.

Step 3: Preparation of 7-Allyl-2-amino-9-(2°,3",5 -tri-O-aacetyl-f-D-ribofuranosyl)-7,9- dihydro-purin-8-one (42)

[00227] ’ Compound 41 (0.27 g, 0.56 mmol) was dissolved in acetic acid and Zn-Cu couple was added to the solution. The mixture was heated at 70 °C for 18 h. The suspended particles were filtered off and the filtrate concentrated uneder vacuum. The residue was purified by flash chromatography using 10% to 100% gradient of ethyl acetate in hexanes.

The solvent was removed to give 150 mg (60 %) of 42 as off-white solid: "H NMR (400

MHz, CDCl;) § 6.05 (t, J = 4.0 Hz, 1H), 6.03 (d, J = 4.0 Ez, 1H), 5.87 (t, J = 6.0 Hz, 1H), 5.83 (m, 1H), 5.48 (br s, 2H), 5.33 (5, 1H), 5.29 (d, J = 5.66 Hz, 1H), 4.49 (d, /=3.2 Hz, 1H), 4.46 (d, J = 3.2 Hz, 1H), 4.41 (d, J = 5.6 Hz, 2H), 4.27 (m, 2H), 2.12 (5, 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-p-D-ribofuranosyl-7,9-dihydro-purin-8-one (43)

[00228] To a solution of 42 (0.13 g, 0.29 mmol) in methanol (4 mL) was added solid

K,COj; (0.024g, 0.17 mmol) and the reaction stirred at am_bient temperature for 18 h. To the cloudy mixture was added Amberlite CG-50 (0.5 g) and stirred till neutral and filtered. The filtrate was concentrated to give an off-white solid, which was washed with water and dried under high vacuum to give 93.5 mg of pure 43 in quantitative yield as an off white solid: 'H

NMR (400 MHz, d;-DMSO) & 7.88 (s, 1H), 6.33 (br s, 2HK), 5.85 (m, 1H), 5.66 (d, /=6.0

Hz, 1H), 5.30 (d, J= 5.6 Hz, 1H), 5.20 (s, 1H), 5.16 (d, J = 8.4 Hz, 1H), 5.01 (d, /= 4.8 Hz, 1H), 4.89 (q, /= 5.6 Hz, 1H), 4.75 (brs, 1H), 4.35 (d,J= 52 Hz, 2H), 4.10 (t, /=8.4 Hz, 1) 3.80 (q, J = 3.6 Hz, 1H), 3.57 (m, 1H), 3.44 (m, 1H). MES (+)-ES [M+H]" 324.1 m/z.

Example 2: 7-Allyl-2-amino-6-ethoxy-9-B-D-ribofuranosyl-7,9-dihydro-purin-8-one 5)

CH,;CH;0 A

NZ Sr

Bess ~

HO

FY

Step 1: Preparatison of 7-Allyl-2-amino-6-ethoxy-9-(2 "3 ’ 5°-tri-O-acety.l-f-D- ribofuranosyl)- 7, P-dihydro-purin-8-one (44)

[00229] To a solution of 40 (0.30 g, 0.64 mmo) in dry THF (15 nL) was added polymer-supportecd triphenylphosphine (0.89 g, 1.93 mmol) and EtOH (O.11mL, 1.93 mmol) at room termperature. To the stirring mixture was added diethyl a= odicarboxylate (0.12 mL, 0.77 mmol) and stirring continued for 18 h. The spent polymer support was filtered off and the= solvent removed under vacuum. The residue was thera purified by flash chromatography ussing 10 to 50% gradient of ethyl acetate in hexanes. Reemoval of the solvent afforded 8=5 mg (26%) of the desired product 6 as a clear oil: 'H BNMR (400 MHz,

CDCl) § 6.07 (d, =/ = 4.0 Hz, 1H), 6.06 (d, J=4.0 Hz, 1H), 6.01 (d, J= 33.6 Hz, 1H), 5.96 (t,

J=6.0 Hz, 1H), 5.87 (m, 1H), 5.14 (d, J= 2.2 Hz, 1H), 5.15 (m, 1H), 4.80 (br s, 2H), 4.46 (m, 4H), 4.37 (q, J~ = 7.2 Hz, 2H), 4.29 (m, 2H), 2.09 (s, 3H), 2.08 (s, 3H), 2.04 (5, 3H), 1.35 (t, J= 17.6 Hz, 3H)5 MS (H-ES [M+H]" 494.1 m/z.

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

[00230] To za solution of 44 (0.084 g, 0.17 mmol) in methanol (4 nL) was added solid K2COs (0.01=%g, 0.17 mmol) and the reaction stirred at ambient temperature for 1 h. To the cloudy mixture= was added Amberlite CG-50 (0.5 g) and stirred till ne=utral and filtered.

The filtrate was comncentrated and purified by flash chromatography using 100% DCM to 10 % of methanol in TDCM. Removal of the solvent afforded 62 mg of 7 (99%) as a clear oil: 'H NMR (400 MH=z, CDCL3) 8 5.97 (d, J = 8.0 Hz, 1H), 5.93 (m, 1H), 5.25 (d,J= 32.4, Hz, 1H), 5.21 (s, 1H), 5.02 (t, J= 8.0 Hz, 1H), 4.62 (br s, 2H), 4.47 (d, J = 5.e6 Hz, 2H), 4.25- 4.45 (m, 3H), 4.21 (q, /= 6.8 Hz, 2H), 3.77 (ABq, Avap =0.14, Jap = 12.4 Hz, 2H), 1.37 (t,

J=6.8 Hz, 3H), 1.227 (, 7.6, 2H); MS (+)-ES [M+H]" 368.0 m/z.

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

CHyCH,0

NZ Br

SN

Step 1: Preparation of 5-Bromo-4-ethoxy-6-phenyl-p>yrimidin-2-ylamine (37

[00231] In a manner similar to step 2 of Exampsle 2 was prepared the title compound as a white solid from 9-amino-5-bromo-6-phenyl-3H- pyrimidin-4-one 35 (Wierenga, et al.,

JMC, 23, 239-240 (1980)) in 13% yield: 'H NMR (4080 MHz, CDCl) & 7.61 (m, 2HO, 7.42 (m, 3H), 5.15 (br 5, 2H), 4.23 (4, /= 7.2 Hz, 2H), 1.4=4 (t, 6.8 Hz, 3H); MS (H-ES MY* 294.1 [M+2] "296.0 m/z. Elemental analysis for CoH 12BIN;O: calc’d: 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-pyrimiadin-4-yloxymethyl)-S-methyl- [1,3]dioxol-2-one (46)

Vea

C

Q

NS a

Step 1: Preparation of 4-(2-Amino-5-bromo-6-pheny sl-pyrimidin-4-yloxymethyl)-5-methyl- [1.3]dioxol-2-one (46)

[00232] In a manner similar to step 2 of Examgple 2 was prepared the title compound as a white solid from 2-Amino-5-bromo-6-phenyl-3FI-pyrimidin-4-one 35 in 4% yield: 'H

NMR (400 MHz, CDCl5) § 7.62 (m, 2H), 7.45 (m, IE), 5.18 (s, 2H), 5.07 (s, 2H), 2.26 (s,

H); MS (+)-ES [M]*3782 [M+2]* 380.1 m/z. Elemeratal analysis for CysH12BrN;O4: calc’d:

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-ylamione (48)

NF Br

Is

Step 1: Preparation of 4-Phenyl-pyrimidin-2-ylaminez (47)

[00233] To a solution of bromobenzene (4.43 : mL, 42.06 mmol) in dry THF (100 mL) at -78 °C was added BuLi (394 mL, 63.08 mmol) anc the mixture left to stir at—78 °C for 2 h. To this was added 2-aminopyrimidine (2.0 g, 21.0 3 mmol) in hot toluene (30 mL) over a minutes period. The mixture was refluxed for 16 Bh and allowed to cool to room temperature and carefully quenched with aqueous N=aHCO;. The mixture was filtered and the filtrate concentrated under vacuum. The residue wvas then dissolved in DCM and washed with aqueous NaHCO;, brine and dried (MgSO04). T he solvent was removed to afford 350 mg of 47 (10%) as a pale yellow solid: "H NMR (400 MHz, CDCL;) 5 8.32 (4, J=4.8 Hz, 1H), 7.97 (m, 2H), 7.45 (m, 3H), 7.02 (J = 4.8 Hz, 1H). 5.27 (br 5, 2H); MS ()-ES M+HT" 172.2 m/z.

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

[00234] Compound 47 (0.30 g, 1.75 mmol) was dissolved in glacial acetic acid (15 ml) and warmed to 45 °C. Br, (0.09 mL, 1.75 mmol) was added slowly. Thee resulting mixture was ther allowed to stir at room temperature for 3 h. The solvent was removed under vacuum to a solid residue. This was then transferred onto a filter funrael and washed with DCM, folloswed by water. The remaining solid was then dried under high vacuum for 16 h to give 197 mg of 13 (45%) as a pale yellow solid: "H NMR (400 MHz, ds-DMSO) & 8.40 (s, 1H), 7.6 1 (m, 2H), 7.45 (m, 3H), 6.96 (s, 2H); MS (+)-ES [M]* 250.0 [M+2]" 252.0 m/z. Elemental analysis for CigHBrNs: calc’d: C, 48.02; H, 3.22; Br, 31.95 ; N, 16.80; found: C, 47.91; H, 3.28; Br, 32.15; N, 16.80.

Example 6: (5-BBromo-6-oxo-4-phenyl-1,6-dihydro-pyrimidin-2-yl)-carbesamic acid ethyl ester (36) : o} 0 HN Br

FEES oN

Step 1: Preparation of (5-Bromo-6-oxo-4-phenyl-1,6-dihydro-pyrimidin-2-y 1)-carbamic acid ethyl ester (36)

To a solutzion of 35 (0.25g, 0.94 mmol) in DMF (8 mL) was added N Et; (0.14 mL, 0.99 mmol) and diethyl pyrocarbonate (0.27 mL, 1.89 mmol). The reaction rnixture was maintained at 65 *°C for 20 h. The solvent was removed and the residue treatesd with DCM.

The resulting mix=ture was filtered to remove the remaining starting material 35 and the filtrate washed with aqueous NaHCO, brine and dried (MgSOs). The filtrate was concentrated and —purified by HPLC (Thomson ODS-A 100A 5p 150 x 21.2 mum column; flow rate = 30 mI_/min; CH3CN with 0.05 % TFA (A), Water with 0.05 % T_FA (B); Make up pump flow = 0».9 mL/min; Make up pump mobile phase; MeOH with 0.05% TFA using a gradient system ass follows: t=0; 15% A, 85% B;t=3.0 min; 15% A, 85 ®4B;t=9.5 min; 70 % A, 30 6B; t= 10.0 min; 100% A, 0 % B; t= 12.0 min; 100% A_ 0% B; t= 12.5 min; 15 % A, 85 % B; t= 15.0 min; 15 % A, 85 % B.) to afford 54 mgs of 36 (17%) as a clear oil: 'H NMR (400 MHz, CDCl) 57.66 (m, 1H), 7.44 (m, 3H), 4.26 (<},J/= 7.6 Hz, 2H), 1.32 t, J= 6.8 Hz, 3H); MS (+)-ES (M]" 338.1 [M+2] © 340.0 m/z. Elemental analysis tor CH sBiNOy: calc’d: C, 46.17; H, 3.58; N, 12.43; found: C, 46.43; H,3.74N, 11.95.

Exammple 7: (5-Bromo-6-0x0-4-phenyl-1,6-dihydro-pyrimidin—2-yl)-carbamic acid penstyl ester (49) [o} o WN

Step 1: Preparation of (5-Bromo-6-oxo-4-phenyl-1, 6-dihydro-pysimidin-2-yl)-carbamic acid pentyl ester (49)

[002235] In 2 manner similar to step 1 of Example 6 the title compound was prepared frormn 35 and dipenty] pyrocarbonate as a clear oil in 9% yield afteer HPLC purification (Theomson ODS-A 100A 5p 150 x 21.2 mm column; flow rate = 30 mL/min; CH3CN with 0.05 % TFA (A), Water with 0.05 % TFA (B); Make up pump fl.ow = 0.9 mL/min; Make up pump mobile phase; MeOH with 0.05% TFA using a gradient system as follows: t= 0; 35 % A, 65 % B; t= 3.0 min; 35 % A, 65 % B; t= 10 min; 100 % As, 0 % B; t = 12.0 min; 100 % A, 0% B; t=12.5 min; 35 % A, 65 % B; t= 15.0 min; 35 % #4, 65 % B.). "H NMR (400

MIz, CDCl) 8 7.69 (brs, 1H), 7.67 (m, 2H), 7.43 (d, J= 2.0 H=, 3H), 4.17 t J=72Hz, 2H, 1.64 (t, J= 6.8 Hz, 2H), 1.34 (m, 4H), 0.92 (t, J= 6.4 Hz, 3 H); MS (+)-ES MM] 380.1

M—+2]" 382.1 m/z.

Example 8: (1-Isobutyl-1H-imidazo{4,5-c}quinolin-4-yl)-carbeamic acid pentyl ester 34D 0

NOENGEN NW

ZN

~

Step 1: Preparation of (1-Isobutyl-1H-imidazo[4,5-c]quinolin-4—yl)-carbamic acid pentyl ester (34)

[002236] To a suspension of 1-Isobutyl-1H-imidazo[4,5-c]equinolin-4-ylamine 31 (0.15 g, 3.62 mmol, prepared according to the procedure given in WO: 94/17043) in CHCl3 (5 mL) wass added NEt; (0.09 mL, 0.65 mmol) and dipentyl pyrocarbonaate (0.231 g, 0.94 mmol).

Thes mixture was stirred at 40 °C for 60 h. The reaction mixture wwas washed with aqueous

NalFICO;, brine and dried over MgSOy. The filtrate was concentarated and purified by flash chreomatography using a 10% to 70% gradient of ethyl acetate in- hexanes to give 50.5 mg of 34 23%) as a white solid: 'H NMR (400 MHz, CDCl) § 8.31 (tbrs, 1H), 8.15 (t, J=8.0

Hz, 2H), 7.85 (t, J=7.2 Hz, 1H), 7.77 (t, J= 8.0 Hz, 1H), 4.43 Cd, J=7.6 Hz, 2H), 4.36 (t, J

"=172 Hz, 2HD), 2.31 (m, 1H), 1.75 (t, J= 6.8 Hz, 2H), 1.36 (m, 4H), 1.06 (d, J= 6.4 Hz, 6H), 0.89 (t, J = 6_8 Hz, 2H); MS (+)-ES [M+H]" 355.3 m/z.

Example 9: q1-Isobutyl-1H-imidazo[4,5-c]quinolin-4-yl)-carbamic acid ethyl ester (50) o

EN

ANN i >

Step 1: Prepcaration of (1-Isobutyl-1H-imidazo[4,5-c]quinolin-4-yl)-carbam ic acid ethyl ester (50)

[00237] In a manner similar to step 1 of Example 8 was prepared the ®itle compound as a white sollid from 31 and diethyl pyrocarbonate in 67% yield as a white solid: 'H NMR (400 MHz, CDCls) § 9.32 (br s, 2H), 8.2 (d, J = 8.0 Hz, 2H) 8.12 (d, /= 8.0 Hz, 1H), 7.83 (t,J=17.2 Hz, 1H), 7.74 (1, J = 8.0 Hz,1H) 4.43 (m, 4H), 2.35 (m, 1H), 1.39 (,J=72Hz, 3H), 1.08 (d, J=6.4 Hz, 6H); MS (+)-ES [M+H]" 313.2 m/z.

Example 10: Carbonic acid 6-amino-9-benzyl-2-(2-methoxy-ethoxy)-9 H—purin-8-y] ester ethyl ester (51)

NH,

I had

PN he

Illy!

Step 1: Prepa=ration of Carbonic acid 6-amino-9-benzyl-2-(2-methoxy-ethoxw)-9 H-purin-8- yl ester ethyl ezster (51)

[00238] 6-Amino-9-benzyl-2-(2-methoxy-ethoxy)-9H-purin-8-ol, 29 (11.75 mg, 0.027 mmol, prepared according to the procedure described by Kurimota, et «al, Bioorg.

Med. Chem., 12, 1091-109 (2004) was suspended in CH,Cl, (0.6 mL) and cawoled to 0 °C.

DIEA (11.96 guL, 0.068mmol) and ethyl chloroformate (3.86 mg, 0.036 mmol, added as a 10% by volume solution in dichloromethane) were then added to the suspenssion. The reaction mixtuare was stirred at 0 °C for 10 minutes then allowed to warm to room temperature for 15 minutes. TLC of the reaction mixture shows that starting material remained. Thes reaction mixture was heated to 35 °C, DMAP (cat.), methanol (60 uL portions) was added to dissolve 29, and additional aliquots of ethyl chlorofor-mate (3.86 mg, 0.036 mmol, aadded as a 10% by volume solution in dichloromethane) were a«dded until the reactions was complete. The crude mixture was purified by flash chromatography using a 0 oo Co to 100% gradient of ethyl acetate in hexane. The desired peaks vere collected and concentrated in-vacuo to yield 8.5 mg (80%) of compound 51 ass a white solid: "HNMR (400 Mz, CDCls) § 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.

[00239] Examples 11-20 were prepared from 6-amino-9-t>enzyl-2-(2-methoxy- ethoxy)- 9H-purin-8-ol, 29 and the appropriate chloroformate according to the procedure describeed in Example 10.

Example= 11: Carbonic acid 6-amino-9-benzyl-2-(2-methoxy-eethoxy)-9 H-purin-8-yl ester preopyl ester (52)

NH, fe) ~

Seo o ia \ . cs 86% yiel_d as a white solid: 1H NMR (400 MHz, CDCl3) 8 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 (5, 3H), 1.46 (1, J=T7.6

Hz, 3H); MS [M+H]" m/z 402.2.

Example 12: Carbonic acid 6-amino-9-benzyl-2-(2-methoxy-e=thoxy)-9H-purin-8-yl ester isobutyl ester (53)

NHz o {

CI Md 0 ba . » cs

[00240] 92% yield as a white solid: 'H NMR (400 MHz, C DCs) 3 7.45 (d, T= 6.4 Hz 2H), 7.27 (m, 3H), 4.99 (s, 2H), 4.45 (m, 2H), 4.17 (d, J =7.4 Hz, 2H),3.73(t,J=4.8 Hz, 2H), 3.4 (:s,3H), 2.15 (m, 1H), 1.06 (d, J = 6.8 Hz, 6H); MS [M+EI}+ m/z 416.3. "Example 13: Carbonic acid 6-amino-9-benzyl-2-(2-methoxy-e®thoxy)-9H-purin-§-yl ester pentyl ester 54)

NEN > - 0” N

Je cs

[00241] 92% yield as a white solid: ‘4 NMR (400 MHz, CDCl) 8 7.45 (d, J = 6.4

Hz, 2H), 7.27 (m, 3H), 4.99 (s, 2H), 4.45 (t, J = 4.8 H=, 2H), 439 (t,J=7.2Hz, 2H), 3.73 (¢, J =5.2 Hz, 2H), 3.4 (s, 3H), 2.15 (m, 1H), 1.82 (mx 2H), 1.40 (m, 4H), 0.93 (t, J=6.8

Hz, 3H); MS [M+H]+ m/z 430.2.

Example 14: Carbonic acid allyl ester 6-amino-9-b enzyl-2-(2-methoxy-ethoxy)-9H- purin-8-yl ester (55)

NH 0. ~~

NF N »—d

N jess

J N

~ 5

[00242] 93% yield as a white solid: 'H NMR (4400 MHz, CDCl3) § 7.46 (d, J = 6.0

Hz, 2H), 7.25 (m, 3H), 6.0 (m, 1H), 5.5 (m, 1H), 5.35 (m, 1H), 499 (s,2H),489(d, J=24

Hz, 2H), 4.46 (t, J = 4.8 Hz, 2H), 3.74 (d, J = 5.2 Hz, 2H),3.42 (s, 3H); MS (M+H}+ m/z 400.2.

Example 15: Carbonic acid 6-amino-9-benzyl-2-(2~methoxy-ethoxy)-9H-purin-8-yl ester 4-chloro-butyl ester (56) cl

NH ls) ad . 2 N o jess

NY

J N

» cs

[00243] 98% yield as a white solid: 1H 'H NMR (400 MHz, CDCls) 6 7.44 (d, J = 6.0 Hz, 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: Carbonic acid 6-amino-9-benzyl-2-(2~methoxy-ethoxy)-9H-purin-8-yl ester butyl ester (57) ,

NH, 0 ~ -d pg Sha

J N

A oS

[00244] 100% yield as a white solid: "H NMR (400 MHz, CDCl) 5 7.45 (d, J = 6.0

Hz, 2H), 7.27 (m, 3H), 4.99 (5, 2H), 4.46 (mm, 2H), 4.41 (t, ] = 4.4 Hz, 2H), 3.73 (t, ] = 7.2

Hz, 2H), 3.42 (s, 3H), 1.79 (m, 2H), 1.48 (ma, 2H), 0.96 (t, J = 7.6 Hz, 3H); MS [M+H}+ m/z 416.2,

Example 17: Carbonic acid 6-amino-9-bemzyl-2-(2-methoxy-ethoxy)-9H-purin-8-yl ester phenyl ester (58) 0

NZ N as or =

J : % oS

[00245] 100% yield as a white solid: *H NMR (400 MHz, CDCls) 6 7.52 (d, J = 6.8

Hz, 2H), 7.41 (m, 2H), 7.30 (m, 3H), 7.25 (1m, 3H), 4.99 (s, 2H), 4.47 (t, ] = 4.8 Hz, 2H), 3.75 (t, ] = 4.8 Hz, 2H), 3.43 (s, 3H); MS [M+H]+ m/z 436.2.

Example 18: Carbonic acid 6-amino-9-bemzyl-2-(2-methoxy-ethoxy)-9H-purin-8-yl ester 2,2-dimethyl-propyl ester (59)

NH, —~

NF N )

KA

J N N

A cs

[00246] 100% yield as a white solid: © HNMR (400 MHz, CDCl3) § 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: Carbonic acid 6-amino-9-bemzyl-2-(2-methoxy-ethoxy)-9H-purin-8-yl ester hepty! ester (60)

NHz o. ea

NZ N > jens 0” ON

J ®

[00247] 100% yield as a white sol id: I NMR (400 MHz, CDCly) 7.45 (d, J = 6.0 :

Hz, 2H), 7.27 (m, 3H), 4.99 (s, 2H), 446 (t, ] = 5.2 Hz, 2H), 4.39 (t, J = 7.2 Hz, 2H), 3.74 (t, J =5.2 Hz, 2H), 3.42 (s, 3H), 1.8 (m, 2H), 1.4 (m, 2H), 1.3 (m, 6H) 0.87 (, J =72 Hz, 3H); MS [M+H]+ m/z 458.3.

Example 20: Carbonic acid 6-amino-9 -benzyl-2-(2-methoxy-ethoxy)-9H-purin-8-yl ester hexyl ester (30) " ] pad enya ia ; » <

[00248] 74% yield as a white solic: 4H NMR (400 MHz, CDCls) § 7.45 (d, J = 5.6

Hz, 2H), 7.27 (m, 3H), 4.98 (s, 2H), 4.45 (t, J= 4.8 Hz, 2H), 441 (t, T= 6.8 Hz, 2H), 3.73 (t, J =4.4 Hz, 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 = 2 Hz, 3H); MS [M+tHI+ m/z 444.4.

[00249] Examples 22 and 23 were prepared 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 methoxide re spectively according to the procedures of Kurimota et al., Bioorg. Med. Chem., 12, 1091-1099 (2004).

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

NHz

NF SN or 0

J > oe o

{00250} 1009% yield as a brown solid: 1H NMR (400 MHz, CDCl3) 8 7.59 (s, 1H), 727 (m, SH), 5.83 (s, 2H), 5.26 (5, 2H), 4.49 (t J = 4.8 Hz, 2H), 3.75 (t, J=5.2 Hz, 2H), 3.43 (s, 3H); MS [IM+H]* m/z 300.2.

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

NHz

NZ /

Illv! [oo251} 91S yield as a brown solid: 1H NMR (400 MHz, d6-DMSO0) 8 7.24 (rm, 2H), 7.24 (m, 3H), 5.00 (s, 2H), 4.42 (m, 2H), 4.27 (t, J=4.8 Hz, 2H), 3.58 (1, J= 4.8 Hz, 2H), 3.26 (s, 3H), 1.32 (1,J=7.2 Hz, 3H); MS [M+H] m/z 344.1.

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

NH,

NEN jens

Illy

[00252] 914 yield as a brown solid: 1H NMR (400 MHz, dg-DMSO) & 7.28 (rn, 2H), 7.22 (m, 3H), 6.86 (5, 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 (5, 3H); MS [M+H]" m/z 330.2.

Example 24: 7-A_llyl-2-Amino-9-(5-O-L-valinyl-B-D-ribofuranosyl)-7,9-dihydro—1H- purine-6,8-dione (68)

A

HN N ens 0 “ Ho BH

Step 1: Preparation of 7-Allyl-2-amino-9-(2°,3 ».Q-isopropylidene--D-ribofuranosyE)-7,9- dihydro-1H-purirme-6,8-dione (66)

[00253] Cosmpound 17 (0.17g, 0.49 mmol) was dissolved in DMF (4.0 mL) and acetone (3.0 mL) was added to the solution. To the mixture was added 2,2- dimethoxypropan € (0.18 mL, 1.47 mmol) and MeSO3H (0.02 mL, 0.05mmol). The reaction mixture was stirrec] at ambient temperature for 20 rand quenched with saturated aqumeous

NaHCO. The aquaeous phase was then extracted (4x) with CHCl. The combined osrganic phase was dried (MgSOx), filtered and concentrated under vacuum to afford 130 mg of 66 in 70% yield as a white solid: 'H NMR (400 MHz, CD;0D) § 5.97 (d, J = 2.4 Hz, 13H), 5.93 (m, 1H), 5.34 (dd, J=4.4,2.0 Hz, 1H), 5.15 (m, 1H), 5.12 (dd, J=7.6, 1.2 Hz, 1H), 4.98 (m, 1H), 4.52 (d, J = 5.6, 2H), 4.16 (m, 1H), 3.71 (m, 2H), 1.56 (5, 3H), 1.36 (s, 3H) ; MS (+)-ES M+H]' 380.0 m/z.

Step 2: Preparation of 7-Allyl-2-amino-9-(2',3 ".Q-isopropylidene-5’-N-tert- butoxycarbonyl-L—valinyl)-B-D-ribofuranosyl)-7, 9-dihydro-1H-purine-6,8-dione (677)

[00254] A mixture of 66 (0.13 g, 0.34 mmol), BOC-Valine (0.08 g, 0.36 mmol), EDC (0.07g, 0.38 mmok), DMAP (0.05g, 0.38 mmol) in THF (8.0 mL) and pyridine (0.8 rnL) under N, atmosphere was stirred at ambient temperature for 16 h. The solvents were= removed under vacuum and the residue dissolved in EtOAc. The organic phase was washed with saturated aqu eous NaHCO3, brine, dried (MgSO4) and filtered. The filtrate was : concentrated under vacuum and the purified by flash chromatography using a 2% to 5% gradient of MeOH in CH,Cl, to give 180 mg of 67 (91%) as pale yellow solid: "H NEMR (400 MHz, CDCl3) § 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 5, 1H), 4.53 (br s, 2H), 4.46 (m, 1H), 4.32 (m, 2H), 420 (m, LH), 2.11 (m, 1H), 1.56 (5, 3H), 1.44 (s, 9H), 1.36 (5, 3H), 0.93 (d, J= 6.8 Hz, 3H), 0.84 «d, J = 6.8 Hz, 3H); MS (—H)-ES [M]* 578.9 m/z.

Step 3: 7-Allyl-2-Amino-9-(5 *.Q-L-valinyl-B-D-ribofuranosyl)-7,9-dihydro-1H-purime-6,8- dione (68)

[00255] To asolution of 67 (0.18 g, 0.311 mmols) in MeOH (10 mL) was adTed

AcCl (0.86 mL, 12.07 mmol) under N; atmosphere. The reaction mixture was left tow stir at ambient temperature for 18 h and thereafter carefully neutralized with saturated aqueous

NaHCOs. To the maixture was added silica gel and concentrated under vacuum. The residue was purified by flash chromatography using a 10% to 20% gradient of MeOH in CHCl, to give 80 mg of 68 (59%) as a white solid: 'H NMR (400 MHz, de-DMS0) 8 6.72 (brs, 2H), 5.84 (m, 1H), 5.5% (d, J=4.8 Hz, 1H), 5.43 (d, J=5.6 Hz, 1H), 5.15 (br 5, 1H), 5.07 d,J= 12 Hz, 1H), 5.0 (d, J = 18.8 Hz, 1H), 4.77 (q, J = 4.8 Hz, 1H), 4.36 (m, 3H), 426 (t, J=44

Hz, 1H), 4.20 (m, 1H), 3.93 (m, 1H), 3.57 (br s, 1H), 2.01 (m, 1H), 0.86 (d, J=4.4 Elz, 3H), 0.85 (d, J = 5.2 Hz, 3H); MS (+)-ES [M+H]" 439.1 m/z.

Example 25: 7.AKlyl-2-amino-9-B-D-ribofuranosyl-6-(5-methyl-2-oxo-{1,3]dioxowl-4- ylmethoxy)-7,9-d&hydro-purin-8-one (71)

0 fo}

Ra ° A

NN

Bess od on

Step 1: Preparation of 7-Allyl-2-amino-9-«2",3",5 tris-O-triethylsilanyl--D- ribofuranosyl)-7,9-dihydro-1H-purine-6,8 -dione (69)

[00256] To a solution of 17 (0.46 g,. 1.36 mmol) and imidazole (0.93 g, 13.64 mol) En

DMF (13 mL) was added chlorotriethylsilzane (0.92 mL, 5.46 mmol) dropwise and stirred at ambient temperature for 2.5 h. The reaction mixture was treated with saturated aqueous

NaHCO; and the resulting two phases separated. The aqueous phase was washed with diethyl ether (2x). The organic layers were combined and washed with water, dried (MgSOs) and concentrated after filtration. The residue was purified by flash chromatography using a 2% to 10% gradient of MeOH in CH2Cl, to give 830 mg of 69 (89%) as pale yellow oil: TH NMR (400 MHz, CDCl3) 8 5.92 (m, 1H), 5.85 (d,J=6.8 Hz, 1H), 5.3 (m, 1H), 5.15 - 5.23 (m, 2H), 5.09 (br 5, 2H), 4.54 (d, J= 4.4 Hz, 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 (H)-ES MJ" 682.6 m/z.

Step 2: Preparation of 7-Allyl-2-amino-9-«2",3",5 "_tris-O-triethylsilanyl- -D- ribofuranosyl)-6-(5-methyl-2-oxo-[1,3 I dicaxol-4-yImethoxy)-7,9-dihydro-purin-8-one (10)

[00257] In a manner similar to Step 1 of Example 2, compound 70 was prepared fro-m compound 69 and 4-hydroxymethyl-S-megthyl-[1,3]dioxol-2-one (prepared according to thee procedure of Alepegiani, Syn. Comm., 22(’9), 1277-82 (1992) in 5% yield as a white solid after HPLC purification (Thomson ODS-/A 100A 5u 50 x 21.2 mm column; flow rate = 30 mL/min; CHsCN with 0.05 % TFA (A), W/ater with 0.05 % TFA (B); Make up pump flow = 1.0 mL/min; Make up pump mobile phase ; MeOH with 0.05% TFA using a gradient systemm as follows: t=0; 50 % A, 50 % B; t=2.0 min; 50 % A, 50 % B; t = 5.0 min; 100% A, 0 %%

B;t=9.5 min; 100 % A, 0 % B; t = 10.0 min; 50 % A, 50 % B; t=13.0 min; 50 % A, 50 4

B.); MS (H)-ES MI" 794.1 m/z.

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

[00258] To a solution of 70 (9.0 mg, 0.012 mmol) in MeOH (1.5 mL) was added

SHFNE; (0.01 mL, 0.07 mmol) and stirred at ambient temperature for 16h. The solvent was removed urader vacuum and the residue purified by flash chromatogragphy. The desired product was elu-ted with 2% to 5% gradient of MeOH in CH,Cl to afford 3.26 mg of 71 (64%) as white solid: 'H NMR (400 MHz, CDCl) 5.96 (d,J=17.6 Hz, IHD, 5.85 (m, 1H), 5.17 (m, 4H), 4.96 (t, J=7.2 Hz, 1H), 4.47 (d,J=6.0 Hz, 2H), 4.25 (J= 5 2 Hz, 1H), 4.24 (s, 1H), 3.81 (A_Bq, Avap = 0.17, Jas = 11.6 Hz, 2H), 2.22 (s, 3H): MS (+) -ES [M+HY 452.4 m/z.

Example 26: (7-Allyl-2-amino-9-B-D-ribofuranosyl-8-0xo-8,9-dibydro—7H-purin-6- yloxymethyl)-mmethyl-carbamic acid ethyl ester (73)

Q co” A

NZS

Bess

HS on

Step 1: Preparaation of (1-Allyl-2-amino-9-(2°3’,5 "“tris-O-triethylsilanyl-/3-D- ribofuranosyl)-B-0x0-8,9-dihydro-7H-purin-6-yloxymethyl)-methyl-carbammic acid ethyl ester (72)

[00259] In a manner similar to Step 1 of Example 2 compound 72 vwas prepared in 4% yield from compound 69 and N-methyl-N-(hydroxymethyl)urethane (K_elper, JOC, 52, 1987, p.453-4555) as a white solid after HPLC purification: "H NMR (400 EIMHz, CDCl) 8 5.94 (m, 1H), 5-82 (d, J= 6.4 Hz, 1H), 5.53 (br 5, 1H), 5.23 - 5.29 (m, 2H), 5.15 ¢, J=9.6

Hz, 1H), 4.57 (cl, J =5.2 Hz, 2H), 4.35 (br s, 1H), 4.21 (m, 2H), 3.96 (br 5 1H), 3.73 (m, 2H), 3.06 (s, 3E3), 1.30 (m, 4H), 0.87 — 1.01 (m, 24H), 0.57 — 0.68 (m, 19F3): MS (+)-ES [M+H]* 797.7 rm/z.

Step 2: Preparaution of (7-Allyl-2-amino-9-p-D-ribofuranosyl-8-oxo-8,9-dE hydro-7H-purin- 6- yloxymethyl)—methyl-carbamic acid ethyl ester (13)

[00260] 'n a manner similar to Step 3 of Example 25 was prepared the title compound 73 ira 64% as a white solid after HPLC purification (Thomson (ODS-A 100A 5p 50 x 21.2 mm ceolumn; flow rate = 30 mL/min; CHsCN with 0.05 % TFA A), Water with 0.05 % TFA (BD; Make up pump flow = 1.0 mL/min; Make up pump mobiile phase; MeOH with 0.05% TFA using a gradient system as follows: t= 0; 50 % A, 50 % E3;t =2.0 min; 50 % A, 50 % B; t =5.0 min; 100 % A, 0 % B; t = 9.5 min; 100 % A, 0 % B; t=10.0 min; 50 % A, 50 % B; t =13.0 min; 50 % A, 50 % B.). '"H NMR (400 MHz, CDCl=) 8 5.92 (m, 1H),

$9274, J= 7.6 Hz, 1H), 5.51 (m, 2H), 5.22 (d, J= 1 5.6 Hz, 1H), 5.16 (d, J=9.2 Hz, 1H), 4.92 (1, J="7.2 Hz, 1H), 4.57 (4, J= 6.0, 2H), 4.40 (d, J = 6.0, 1H), 4.21 (m, 3H), 3.79 (ABq, Avas = 0.178, Jap = 14.0 Hz, 2H), 3.06 (s, 3H), 1.31 (t, J=7.2 Hz, 3H): MS (+)-ES

M]" 455.4 m/z.

Example 27: 5-Amino-3-(5’-0-L-valinyl--D-ribofuranosylthiazolo[4,5-d]pyrimidine- 2,7-dione Dihydrochloride (24) 0

OD

HN ON

0 “honed we ow

Step 1: Preparation of 5-Amino-3-(2,3 .O-isopropy/lidene-f-D-ribofuranosyl) thiazolo[4,5-d]pyrimidine-2,7-dione (22)

[00261] To a heterogeneous mixture of 21 (5. 37 g, 17.0 mmol, prepared according to the procedure given in U.S. Patent No. 5,041,426 (Example 2), which is incorporated by reference in its entirety) in acetone (40 mL) contained in a 250 mL Morton flask was added successively 2,2-DMP (6.26 mL, 50.9 mmol), DMSO (6.6 mL), and MeSO;H (220 pL, 3.39 mmol) at room temperature. The reaction mixture was stirred vigorously, becoming homogeneous and golden yellow as the diol was consumed. TLC analysis (SiOz, 10%

MeOH-CHCl3) indicated reaction completion after 6 h. Undissolved solids were removed via gravity filtration using fluted Whatman type 1 filter paper. This was followed by pouring of the filtrate into 10 volumes of ice water (—~400 mL), resulting in immediate precipitation of a white solid. After a brief period of stirring, NaHCO5 (285 mg, 3.39 mmol) dissolved in water (10 mL) was added to newtralize the MeSOsH. Vigorous stirring in the Morton reactor was continued for 15 min, whereupon the mixture was filtered through a coarse scintered glass funnel. The solid material was washed with ice water (100 mL), air dried, then dried further under high vacuum at 65 °C, affording 5.36 g (88%) of the acetonide 22 as a white solid: mp 280-81 °C; 'H (DIMSO-ds) 5 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 (brs, 2H), 11.25 (s, IH).

Step 2: Preparation of 5-Amino-3-(2°,3 -O-isopropylidene —5 "-N-tert- . butoxycarbonyl-L-valinyl)-f-D-ribofuranosyl)-thiazolo(4,5-d]pyrimidine-2,7-dione 3)

[002621 ~~ “To aSolition 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). The resultant homogeneous mixture was stirred 45 min at 0 °C, at which point it had become heterogeneous, and solid acetonide 2 from Step 1 above (1.00 g, 2.81 mmol) was added as one portion. Subsequently added was solid DMAP (522 mg, 4.27 mmol). The reaction mixture was permitted to reach room temperature, and stirred an additional 5 h, whereupon it was concentrated at 25 °C via rotary evaporation to a yellow syrup. The residue was dissolved in EtOAc (50 mL), partitioned with 1 N HC (10 mL) followed by neutralization of acid with saturated aqueous

NaHCO; (10 mL). The acidic aqueous phase was further extracted with EtOAc (2 x 50 mL), and then partitioned with the basic aqueous phase. The combined organic phases were dried over Na,SO,, filtered through a short pad off 8iOa, and concentrated, affording 1.480 g (96%) of Boc-protected amino acid ester 23 as a foam: mp 158 °C (dec); 'H (CDCl3) 8 0.86 (@, J=17.0, 3H), 0.95 (d, J= 7.0, 3H), 1.35 (5, 3H), 1.44 (s, 9H), 1.56 (s, 3H), 1.75 (br s, 1H), 2.08-2.19 (m, 1H), 4.20-4.24 (m, 2H), 4.30-<4.37 (m, 1H), 4.56 (dd, J=11.0, 5.9, 1H), 4.96 (dd, J=62,3.7, 1H), 5.11 (br d,J=8.8, 1H(),5.29 (br d, J = 6.6, 1H), 5.88 (brs, 2H), 6.23 (s, 1H).

Step 3: Preparation of 5-Amino-3-(5 *.O-L-valinwl-B-D-ribofuranosyl)thiazolo[4,5- d]pyrimidine-2,7-dione Dihydrochloride (24) {00263} A stream of HCI gas was passed through a bubbler of concentrated H,SOs, and subsequently directed (via fritted dispersion #ube) into a 250 mL 3-neck Morton flask containing dry isopropyl acetate (80 mL) at 0 °C until a saturated solution was obtained. To this was added a solution of the Boc-amino acid ester from Step 2 above (5.53 g, 9.95 mmol) in isopropyl acetate (30 mL), resulting in the formation of a white solid precipitate within 5 min. To this was added 10% (v/v) IPA. {11 mL). The reaction mixture was warmed to room temperature, then stirred 12 h. "The heterogeneous reaction mixture was diluted with dry toluene (100 mL). Filtration using a medium pore scintered glass funnel under N; provided an off-white, amorphous solid. Trituration of the solid in dry THF was ‘ followed by filtration and vacuum drying at 65 °«C, affording 3.677 g (81%) of the title compound 24 as a white solid: mp 166-68 °C (dec); 'H (DMSO0-ds) § 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), 4234.28 (m, 2H), 4.42 (dd, J=117, 34, 1H), 4.75 (dd, J =10.3, 5.5, 1H), 5.81 (d, J=44, 1H), 6.46 (brs, 3H), 7.23 (br s, 2H), 8.47 (s, 3H), 11.5 (br s, 1H). Elemental analysis for

C1sH2NsO;S « 2HCI: cale’d: 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-Acetylamirao-3-(2’,3",5-tri-O-acetyl-B-D-ribofuranosyDthiazolo[4,5- d]pyrimidine-2,7(6H)-dione (74) 0 s

LI

~~

Ac “OAc

Step 1: Preparation of 5-A cetylamino-3-(2',3",5 "tri-O-acetyl-f3-D- ribofuranosyl)thiazolo[4,5—d]pyrimidine-2, 7(6H)-dione (14) [00264}) Anhydrous 21 (8.0 g, 39.5 mmol) was dissolved in dry pyridine (65 mL).

DMAP (3.1g, 25.3 mmol) and acetic anhydride (19.1 mL 202.4 mmol) were added sequentially. The reaction “was allowed to progress for 2 h at room temperature, whereupon it was quenched with saturated NaHCO; (100 mL) and extracted with DCM (3 x 200 mL).

The organic phase was concentrated, and then triturated with ether. This provided 12.5 g (103%) of slightly impure S-acetylamino-3 -(2,3,5-tri-O-acetyl-B-D-ribofuranosyl)thiazolo- {4,5-d}pyrimidin-2,7(6H)-clione as a white solid 74: mp 246.7-248.1 °C; Re= 0.20 (SiOz, 50% EtOAc-CHCly); "H NIMR (400MHz, ds-DMSO) 8 12.23 (s, 1H), 11 85 (s, 1H), 53.97 (m, 2H), 5.48 (t, /=6, 1H) , 4.35-4.40 (m, 1H), 4.254.31 (m, 1H), 4.08-4.18 (m, 1H), 2.49 (s, 3H), 2.07 (s, 3H), 2.01 Cs, 3H), 2.00 (s, 3H).

Example 29: 5-Amino-3-(2,3,5°-tri-O-acetyl-B-D-ribofuranosyl)thiazolof4,5- d)pyrimidine-2,7(6H)-diome (75) lo}

HN $

JL

~~

Al OAc

Step 1: Preparation of 5-A mino-3-(2°,3",5 '.tri-O-acetyl-p-D-ribofuranosyl)thiazolo[4,5- d]pyrimidine-2,7(6H)-dione (75)

[00265] To a suspension of 21 (5.00 g, 15.8 mmol) in acetonitrile (160 mL) at 0 °C was added successively EtaN (11.0 mL, 79.0 mmol), DMAP (195 mg, 1.59 mmol), and

Ac,0 (4.47 mL, 47.4 mmo1). The reaction mixture was stirred at room temperature for 2 h, whereupon it was concentrated to a brown syrup. The residue was purified by flash column chromatography (silica, MeOH/CHCl; = 1-10%) to afford 6.22 g (89%) of triacetate 7S as a white solid: mp 198-199 °C; 'H (400 MHz, ds-DMSO) 5 11.34 (s, 1H), 7.02 (br s, 2H), 5.90

(m2E).5.51 (t, J= 6-0 Hz, 1H), 4.36 (dd, J= 12.4, 32 Hz, 1H), 4.21 (m, 1H), 4.08 (, J= 6.0 Hz, 1H), 2.06 (s, 3H), 2.06 (s, 31), 2.00 (s, 3H); MS (+)-ES [MH] m/z 443.3.

Example 30: S-Amin o7-cthoxy-3-p-D-ribofuranosyl-thiazolof4;5-d]pyrimidin-2-on e an

CH3CH20

NZ NS

Bees ~~ wo on

Step 1: Preparation «of 5-Acetylamino-7-ethoxy-3-(2 "35 ».tri-O-acetyl-B-D-ribofuranesyl)- thiazolo[4,5-d]pyrim ddin-2-one (76) {00266} In a re anoer similar to Example 2, step 1, 76 was prepared from 74 and ethanol in 72% yield as a white foam: MS (+)-ES [M+H] m/z 513. Re= 0.45 (75% Ethyl acetate-CHCl,).

Step 2: Preparation of S-Amino-7-ethoxy-3-B-D-ribofuranosyl-thiazolo[ 4,5-d]pyrimidine- 2-one (77)

[00267] In a manner similar to Example 2, step 2, the title compound was prepamred from 76 in 65% yield as a white solid: 'H NMR (400 MHz, ds-DMSO) 3 6.87 (s, 2H),. 5.85 (d,J=4.8 Hz, 1H), S527 (4, J=5.6 Hz, 1H), 4.96 (4, J= 5.2 Hz, 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 (mn, 3H); MS (+)-ES [M-+H]" m/z 445, [2M+H]" m/z 689. Ry=0.2 (50% THF-CHC).

Elemental Analysis or Cy2H;6N40S-0.25 H20: calc’d: C, 41.31; H, 4.77; N, 16.06; S 9.19.

Found: C, 41.24; H, 471;N, 15.89; S, 9.06.

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

CH fees

NT NTN

~~ no oH

Step 1: Preparatiors of 5-Acetylamino-7-methoxy-3-(2",3 * 5°-tri-O-acetyl-p-D- ribofuranosyl)-thiazolo[4,5-d]pyrimidin-2-one (78)

' [60268] In a mnanner similar to Example 2, step 1, 77 was prepared from 74 and methanol in 65% yield as a white foam: MS (+)-ES [M+H]" 499. Re=0.5 (75% Ethyl acetate-CHCl3).

Step 2: Preparatiora of 5-Amino-7-methoxy-3-p-D-ribofuranosyl-thiazolo[: 4,5-dJpyrimidin— 2-one (79)

[00269] In a xnanner similar to Example 2, step 2, the title compound was prepared from 78 in 78% yield as a white solid: 'H NMR (400 MHz, ds-DMSO) 8 6.91 (s, 2H), 5.86 (d, J=5.2 Hz, 1H), 5.28 (d, J=5.2 Hz, 1H), 4.96 (d, /=5.2 Hz, 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. Re= 0.2 (50% THF-CHCl3). Elemental Analysis for C11H14N4068:0.25 HO: calcd: 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-Ammino-2-oxo-3-B-D-ribofuranosyl-2,3-dihydro-thiazolo[4,5- d)pyrimidin-7-yloxymethyl)-carbamic acid ethyl ester (82) ~ He

NZ NS

Bess

We om

Step 1: Preparation of 5-Amino-3-(2°,3",5 tris-O-triethylsilanyl-f-D-ribofuranosyl)- thiazolo[4,5-d]pyr3dmidin-2,7-dione (80)

[00270] To a suspension of 21 (1.00 g, 3.16 mmol) in DMF (20 mL) at room temperature was aclded successively imidazole (753 mg, 11.06 mmol), DMAP (39 mg, 0.32 mmol), and chlorotriethylsilane (1.64 mL, 9.80 mmol). The reaction mixture was stirred aat room temperature for 2 h, whereupon it was quenched by saturated NaHCO; solution 20 mL). The mixture was extracted with CHC; (3 x 20 mL), dried over MgSO, and concentrated. The residue was purified by flash column chromatography (silica,

MeOH/CHCl; = 1~5%) to afford 1.91 g (92%) of compound 80 as a white solid: "H (400

MHz, ds-DMSO) S 5.99 (s, 1H), 5.62 (brs, 2H), 5.19 (dd, J = 4.4, 6.0 Hz, 1H), 435 (dd, J= 2.8, 4.4 Hz, 1H), 3.99 (m, 1H), 3.77 (dd, J = 7.6, 10.8 Hz, 1H), 3.68 (dd, J = 4.8, 10.4 H=, 1H), 1.10 (t, J = 7-1 Hz, 3H), 0.96 (t, J=7.1 Hz, 3H), 0.89 (t, J=7.1 Hz, 3H), 0.68 (q, J = 7.1 Hz, 2H), 0.61 «gq, J = 7.1 Hz, 2H), 0.54 (m, 2H); MS (+)-ES [M+H]" m/z 660.0.

Step 2: Preparation of 5-Amino-3-2°,3',5 .tris-O-triethylsilanyl--D-ribofuranosyl)- 2, 3- dihydro-thiazolo[<,S-d]pyrimidin-7-yloxymethyl)-carbamic acid ethyl ester (81)

[002721] In a manner similar to Step 1 of Example 2, compound 81. was prepared from 80 anmd N-ethylurethane as a white solid in 31% yield: [M+H]" 760.5; 'H NMR (400 MHz,

CDC 13) 5 6.43 (br s, 2H), 6.09 (t, J=7.6 Hz, 1H), 5.94 (4, J= 6.0 Hz, 1H), 5.31 (d, J=4.38

Hz, 2H), 5.19 (dd, J=6.0, 4.8 Hz, 1H), 4.35 (dd, J=4.8,2.8 Hz, 1H), 4-19 (q, J=6.4 Hz, 2H), 3.98 (m, 1H), 3.76 (dd, J=10.8, 7.6 Hz, 1H), 3.68 (dd, J=10.4, 4.8 Hz, 1H), 1.29 (tJ =6.8 Hz, 3H), 1.02 (t, J = 8.0 Hz, 3H), 0.96 (t, J= 7.6 Hz, 3H), 0.90 (t, J = 8.0 Hz, 3H), 0.69 (q,J= 8.0 Hz, 2H), 0.61 (q, J = 8.0 Hz, 2H), 0.55 (m, 2H); [M+H]"™ 760.5.

Step 3: Preparation of (5-Amino-2-0x0-3-p-D-ribofuranosyl-2, 3-dihydr-o-thiazolof’ 4,5- d]pymimidin-7-yloxymethyl)-carbamic acid ethyl ester (82)

[002772] A solution of 81 (244 mg, 321 pmol), SM HF in pyridine (321 pL, 1.60 mmosl) and THF (3.20 mL) were stirred at room temperature for 3 h. Rernoval of the solve=nts under vacuum left a residue that was purified by flash chromatosgraphy (SiOs, 10%

MeOsH-CHCl) to afford 82 (119 mg, 90%) as a white solid: 'H NMR (4-00 MHz, ds-

DMSO) 6 8.43 (br s, 1H), 7.76 (br s, 2H), 5.82 (d, J =5.2 Hz, 1H), 5.78 (5, 2H), 532(d,J= 5.6 Hz, 1H), 5.24 (dd, J= 6.0, 4.8 Hz, 1H), 5.00 (d, J= 5.6 Hz, 1H), 4.82 (q, J= 5.6 Hz, 1H), 468 (t,J=6.0, 1H), 4.11 (q,J=5.2 Hz, 1H), 4.09 (q, J= 7.2 Hz, 2H), 3.78 (q, J = 5.6

Hz, BH), 3.60 (m, 1H), 3.46 (m, 1H), 1.21 (t, J= 7.2 Hz, 3H); [M+H]* 4 18.2.

Example 33: (5-Amino-2-0x0-3-B-D-ribofuranosyl-2,3-dihydro-thiazolo[4,5- d]pywrimidin-7-yloxymethyl)-methyl-carbamic acid ethyl ester (84) fo}

Ao

A

Bess ~~ wo OH

Step 1: Preparation of (5-Amino-2-ox0-3-(2°,3",5 -tri-O-acetyl-p-D-ribaofuranosyl)-2, 3- dihycdro-thiazolo[4, 5-d]pyrimidin-7-yloxymethyl)-methyl-carbamic acid ethyl ester 383

[002773] In a manner similar to Example 2, step 1, compound 83 was prepared from 75 amd N-methyl-N-(hydroxymethyljurethane as a white solid in 24% yield: R= 0.4 (33%

EO c-CHCL;); 'H NMR (400 MHz, CDCls) 8 11.49 (br s, 1H), 6.08 (dw J= 4.0 Hz, 1H), 5.75 «(t, J = 6.0 Hz, 1H), 5.53 (s, 2H), 4.49 (dd, J = 13.5, 8.4 Hz, 1H), 4.30 (m, 5H), 3.62 (q,

J=17 .2Hz, 2H), 2.30 (s, 3H), 2.12 (5, 3H), 2.09 (s, 3H), 2.08 (s, 3H), 1.36 (t, /= 6.8 Hz, 3H), 120 (t, J=6.8 Hz, 3H); [M+H]" 614.2.

Co So

Stepi— 2" Preparation of (5-Amino-2-0x0-3--D-ribofuranosyl-2,3-dikydr=o-thiazolo[ 4,5- d]py-rimidin-7-yloxymethyl)-methyl-carbamic acid ethyl ester (84)

[00274] In a manner similar to Example 1, step 4, the title compomind was prepared from 83 as a white solid in 20% yield: 'H NMR (400 MHz, ds-DMSO) & 7.86 (br 5, 21), 5.82 (4, J=4.8 Hz, 1H), 5.47 (5, 2H), 5.31 (d, J= 5.2 Hz, 1H), 5.00 (d, «/= 5.6 Hz, 1H), 4.82 (q, J=5.2 Hz, 1H), 4.67 (q, J = 5.6 Hz, 1H), 4.18 (g, J= 6.4 Hz, 2H), 4.12 (m, 1H), 3.78 (gq, J=6.0 Hz, 1H), 3.60 (m, 1H), 3.47 (m, 1H), 3.30 (s, 3H), 1.27 «t, J = 6.8 Hz, 3H); [M+-H]* 432.3.

Exaxuple 34: 5- Amino-7-(5-methyl-2-0x0-{1,3]dioxol-4-ylmethoxy)-3-2*,3°,5"-tri-0- acetyl-B-D-ribofuranosyl)-thiazolo[4,5-]pyrimidin-2-one (85)

Neg %- 0

NES

Bers ~~ : al ome

Stepr 1: Preparation of 5-Amino-7-(5-methyl-2-oxo-[. 1,3]dioxol-4-ylmethoxy)-3-(2°,3,5 ~ tri-O-acetyl-f-D-ribofuranosyl)-thiazolo[4,5-d]pyrimidin-2-one (85)

[00275] To a solution of triacetate 75 (1.55 g, 3.50 mmol) in THF (50 mL) at 0 °C was added polymer supported-triphenylphosphine (4.95 g, 10.50 mmol, Argonaut). To this mixture was added 4-hydroxymethyl-5-methyl-[1,3]dioxol-2-one (0.91 sg, 7.00 mmol), prep-ared according to the procedure of Alepegiani, Syn. Comm., 22(9), 1277-82 (1992)

Diethyl azodicarboxylate (0.73 ml, 4.60 mmol) was then added dropwis=e. The resulting mixture was stirred at room temperature for 48 h, filtered and washed w ith MeOH and

CHC. The filtrate was concentrated and purified by flash column chrornatography (silica, acetone/CHCIs = 10-20%) to afford dioxolone derivative 85 (1.38 g, 714) as white solid: 1H (4400 MHz, ds-DMSOY); & 7.06 (s, 2H), 6.00 (d, J = 4.0 Hz, 1H), 5.92 (dd, J=6.6,4.4 Hz, 1H), 5.56 (t,J = 6.4 Hz, 1H), 5.30 (5, 2H), 4.38 (dd, J= 11.6, 3.6 Hz, 11-1), 4.25 (t, J=3.6

Hz, 1H), 4.10 (q, J= 6.0 Hz, 1H), 2.23 (s, 3H), 2.08 (s, 3H), 2.07 (5, 3H), 2.00 (s, 3H); MS

HES [M+H]" m/z 555.3. Elemental Analysis calc’d for Cy HaaN401.S~MexCO: C, 47.06,

H, 4.61; N, 9.15; S, 5.23. Found: C, 47.25; H, 4.37; N, 9.53; §, 5.52.

Exarmnple 35: 5.Amino-7-(5-methyl-2-0xo0-{1,3]dioxol-4-ylmethoxy)-3~(-D- ribofuranosyl-thiazolo[4,5-d]pyrimidin-2-one (87)

Neg

C

0 jens

HN" TN =

Step 1: Preparation of 5-Amino-7-(5-methyl-2-oxo-[1,3 J dioxol-4-yImethoxy)-3-(2',3 V5 tris-O-triethylsilanyl-p-D-ribofuranosyl)-thiazolo[ ,5-d]pyrimidin-2-one (86) {00276} In a manner similar to Example 34, compound 86 was prepared from 80 and 4-hydroxymethyl-5-methyl-[1,3]dioxol-2-one as a white solid in 45% yield: 'H NMR (400

MHz, CDCl) 5 6.06 (d, J= 6.0 Hz, 1H), 5.21 (dd, J = 6.0, 4.8 Hz, 1H), 5.18 (d, J=3.2 Hz, 2H), 4.94 (brs, 2H), 4.38 (dd, J=4.8, 2.8 Hz, 1H), 4.00 (m, 1H), 3.79 (dd, J=11.2, 8.0 Hz, 1H), 3.69 (dd, J=10.8, 5.2 Hz, 1H), 2.23 (5, 3H), £.02 (t, J = 8.0 Hz, 3H), 0.96 (t,/=7.6

Hz, 3H), 0.89 (1, J = 8.4 Hz, 3H), 0.70 (q, J= 7.6 1Xz, 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-oxoe-{1 ,3]dioxol-4-ylmethoxy)-3--D- ribofuranosyl-thiazolof4,5-d]pyrimidin-2-one C1) ’

[00277] In a manner similar to Steps 3 of Excample 32, the title compound was prepared from 86 as a white solid in 89% yield: "HL NMR (400 MHz, dg-DMSO) 6 7.03 (br 5, 2H), 5.90 (d, J=5.2 Hz, 1H), 5.33 (5, 2H), 5.02 (d, J=4.8 Hz, 1H), 4.83 (q, J= 5.6 Hz, 1H), 4.71 (1, J = 6.0 Hz, 1H), 4.14 (q, J= 5.2 Hz, 1 H), 3.80 (q, J = 4.8 Hz, 1H), 3.62 (m, 1H), 3.47 (m, 1H), 2.27 (s, 3H); [M+H]" 429.2.

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

Lo

WNT ONT ON

Wo OM

Step 1: Preparation of 5-Amino-7-thioxo-3-(2',3°, 5 -tri-O-acetyl-p-D-ribofuranosyl)- thiazolo[4, 5-d]pyrimidin-2-one (88)

[00278] To a solution of 75 (1 g, 2.26 mmo) in pyridine (50 mL) was added at room temperature P2Ss (2.13 g, 4.79 mmol). The solutiom was refluxed gently (bath temperature 130 — 140 °C) for 29 h. The reaction mixture was evaporated to dryness in vacuo. The

“excess P;85 was decomposed by the addition of H;O (40 mL) at 60 °C. The mixture was stirred for 1 h at 60 °C and then cooled to room temperature. The mixture was extracted with CHC; (3 x 40 mL). The dried (MgSOs) organics layer was evaporated to yield a syrup, which was purified by flash column chromatography (silica, acetone/CHCls = 15%) to afford 0.93 g (90%) of 88 as a yellow solid: 'H (400 MHz, d;-DMSO) 3 12.50 (s, 1H), 7.35 (br s, 2H), 5.89 (m, 2H), 5.51 (t, J = 6.4 Hz, 1H), 4.346 (dd, J = 12.0, 4.0 Hz, 1H), 4.24 (m, 1H), 4.10 (q, J= 6.0 Hz, 1H), 2.07 (s, 3H), 2.06 (s, 37¥]), 2.01 (s, 3H); MS (H)-ES MH] m/z 459.3,

Step 2: Preparation of 5-Amino-3-(2',3’,5 "-tri-O-ace2tyl-p-D-ribofuranosyl)-3H- thiazolo[4,5-d]pyrimidin-2-one (89)

[00279] A suspension of Raney® 2800 nickel (3 big spatula, pre-washed with HzO,

MeOH and acetone) in acetone (50 mL) was stirred a_t refluxing for 1 h. Triacetate 88 (0.93 g, 2.03 mmol) was subsequently added into the above suspension at reflux. The mixture was stirred for 5 min, cooled to room temperature over 30» min, The reaction was quenched by bubbling HS (g) into the mixture for 2 h. The resultimg mixture was filtered through a short pad of Celite® and washed with EtOH. The filtrate waas concentrated and purified by flash column chromatography (silica, MeOH/CHCI; = 1-2%24) to afford 0.52 g (60%) of 89 as a white solid: mp 121-123 °C; 'H (400 MHz, ds-DMS() § 8.38 (s, 1H), 6.93 (s, 2H), 6.03 (d,

J=3.6 Hz, 1H), 5.93 (dd, J= 6.4, 3.6 Hz, 1H), 5.58 (t,J=6.0 Hz, 1H), 4.38 (dd, /= 11.6, 3.6 Hz, 1H), 4.26 (m, 1H), 4.11 (gq, J= 6.0 Hz, 1H), 2 .08 (s, 3H), 2.07 (s, 3H), 2.00 (s, 3H);

MS (H)-ES M+HT m/z 427.2. Elemental Analysis ca_lc’d for C1¢H sN4O5S-0.5 CH30H0.25

H20: C, 44.34; H, 4.62; N, 12.54; S 7.17. Found: C, 4-4.54; H, 4.88; N, 12.16; §, 7.17.

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

[00280] 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 at room temperature overnight, then neutralized with AcOH (21 pL, 0.36 mmol). The resialting mixture was stirred at room temperature for additional 30 min, concentrated, and triturated with H,O (2 ml) to afford 0.33 g of compound 90 (89%) as a white solid: mp 220 °C (Dec); 'H (400 MHz, ds-DMSO) 8.34 (s, 1H), 6.85 (s, 2H), 5.90 (d, J=4.8 Hz, 1H), S.31 (d, J= 5.6 Hz, 1H), 4.98 (d, J = 5.6 Hz, 1H), 4.81 (q, J=5.2 Hz, 1H), 4.67 (t, J= 6.0 Ez, 1H), 4.11 (q, J= 5.2 Hz, 1H), 3.77 (dd, J=10.8, 4.8 Hz, 1H), 3.58 (m, 1H), 3.44 (m, 1H) ; MS (H)-ES [M+H]" m/z 301.1.

Elemental Analysis calc’d for C10H12N305S-0.3 HoO: oC, 39.29; H, 4.15; N, 18.33; S 10.49.

Found: C, 39.51; H, 4.18; N, 17.95; S, 10.27.

‘Ex afiple 37: *S-Amino-3-(2”;3’-di-O-acetyl-B-D-ribenfuranosyl)-3H-thiazolo [4,5- d)pyrimidin-2-one (93)

LI

HN" ON ~~ : ald OAc

Step 1: Preparation of 5-Amino-3-(5 '. O-tert-butyl-dirnethylsilanyl-p-D-ribofuranosyl)-3H- thi_azolo[4,5-d]pyrimidin-2-one (91)

[00281] To a solution of 90 (0.68 g, 2.28 mmol.) in DMF (10 mL) was added im _idazole (0.54 g, 7.93 mmol) and tert-butyldimethyl silyl chloride (0.68 g, 4.56 mmol) secjuentially. The reaction mixture was stirred at room temperature for 2 h, at which point it wams concentrated and purified by flash column chromatography (silica, MeOH/CHCl; gradient = 5-20%)) to afford 0.49 g (52%) 91 as a whiste solid: 'H (400 MHz, ds-DMSO) 8 8.33 (s, 1H), 6.87 (s, 2H), 5.90 (d, J= 4.0 Hz, 1H), 5.33 (d, J= 5.6 Hz, 1H), 5.00(d,J= 5.2 Hz, 1H), 4.79 (q, J= 5.2 Hz, 1H), 4.16 (9, /=5.2 Hz, 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-dimethylsilanyl-f-D- ribmofuranosyl)-3H-thiazolo[4, 5-d]pyrimidin-2-one (92)

[00282] To a solution of 91 (0.20 g, 0.48 mmol. ) in acetonitrile (5 mL) at 0 °C was adcded successively Et;N (0.26 mL, 1.86 mmol) and A.c;O (91 pL, 0.96 mmol). The reaction mizxture was stirred at room temperature for 24 h, whesreupon it was concentrated and purified by flash column chromatography (silica, acet-one/CHCls: gradient = 5-10%) to afford 0.22 g (92%) of 92 as a white solid: 'H (400 M_Hz, ds-DMSO) 8.36 (s, 1H), 6.90 (s, 2H), 6.00 (m, 2H), 5.57 (t, J = 6.0 Hz, 1H), 4.07 (q, J = 5.2 Hz, 1H), 3.77 (m, 2H), 2.07 (s, 3HD), 2.06 (s, 3H), 0.83 (s, 9H), 0.00 (d, J = 2.4 Hz, 6H); MS (+)-ES [M+H]" m/z 499.5.

Ste=p 3: Preparation of 5-Amino-3-(2',3 -di-O-acetyl-p3-D-ribofuranosyl)-3H-thiazolo[4,5- d]poyrimidin-2-one (93) [oow283] To a solution of 92 (0.22 g, 0.44 mmol’) in THF (5 mL) in a plastic vial was adcded HF /pyridine (0.70 mL). The reaction was stirread for 2h, concentrated and purified by flassh column chromatography (silica, MeOH/CHCl;: ggradient = 5-10%) to afford 0.17 g _ (1080%) of the title compound as a white solid: mp 109-111 °C; H (400 MHz, ds-DMSO) 8 .37 (s, 1H), 6.91 (s, 2H), 6.00 (m, 2H), 5.48 (t, J= 6.0 Hz, 1H), 4.91 (, J= 6.0 Hz, 1H), 4.0 4 (dd, J= 10.4, 6.0 Hz, 1H), 3.64 (m, 1H), 3.52 (m., 1H), 2.08 (s, 3H), 2.05 (s, 3H); MS

(HES MAH] m/z 385.3. Elemental Analysis cale’d for C14H16N4078-0.5 CH;0H-0.2

CHCl: C, 41.61; H, 4.32; N, 13.21; S 7.56: Found: C, 41.73; H, 429;N, 12.86; S, 7.33.

Example 38: [2-Ethoxymethyl-1-(2-hhydroxy-2-methyl-propyl)-1H-imidazo[4,5- c¢jquinolin-4-yl]-carbamic acid ethyl ester (39)

Q oP

NZ pons

N ne

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

[00284] In a manner similar to step 1 of Example 8 except, substituting MeOH as the solvent, was prepared the title compound from 1-(4-amino-2-ethoxymethyl-imidazo[4,5- c]quinolin-1-yl)-2-methyl-propan-2-oR (38) (prepared according to the procedure given in

International Publication No. WO 94/1 7043) and diethyl pyrocarbonate as an oil in 39% yield: 'H NMR (400 MHz, CDCl;) 3 8.36 (d, J= 8.0 Hz, 1H), 8.05 (d,/=8.0 Hz, 1H), 7.70 (t, J=17.2 Hz, 1H), 7.61 (t, J= 8.0 Hz, 1H), 4.96 (br s, 2H), 4.80 (s, 2H), 4.39 (q, /=7.2

Hz, 2H), 3.62 (gq, J = 7.2 Hz, 2H), 1.40 (t, J= 7.2 Hz, 3H), 1.36 (br 5, 6H), 1.24 (t, J=6.8

Hz, 3H); MS ()-ES [M+H]" 387.4 mr%. 6.4 Masking Effect of TLR 7 Ligand Prodrugs

[00285] A typical experiment would use human peripheral blood mononuclear cells (PBMC) isolated from a healthy donor- and placed in replicate cell culture wells; typically, 2x10° to 5x10° cells are placed in eachn well. The PBMC are incubated in the absence of test compounds at 37 °C in a humidified atmosphere containing 5% CO; for 24 hours to allow stabilization to the culture conditions, sand then 100 micromolar isatoribine, the TLR7 ligand and a corresponding TLR7 ligand prodirug are added to separate wells containing PBMC from the same donor; untreated controls are included. The concentrations of TLR7 ligand and TLR7 ligand prodrug may be varied to suit the particular experiment, and the PBMC cultures are then incubated at 37 °C in a humidified atmosphere containing 5% CO; for a period of time ranging from two hours to 48 hours. Samples of cell culture supernate media are taken during the incubation. These are assayed for cytokine production by ELISA.

Additionally the amount of TLR7 ligarad and TLR7 ligand prodrug remaining at the end of the incubation may be assayed by LC-MS. Cytokine production is calculated relative to production in the isatoribine control, following subtraction of the cytokine production in untreated controls. The cytokine results are compared to determine the extent that the TLR7 ligand is more active than the corresponding TLR7 ligand prodrug.

[00286] * " Thus, if the TLR7 ligand generates more interferon alpha (a conveniently measured cytokine) than does the corresponding TLR7 ligand prodrug after a similar duration of exposure and concentration, the TLR7 ligand prodrug may be deemed a “masked” TLR7 ligand prodrug. The magnitude of reduction in cytokine production that constitutes “masking” may be as little as a 25% reduction relative to the parent TLR, sinc € this would afford a corresponding increase in administered dose for a given level of tolerability.

[00287] Tables 9 through 14 provide data illustrating that TLR7 ligands of multiple chemical classes <an be masked. The examples shown demonstrate substantial masking relative to the parent TLR7 ligand. The chemical substitutions shown are exemplary, and in no way restrictive of the invention, since additional chemical substitutions may also exhibit masking and are contemplated in the invention. Masking can be achieved by introductiom of substitutions at a range of locations on any TLR7 ligand, and as shown can incorporate a variety of chemical linkages. It will be appreciated that the preferred substitution and linkage may vary for different parent TLR7 ligands.

Table 9: Masking of Isatoribine Prodrugs

Compound No. Amount of INFa relative to that molecuale and ' induced by isatoribine at 100 uM, its prosdrugs %

Parent mxolecule 21 100

Isator-ibine

Prodxug:

Amino 24 1 acid ester

Prodxug:

Deoxy 93 0

Prodrug: 6-Eth oxy 77 0

Prodrug: 6-Methoxy 79 0

Prodrug:

Amimal 84 0

Prodruag:

Aminal 82 0

Prodrnag:

Dioxolemone 85 0

[00288] The masked property of isatoribine prodrugs can be demonstrated in a

PBMC assay. The results of the PBMC assay (Table 9) show the amount of INFa released after exposure of the parent compound and its prodrugs for either 8 hours (Cval-isatoribine, 24) or 24 hours (other prodrugs) at the initial concentration of 100 uM. The amount of the released INFa was normalized to that induced by 100 pM of isotoribine at 100 uM inthe same blood donor with the same exposure time.

Table 10: Masking of Loxoribine Prodrugs

Co Parent molecule Compound No. Amount of INFa relative to that

Er aa %

Parent molecule:

Loxoribine 17 50

Prodrug: 6-Ethoxy 45 0

Prodrug:

Deoxy 43 0

Prodrug:

Valyl ester 68 0

[00289] The masked property of loxoribine prodrugs can be demons-trated in a PBMC assay. Thue results of the PBMC assay (Table 10) show the amount of INFa released after exposure -of the parent compound and its prodrugs for 24 hours at the initia_1 concentration 100 uM. The amount of the released INFa was normalized to that induced by 100 uM of isotoribinee at 100 pM in the same blood donor with the same exposure times.

Table 11: Masking of Imiquimod Prodrugs

Amount of INFa relative tow that phyA Compound No. induced by isatoribine at 100 kM,

Parent molecule: .

Imiquimod 60-76 ’ Prodrug: 0

Pentyl carbamate

Prodrug: 0

Ethyl carbamate

“Results of two experiments with three different donors :

[002290] The masked property of imiquimod prodrugs can be demonsstrated in a

PBXMC assay. The results of the PBMC assay (Table 11) show the amount of INFa released after exposure of the parent compound and its prodrugs for 24 hours at the Anitial cormcentration 100 pM. The amount of the released INFa was normalized to that induced by 100 pM of isatoribine at 100 uM in the same blood donor with the same ex-posure time.

Table 12: Masking of Resiquimod Prodrugs

Parent Compound No. Amount of INFa relative tc» that molecule and induced by isatoribine at 1080 uM, its prodrugs %

Parent molecule: 39 9S @luM

Resgiquimod

Prodrug: 38 9@ 100 YM

Ethyl

Carbamate

[00291] The masked property of resiquimod prodrugs can be demon. strated in a

PBMC assay. The results of the PBMC assay (Table 12) show the amount of INFa released after exposure of the parent compound and its prodrugs for 24 hours at the initial concentrations of either 1 or 100 pM. The amount of the released INFa was normalized to that induced by 100 pM of isatoribine at 100 uM in the same blood donor wvith the same exposure time.

Table 13: Masking of Bropirimine Prodrugs

Parent molecule Compound No. Amount of INFa relative to that %

Fi te 3 ”

Prodrug:

Prodrug:

Ethoxy 37 0

Prodrug: 0

Ethyl 36 carbamate

Pr

Prodrug: 0

Pentyl 49 carbamate

[00292] The masked property of bropirimine gprodrugs can be demonstrated in a

PBMC assay. The results of the PBMC assay (Table 13) show the amount of INFa released after exposure of the parent compound and its prodrugs for 24 hours at the initial concentration of 100 pM. The amount of the releasesd INFa was normalized to that induced by 100 pM of Isatoribine at 100 uM in the same blosod donor with the same exposure time.

Table 14: Masking of Adeenine Prodrugs

Parent molecule Compound No. Amount of INFa relative to that

Ea %

Parent molecule 29 128 @ 0.1 M

Prodrug:

Methoy 65 0 @ 100 uM

Prodrug:

Ethoxy 64 0@ 10 pM

Deoxy 62 : 0@0.1 pM

BE pn 51 18@ 32 uM carbonate pentyl 54 15@ 10 pM carbonate

[00293] The masked property of adenine prodrugs can be demonstrated in a PBMC assay. The results of the PBMC assay (Table 14) skaow the amount of INFa released after exposure of the parent compound and its prodrugs £or 24 hours at different initial concentrations specified in the table. The amount ofthe released INFa was normalized to that induced by 100 uM of isatoribine at 100 pM in the same blood donor with the same exposure time.

[060294] TLR7 ligand prodrugs can also be asssessed in vitro for their conversion to the active parent TLR7 ligand. This can be measureed by incubation of the prodrug in blood, plasma, or in a cell culture of hepatocytes. At selecsted time intervals, samples are taken to determine the amount of prodrug remaining and the amount of TLR7 ligand produced.

Such determinations ere readily made by use of analytical tools known in the art such as

LC-MS. The determination of the extent of conversion of a masked TLR7 ligand prodrug to the parent TLR7 ligand is useful in interpreting data wherein masking is apparent at shorter times but diminishes upon long incubations irm the PBMC assay described herein.

The rate of conversion of the prodrug to the TLR7 liggand may be determined to ensure that the cytokine results arise predominately from exposure to prodrug rather than from exposure to TLR7 ligand generated by rapid conversion of the prodrug under the conditions of the experiment. 6.5 Biological Testing of TLR7 Ligand Prodrugs Demonstrating

Increased Oral Availability and Decre ased Side-Effects

Oral Availability {00295] The improved bioavailability of TLR 7 ligand prodrugs can be assessed by performing studies in vivo. In such experiments, the candidate prodrugs are administered by oral administration to mice, rats, monkeys, and/or do gs, and blood samples are taken at selected intervals. The blood samples are analysed for both the prodrug and the desired

TLR7 ligand. Additional blood or liver samples may~ be analyzed for the presence of interferons and other cytokines that indicate function al activation of the TLR7 pathway in vivo. Desired candidates will demonstrate a blood exposure to the prodrug and also demonstrate a blood exposure to the desired TLR7 liggand of about 10% to 99% of the applied dose, as measured on a molar basis.

[00296] A representative example is the result obtained with the TLR7 ligand prodrug val-isatoribine (24), which as described below in the mouse and dog generated significant amounts of the parent TLR7 ligand isator@bine (21) in the blood. See U.S. Patent

Application No. 10/305,061 (incorporated herein by reference in its entirety).

Q 0 s N s aes Bass ~~) HN Jone)

Ho OH AN wd om 21 24

Interferon Alpha (Mu-IFN-a) Concentrations in Mice

[00297] The normal mouse provides a useful system for the assessment of the degree to which the inventions described herein provide material improvement in the oral delivery of 21 (isatoribine). Not only can one measure the plasma concentrations of isatoribine arising from oral adminis tration of the said prodrug(s) but also the extensive immumnological research conducted in thes mouse has provided reagents suitable for measuring the Jevels of interferon alpha, a cytokine of interest reflecting one of the desired biologic activities of isatoribine.

[00298] We have wased the murine system in a series of experiments that dermonstrate that 24, the 5°-valine ester of 21 (val-isatoribine) elicits an interferon response sub-stantially improved over that resulting from administration of isatoribine itself.

[00299] Table 15 records the results of an assay for murine interferon alpha in the plasma of mice that were dosed two times with isatoribine, formulated in bicarbormate, at a level of 50 mg/kg by the oral route. It is evident that no interferon was measurablee even when the dose was repeated after an interval of four hours.

Table 15: Interferon Alpha (Mu-IFN-o) Plasma Concentration (pg/mL) in Mice WFollowing

Two Oral 50 mg/kg Doses of Isatoribine 4 Hours Apart

Teen Idviamlvawe Mem SD

First Dose 0.00 BQL” BQL'# BQL* 0.00 0.000 0.03 BQL* BQL*® BQL® 0.00 0.00 0.08 BQL* BQL¥ BQL® 0.00 0.000 0.25 BQL® BQL?® BQL? 0.00 0.000 0.50 BQL* BQL? BQL® 0.00 0.080 1.00 BL”? BQL* BQL® 0.00 0.080 1.50 BQL'® BQL® BQLY 0.00 0.00 2.00 BQL” BQL” BQL* 0.00 0.080 3.00 BQL? BQL? BQLY 0.00 0.080 4.00 BQL¥ BQL? BQL® 0.00 0.080

Second Dose 4.03 BQL* BQL? BQL? 0.00 0.080 4.08 BQL” BQL® BQL® 0.00 0.060 4.25 BQL* BQL* BQL* 0.00 0.080 4.50 BQL® BQLY* BQL® 0.00 0.080 5.00 BQL® BQLY BQLY® 0.00 0.080 5.50 BQLYS BQLYS BQL*? 0.00 0.00 6.00 BQL” BQLY? BQLY? 0.00 0.00 7.00 BQL® BQLY® BQL%® 0.00 0.00 8.00 BLY BQL® BQLY 0.00 0.000

TBQL"- Below Blevewed Quanifiable Limit <n pg/ml.

[00300] Table 16 records the results of assays for~ murine interferon alpha in the plasma of mice that first were dosed with bicarbonate amnd then four hours later were dosed

Orally with isatoribine, formulated in bicarbonate, at a level of 50 mg/kg. Interferon was reported in the plasma from four mice, including two thuat had received the bicarbonate wehicle dose. All the values reported in this experiment were low, and the reported

Enterferon levels were not consistently reported for all thhree mice assessed at each time + point, suggesting that these signals may be artifacts arissing from measurement near the

Bower limits of the assay. “Table 16: Interferon Alpha (Mu-TFN-a) Plasma Conce=nration (pg/mL) in Mice Following «One Vehicle Dose and One 50 mg/kg Doses of Isatorib -ine 4 Hours Later

TT Tmeh Indvidwi vale Mem SD ve 0.00 BQL¥® BQL'® BQLS?? 0.00 0.00 0.03 BQL® BQLY® BQL™"¢ 0.00 0.00 0.08 BQL® BQL*® BQL.* 0.00 0.00 0.25 BQL¥® BQL®* BQL.% 0.00 0.00 0.50 BQL® BQL¥ BQL_* 0.00 0.00 1.00 BQL® BQL*® BQL 1% 0.00 0.00 1.50 BQL® BQL'® BQL*® 0.00 0.00 2.00 34.9 BQL?® BQL.* 11.6 20.15 3.00 BQL* BQL® BQL_* 0.00 0.00 4.00 BQL® 35.4 BQL-'% 11.8 20.44

Second Dose 4.03 BQL* BQL* BQL_* 0.00 0.00 4.08 BQL* BQL® BQL_* 0.00 0.00 4.25 BQL* BQL® BQL_* 0.00 0.00 4.50 BQL'® BQL* 133..2 44.4 76.90 5.00 74.9 BQL® NR 37.5 52.96 5.50 BQL* BQL” BQL_Z 0.00 0.00 6.00 BQLY BQL"™ BQL_." 0.00 0.00 7.00 BQL* BQL¥ BQL_* 0.00 0.00 8.00 BQL¥ BQL® BQL_% 0.00 0.00

TBQL- Below Elevated Quantifisble Limit <n pgmil.

NR - Not reportable.

[00301] Table 17 records the results of assays for murine interferon alpha in the plasma of mice that were dosed orally with val-isatoritoine, dissolved in bicarbonate, at a dose that is equivalent to 50 mg/kg of isatoribine on a mmolar basis. It is evident that interferon was readily measurable at 1.0 hour, 1.5 hours, and 2.0 hours after dosing.

Interferon was detected in all mice assayed at a given time point, indicating the reliability of the effect following val-isatoribine administration. Thus a single administration of val- isatoribine was superior to either a single dose or a repeated dose of isatoribine.

Table 17: Plasma Concentration (pg/mL) of Interferon Alpha (Mu-TFN-q) in Mice

Following a Single 73.0 mg/kg Dose of Val-Isatoribine mn ndvdmvaw Mem SD 0.00 BQL BQL'® BQL? 0.00 0.00 0.25 BQL BQL BQL 0.00 0.00 0.50 BQL® BQL? BQL 0.00 0.00 0.75 BQL BQL BQL?® 0.00 0.00 1.00 173.2 125.1 89.0 129.1 4224 1.50 202.9 145.9 294.8 214.5 75.13 2.00 49.2 137.9 138.3 108.5 51.33 3.00 BQL¥ NR NR 0.00 0.00 4.00 BQL¥ 27.6 BQL 9.20 15.90 5.00 BQL BQL® BQL® 0.00 0.00 “BOL Below Be Quantfeble Limit <125pgml

BOL" - Below the Elevated Quantifiable Limit <n pg/ml

NR - Not Reportable

[00302] The data tabulated in Tables 15, 16, and 17 may be also considered from the point of view of the incidence of measurable interferon levels. Interferon was detected in the plasma of only 4 of the 114 mice used in the studies of isatoribine, whereas 10 of the 30 mice dosed with val-isatoribine had detectable interferon in their plasma. Thus, the prodrug increased the proportion of mice exhibiting an interferon response from 4% to 30 % and the magnitude of both the average and peak response was increased twofold (100%).

[00303]} In other experiments, plasma levels of isatoribine and interferon alpha were measured in mice that were dosed with isatoribine by the intravenous route, and these levels were compared to the levels of isatoribine and interferon alpha arising after oral administration of val—isatoribine. These data are summarized in Figure 1. In this figure itis evident that the levels of interferon alpha induced by oral val-isatoribine (“val-isator”) (ant 50 mg/kg isatoribine molar equivalent) was similar to that from intravenous isatoribine (“isator”) at 25 mg/kg. Thus, oral val-isatoribine provides levels of isatoribine and interferon that are approximately 50% of those observed after intravenous administration_ of isatoribine itself.

Beagle Dog

[00304] The effect of a prodrug (val-isatoribine, 24) on the systemic exposure to isatoribine (21) after oral administration to beagle dogs was investigated. Isator-ibine was prepared in sodium bicarbonate solution. Val-isatoribine and isatoribine were p-repared as the following formulations, which were chosen to ensure solubility:

[00305] FormulXation 1: Isatoribine in sodium bicarbonate solution, 1 ancl 4 mg/mL.

[06306] Formulation 2: Val-isatoribine in phosphate buffered saline, 1.622 and 6.48 mg/mL, equivalent to 1 and 4 mg/mL of isatoribine on a molar basis.

[00307] Four male and four female adult beagle dogs weighing between 715 to 27 kg and approximately 1-2 years old were used at the beginning of the study. The animals were divided into 2 groups of 2 males and 2 females each. The test material was administered by gavage on Days 1 and 8, allowing a 7 day washout period between administrati<ons. Blood samples (2 mL) were collected from each animal at predose, 15, 30 minutes, 1, 2,3,4,6,8 and 10 hours into lithium heparin tubes after each dosing. The plasma was froz=en at —70 °C until analysis. The pl asma was analyzed for isatoribine by an HPLC-MS/MS as=ssay.

[00308] The pharmacokinetic parameters for isatoribine arising from isat-oribine or val-isatoribine in each dog are summarized in Tables 18 and 19. The ratios for the key pharmacokinetic parameters defining the maximum concentration (Cmax) and -total exposure as measured by the area under the time-concentration curve (AUC) for the prodrug and the bicarbonate solution at the 50 mg/kg dose are summarized in Table 20. For the prodrug 24, the Cmax ratio was 2.98 & 0.695 and the AUC ratio was 2.38 + 0.4 85. These results indicate that at 50 mg/kg dose, the prodrug val-isatoribine provided subsstantially higher Cmax and greater bioavailability than isatoribine in bicarbonate solutiora.

[00309] The ratios for the Cmax and AUC for the prodrug to the bicarbo-nate solution for the 10 mg/kg dose 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 indicate that a-t 10 mg/kg dose, the prodrug val-—isatoribine provided higher Cmax and greater bioavailabi_lity than isatoribine in bicarbonate solution.

[00310] Thus, the maximum concentrations of isatoribine achieved after oral dosing are at least doubled, and the systemic exposure to isatoribine is enhanced by apsproximately 2-fold following oral administration of the prodrug val- isatoribine, compared to isatoribine itself, at both 10 and 50 mg/kg dose.

Table 18: Pharymacokinetic Parameters of Isatoribine in Dogs dosed at SO mg/kg © DosingPeriold I 2 ee

Formulation Isatoribine ~Val-

Asatoribine

I —— ee —————————————————————

Animal Number Dose, mg/kg 50 50 molar equivalent isatoribine

J

Dog 3517322 Cmax, ng/mL 3038.7 1 1741.5

Tmax, h 0.50 0.50

AUC(0-inf), 15227.0 3 3038.1 ng'h/mL

T1/2,h 6.4 2.4 ee

Dog 3521451 Cmax, ng/mL 3354.0 10652.1

Tmax, h 1.00 1.00

AUC(0-inf), ng/mL 9422.2 6552.7

Tz, b 19 1.6

Dog 3528707 Cmax, ng/mL 8915.3 0340.6

Tmax, h 0.50 0.50

AUC(0-inf), ng'hymL 29701.7 532730

Twas h 2.2 2.3

Dog 3532828 Cmax, ng/mL 6134.7 159879

Tmax, h 0.50 Q50

AUC(0-inf), ng-hvmL 12069.7 32987.0

Tay hb 1.4 16

Table 19: Pharmacokinetic Parameters of Isatoribine in Dogs Dosed at 10 mg/ke

Dosing Period 1 2

-

Formulation Isatoribine vall- isatori bine -

Dose, mg/kg molar 10 10

Animal Number equivalent isatoribine —

Dog 3524523 Cmax, ng/mL 4091.5 8594-.6

Tmax, h 1.00 0.50

AUC(0-inf), ng'/mL 13305.8 17166.2

Tin, h 2.1 1.7

Dog 3526402 Cmax, ng/mL. 1859.5 4047. 0

Tmax,h 1.00 1.00

AUC(0-inf), ng/mL. 5774.4 10548..9

Tie, bh 1.6 22

Dog 357450 Cmax, ng/mL. 1620.3 4228.27

Tmax, h 0.50 1.00

AUC(0-inf), ng-h/mL. 4387.3 11158.«0

Tz, h 1.5 2.3

Dog 354708 Cmax, ng/mL 2781.2 5784.8

Tmax, h 0.50 0.50

AUC(0-inf), ngh/mL ~~ 7522.1 12259.1

Tz, h 1.6 2.0

Table 20: Rati o of Pharmacokinetic Parameters of Isatoribine in Dogs Doosed at 50 mg/kg isatoribine

Animal Number

Dog 3517322 Cmax Ratio 1.00 3.86

AUC Ratio 1.00 2.17

Dog 3521451 Cmax Ratio 1.00 3.18

AUC Ratio 1.00 2.82

Dog 3528707 Cmax Ratio 1.00 2.28

AUC Ratio 1.00 1.79

Dog 3532828 Cmax Ratio 1.00 261

AUC Ratio 1.00 273 a

Mean Cmax Ratio NA 2.98

SD Cmax Ratio N/A 0.695

Mean AUC Ratio N/A 2.38

SD AUC Ratio NA 0.485

Table 21: Ratio of Pharmacokinetic Parameters of Isatoribine in Dogs Dosed at B 0 mg/kg

Formulation Isatoribine Val- isatoribine

Animal Number

Dog 3524523 Cmax Ratio 1.00 2.10

AUC Ratio 1.00 1.29

Dog 3526402 Cmax Ratio 1.00 2.18

AUC Ratio 1.00 2.20

Dog 3527450 Cmax Ratio 1.00 2.61

AUC Ratio 1.00 2.54

Dog 355708 Cmax Ratio 1.00 2.08

AUC Ratio 1.00 1.63

Mean Cmax Ratio N/A 2.24

SD Cmax Ratio N/A 0.249

Mean AUC Ratio N/A 1.82

SD AUC Ratio N/A 0.529

J

[00311] The prodrug val-isatoribine is preferred for several reasons. First, the prodmrug is easily formulated to provide a high proportion of active agent. This results in smal capsule sizes for a given dose, which is an advantage for an orzal product. Second, at the doses tested, val-isatoribine provides plasma levels of isatoribine= that are well within the range desirable for biologic effect after oral administration, which is not the case for isato-ribine itself.

Cynomolgus Monkey [003 12] From two to four male or female cynomolgus monkeys were used on the anirmal testing study. The study compound was formulated in a vehi cle appropriate for animal oral or intravenous administration. The vehicles used were e=ither as aqueous buffers or a solutions containing Cremophor. Animals were dosed via oral gavage or intravenous bolus injection for each test article. Blood samples (approximately O.5 mL) were collected at preedetermined time points (usually, pre-dose, 15, 30, and 45 minumtes and at 1, 1.5, 2, 2.5, 3,4, 8and 24 hours post-dose), placed into tubes containing disodium EDTA. The samples were placed on wet ice following collection, and plasma separated a=s rapidly as possible.

The plasma samples were aliquoted into a single vial, and stored fro=zen at approximately - 20°C until shipped on dry ice to the Sponsor. Animals were given feood and water appreoximately 4 hours after the dose. [003 13] The plasma samples were analyzed for a prodrug andl a parent compound usingz well-known LCMS/MS quantitation techniques by triple quad-rupole instruments, i.

Sciex API3000. The quantitation results for the parent compound delivered by oral administration of the parent compound itself or by its prodrug admiristered orally were used to caulculate the area-undér-the-cureve (AUC) values from time zeto to 24 hours (PO AUCO- 24h) . The comparison of the AUC values for the parent compound elelivered into systemic circualation with that delivered by the prodrug allowed calculating re ative oral bioavalibility of th e prodrug. See results provided in Tables 22-26. When the AUC data for the parent comppound delivered by the parent compound itself after its intraveneous administration were availlable (AUC IV), it allowed calculating the absolute oral bioavaillability by deviding the

PO AUC (0-24h) for the prodrug by the IV AUC (0-24h) for the par ent moleclule.

Table 22: Oral bioavailability of Isatoribine and its Prodrnags in Monkeys

W © 2005/025583 P&CT/US2004/028236 ee aes Structure Oral bioavailability in cynomolgus monkey, %

Parent molecule: 21

Isatoribine 3

Prodrug: .

Amino 24 7-9 , acid ester

Prodrug:

Deoxy 93 80

Prodrug: 6-Ethoxy 77 28

Prodrug: 6-Methoxy 79 21

Prodrug:

Aminal 84 14

Prodrug:

Aminal 82 4

Prodrug:

Dioxolenons 8s 17

Average of multiple experiments at different doses.

Table 23: Oral bioavailability of Loxoribine and its Prodrugss in Monkeys ond 1s marae | Compound No. Oral bioavailability in cynomolgus meonkey, %

Parent molecule:

Loxoribine 17 2

Prodrug: 6-Ethoxy 45 E

Prodrug: 13

Deoxy 43

Table 24: Oral bioavailability of Imiquimod amd its Prodrugs in Monkeys pln Compound No. «Oral bioavailability in cynomolgus monkey,

Y%

Parent molecule: 100

Imiquimod 31

AUC(0-24h)=9.0

Prodrug: 555

Pentyl 34 carbamate AUC(0-24h) = 50 234 i Prodrug: 50

Ethyl AUC(0-24h) =21.1 carbamate

Table 25: Oral bioavailability of Bropirimine ard its Prodrugs in Monkeys

Be eae | Compound No. Oral bioavailability in cynomolgus monkey, %

Parent molecule: 35 100

Bropirimine prodrug: 48 137

Deoxy

Prodrug: 37 94

Ethoxy

Produg: 36 33

Ethyl carbamate

Prodrug: 49 6

Pentyl carbamate * The oral bioavailability exceeding 100% may be assoc iated with gender differences since 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 pie Compound No. Oral bioavailability ir cynomolgus monkey, %

Parent molecule: 29 46

Prodrug:

Methoxy 65 13

Prodrug:

Ethoxy 64 4.6

Prodrug:

Deoxy 62 0.7

Prodrug: .

Pentyl 54 9.7 carbonate

Reduction of Gastrointestinal Irritancy

[00314] TLR7 ligand prodrugs of the invention also demonstrate unexpected and greatly reduced toxicology effects, and in particular re<duced GI irritancy.

[00315] The gastrointestinal (“GI”) tract is line} with substantial immune tissue (€.g.,

Peyer’s patches, etc.). TLR7 ligand prodrugs offer thes prospect of masking the active structure as the agent passes through lymphoid tissue Rining the gut, which should minimize activation of this tissue and thereby reduce GI irritancy.

[00316] Robins ef al. have shown that eliminati_on of the 5°-hydroxyl of isatoribine nucleoside eliminates activity. See Robins et al., Adv. Enzyme Regul., 29, 97-121 (1989). ‘Without being limited to any particular theory, it was “hypothesized that blockade of this hydroxyl site by an ester substitution would similarly eliminate activity but allow transport in the systemic circulation, where the valine ester would be cleaved and result in exposure to isatoribine.

[00317] We have found that the hypothesis wass confirmed. Formal toxicology studies of intravenously administered isatoribine and orally administered isatoribine and val- isatoribine were conducted in beagle dogs. The toxicology results for orally administered isatoribine are from a study conducted t>y ICN/Nucleic Acid Research

Institute.

[00318] We compared in the dog the oral toxiczology of 21 and 24, and the intravenous toxicology of 21. We observed that the oral toxicology of 24 was much more like intravenous 21 thzan it was like oral 21. In particular, the dose limiting toxicology of oral 3 was similar in raature to that of intravenous 21, and occurred at blood exposures that were similar to those @bserved after intravenous 21. In contrast, oral 21 had a different limiting toxicity (gastrointestinal lesions) and this toxicity was observed ata dose lower than the toxic dose of” either intravenous 21 or oral 24. Also, emesis was observed in dogs treated with oral 21 ak doses lower than the dose of oral 24 that resulted in emesis - See

Table 27. Other systesms for assessment of emesis also are known, such as in ferrets, allowing comparison of oral and intravenous administration of compounds. See, «.g.,

Strominger N. ef al., #Brain Res. Bull, 5, 445-451 (2001).

[00319] In each case the compound was administered as a solution, by gavaage or by intravenous infusion. Multiple parameters were assessed, as is customary in a tox<icology study. Inthe studies providing higher potential exposure to isatoribine, the plastea concentration of isatosribine was assessed by a LC/MS method. The notable GI findings were graded and are Listed in Table 27.

TABLE 27: Effect om GI Tolerance in Dogs after Dosing of Isatoribine (21) or V”al-

Isatoribine (24) Rank ed by Systemic Exposure (AUC) to Isatoribine in Toxicology Studies.

Oral Isatoribine IV Isatoribine Oral Va_l-

I dl Rl

Isatoribine | AUCesnrs | Emesis GI Emesis GI Emesis GI dose loose | Irritation | loose | irritation | loose | irritation (mg/kg) stool stool stool

I EN A

| ond [* [Ne | [|]

IE EL I I I I sr Jus fff [Ne | Me [se] ff [Nes | Mes [195 [ | [Ne | Ne 2 esp [ff [Ne | Me $s [28] [ [Ne [Ne [| #7] [Nee | Ne

ENE EEE

I A I I I

Oral Isatoribine IV Isatoriboine Oral Val-

I id nl

Ic cE FS 0 NL

I 0 FO Hl IL.

WL 2 I Ha a3 HN I

[003220] For orally administered isatoribine the principal findings were related to GI toler-ability as measured by GI irritancy. The clinical signs n_oted in Table 27 were emesis and/or loose stools. These clinical signs were more frequent in the 10 mg/kg group, and in one animal at this dose a bloody stool was noted. Gross histopathologic evaluation of the

GI tract noted multiple, scattered red lesions on the intestinal mucosa in four of eight dogs at 10» mg/kg, which on microscopic evaluation revealed cellu_lar congestion and hemorrhage, as might be expected for an ongoing localized inflammatory process. The GI effects estabwlished the NOAEL as 5 mg/kg.

[003221] Intravenously administered isatoribine resulte«d in emesis and/or loose stools as a common finding in dogs; this effect occurred at substantially higher applied doses than orall=y administered isatoribine. No lesions were seen in the CGI tract either at necropsy or histogpathologic evaluation of tissues. The GI toxicity did not affect the NOAEL, which was estab lished as 12.5 mg/kg on the basis of other findings.

[00322] Orally administered val-isatoribine demonstra—ted a toxicology profile similar to intravenously administered isatoribine. At higher applied adoses, emesis and loose stools were observed. No GI lesions were found, although this was a focus of evaluation in this study. As for intravenously administered isatoribine, the NO_AEL was established on the basis of other findings. The correspondence of observed toxi city to systemic exposures of isator-ibine is of interest in this study; the threshold of isatoribeine AUC for observation of emesis and loose stools is similar for intravenously administe=red isatoribine and orally admimistered val-isatoribine (Table 27).

[003223] The data in Table 27 indicate that orally admiraistered val-isatoribine provicdes an improved toxicity profile over orally administereed isatoribine, and is consistent with the hypothesis that chemical masking of the activity of issatoribine is afforded by chemically substituting an ester at the 5’-hydroxyl position of~ the nucleoside. As illustrated in Tables 9 through 14, it is possible to chemically mask any “TLR7 ligand using a variety of substituents. Engineering this substitution to be cleavable upo-n entry into the body affords systemic exposure to the useful activity of the compound withmout the limiting GI toxicity arising from the anatomical structure of the GI tract. As illust-rated in Tables 22 through 25,

it is possible to design the chennical substitution on a masked TLR7 ligand to be cleavable after administration. Thus, masked TLR7 ligand prodrugs can be generated for any TLR? ligand. This enables administration of doses that are substantially higher on a molar basis than otherwise would be acceptable, with the result of greater efficacy and reduced side effects when compared to administration of the parental “unmasked” compound alone. 6.6 Oral Composition

[00324] Table 28 illustrates a batch formulation and a single dose unit formulation containing 100 mg of val-isatoribine.

Table 28: Formulation for 100 mg tablet

Ce CO LL LL

Microcrystalline 53.5% 133.75 26.75 veal A A

Pluronic F-68 4.0% 10.00 2.00 — co

Croscarmellose 2.0% 5.00 1.00 bore | " \ Magnesium Stearate, [0.5% 1.25 0.25

Cm I

[00325] The microcry stalline cellulose, croscarmellose sodium, and val-isatoribine ) components are passed throtagh a #30 mesh screen (about 430p to about 655). The ) Pluronic F-68® (manufactured by JRH Biosciences, Inc. of Lenexa, KS) surfactant is passed through a #20 mesh screen (about 457 to about 1041p). The Pluronic F-68® surfactant and 0.5 kgs of crosscarmellose sodium are loaded into a 16 qt. twin shell tumble blender and are mixed for about 5 minutes. The mix is then transferred to a 3 cubic foot twin shell turnble blender where the microcrystalline cellulose is added and blended for about 5 minutes. The thalidomide is added and blended for an additional 25 minutes. This pre-blend is passed through aroller compactor with a hammer mill attached at the discharge of the roller compactor and moved back to the tumble blender. The remaining croscarmellose sodium and magnesium stearate is added to the tumble blender and blended for about 3 minutes. The fimal mixture is compressed on a rotary tablet press with 250 mg per tablet (200,000 tablet batch size).

6.7 Mucosal Composition

[00326] A concentrate is prepared by combining jsatoribine, and a 12.6 kg portion of the trichloromonofluoromethane in a sealed stainless steel vessel equipped with a Thigh shear mixer. Mixing is carried ou for about 20 minutes. The bulk suspension is then pre=pared in the sealed vessel by combining the concentrate with the balance of the propellants in a bulk product tank that is temperature controlled to 21° to 27°C. and pressure controllecl to 2.8 to 4.0 BAR. 17 ml aerosol co-ntainers which have a metered valve which is designe to provide 100 inhalations of ®he composition of the invention. Each container is provided with the following: val-isatoribine 0.0120 ¢ trichloromono-fluoromethane 1.6960 g : dichlorodifluoxomethane 3.7028g dichlorotetrafi-uoroethane 1.5766 g total _70000g 6.8 Intravenous Composition

[00327] The intravenous formulation is prepared by reconstituting a compeound of the invention with an appropri. ate liquid medium, such as water for injection (WFI) ora 5% dextrose solution. A desire=d concentration of the intravenous formulation can be obtained } by reconstituting an approwpriate amount of a compound of the invention with an appropriate volume of liquid medium. A desired concentration of the intravenous formulatiomn provides a therapeutically effective amount of a compound of the invention to the patient, preferably a mammal, more preferabRy a human, in need of the intravenous pharmaceutical formulation and maintains a therapeuti_cally effective level of a compound of the invention in_ the patient.

The dose which is therapeutically effective will depend on the rate at which the Lntravenous formulation is delivered to the patient and the concentration of the intravenous formulation.

[00328] For example, one vial containing a composition (e.g., 50 mg of a <compound of the invention per vial) sare reconstituted with a 5% dextrose solution (14 ml off 5% dextrose solution per vial yielding a total of 25 mL of solution. The reconstitute=d solution is incorporated into a dextrose solution in an infusion bag and q.s. to 50 mL, restalting in a solution containing 1 mg/mml of a compound of the invention suitable for intraverous infusion administration. The preferred concentration of a compound of the invermtion in the liquid medium, in the infusion bag, is about 0.001 to about 3 mg/ml, preferzably about 0.75 to about 1 mg/ml.

[00329] The foregoing has demonstrated the pertinent and importan® features of the present imvention. Many modifications and variations of the present inven-tion can be made without «leparting from its spirit and scope, as will be apparent to those skilled in the art.

The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims along with the full scope of equiv-alents to which such claims are entitled.

[00330] All references cited herein are incorporated herein by refereence in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorpeorated by reference in its entirety for all purposes.

Claims (1)

1. \ . Claims: . 1. A substance of composition for use in a method of treatment, said substance of . -. composition comprising & TLR7 ligand or a pharmaceutically acceptable salt, hydrate, or stereoisomer thereof and said method comprising treating a hepatitis C virus infection ir a patient by administering to the patient in need thereof a therapeutically or prophylactica_lly effective amount of said substance or composition.

   2. A substance or co mposition for use in a method of treatment, said substance or composition comprising & TLR7 ligand or a pharmaceutically acceptable salt, hydrate, Or stereoisomer thereof, wherein the TLR7 ligand is selected from analogs and derivatives of guanosine, imidazoquino line, adenine, and pyrimidine and said method comprising trea ting a hepatitis C virus infecti on in a patient by administering to the patient in need thereof a therapeutically or prophylactically effective amount of said substance or composition.

   3. A substance or composition for use in a method of treatment according to claim 2, wherein the TLR7 ligand is selected from (@] rR? Q Rv HN NH NA NS NZ ’ N HN SN HN" ON | a 0) HO © R HO s \_/ ’ R2 ) H = 5 H HO OH Ia ib Ic oO Rr Q N HN r- HN N—y Y N NH HN SH 7 HANS N rt S—0H O 0) R3 Ay | a HO” =~ HO —R! HS BH HG OH , Id le If 0} Ss HN NH; wr )=0 HN NT TN NZ NH z RNA O z , and HON HO OH ° Ig Ih AMENDED SHEET

   Lo ® 115 wherein: each R! is H, or a substituted or unsubstituted alkyl, alkenyl, or alkyrayl, which may be interrupted by one or more O, S, or N heteroatoms, or a substituted oer unsubstituted aryl or heteroaryl; R%is H, OH_, SH, halo, or a substituted or unsubstituted alkyl, alkeny~1, or alkynyl, which may be interrupted by one or more O, S, or N heteroatoms, or a substituted or unsubstituted -O-(alkyl), -#O-(aryl), -O-(heteroaryl), -S-(alkyl), -S-(aryl), -S-(heteroaryl), aryl, or heteroaryl; R?is H, OH_ or SH, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, -#0-(alkyl), -O-(aryl), -O-(heteroaryl), -S-(alkyl), -S-(aryl), -S-(heteroaryl), -NH(alkyl), ~NH(aryl), -NH(heteroaryl), -NH(R4)(alkyl), -NH(R*)(a-ryl), or -NH(R*)(het_eroaryl), wherein R* is a substituted or unsubstituted alk yl; XisOorS; Y is H, halo, OH, OR’, SH, SR*, or a substituted or unsubstituted alkyl or aryl; Z is H, halo, OH, OR", SH, or SR; or a pharmaceutically acceptable salt, hydrate, metabolite or stereoisemer thereof or a pharmaceutiscally acceptable salt or hydrate of said stereoisomer. 4, A substance or composition for use in a method of treatment according to claim 2, wherein the "TLR7 ligand is selected from Formula Ia, Ib, Ic, Id, Ie, IX, Ig, and Ih, wherein R! is H or a substituted or unsubstituted alkyl, alkenyl, or alkynyl; R= is H, OH, halo, or a substituted o-r unsubstituted alkyl, alkenyl, or alkynyl, or ~CH;-O-(alkzyl); R3is H, OH, or SH, or a substituted or unsubstituted -O-(alkyl), -S-(alkyl), or -NH(alkyR); X is O or S; Y is H, halo, OH, OR*, SH, or SR*; and Z is H, halo, OH, OR? SH, or SR.

   5. A substance or composition for use in a method of treatment according to claim 2, wherein the _I'LR7 ligand is selected from AMENDED SHEET

   0] 0 b lo] N Ss aR HN HN [=o [=o Ne EN N hn N EN N (e] a [0] HO HO HO HO ~~ OH, HO oH | HO OH NH, OH NZ" “NH HCO. A, N x o oY he NH, NH, N 2 N N= I NT CH, 0CH,CH; N Ly —( , and OH .

   6. A substance or composition for use in a method of treatment according to claim 2, wherein the patient is human.

   7. A substance or composition for use in a method of treatment according to claim 2, the method further comprising administerin g a pharmaceutically acceptable excipient, carrier, or vehicle.

   8. A substance or composition for use in a method of treatment according to claim 2, the method further comprising administerin_g an additional therapeutic agent.

   9. A substance or composition for use ®n a method of treatment according to claim 8, wherein the additional therapeutic agent is aan antiviral agent.

   10. A substance or composition for use ana method of treatment according to claim 2, wherein the therapeutically or prophylactically effective dose is 0.001 to 100 mg/kg per day.

   11. A substance or composition for use 1. na method of treatment according to claim 10, wherein the therapeutically or prophylactically effective dose is about 0.0] to 50 mg/kg per day. AMENIDDED SHEET

   ® 117 :

   12. A substance or composition for use in a method of treatment according to claim 11, wherein thme therapeutically or prophylactically effective dose is absout 0.1 to 20 mg/kg per day.

   13. A substance or composition for use in a method of treatment according to claim 2, wherein the TLR7 ligand is administered parenterally.

   14. A substance or composition for use in a method of treatment according to claim 2, wherein th.e TLR7 ligand is administered intravenously.

   15. A substance or composition for use in a method of treatment according to claim 2, wherein th.e TLR7 ligand is administered orally.

   16. A substance or composition for use in a method of treatment according to claim 2, wherein th € TLR7 ligand is administered mucosally.

   17. A substance or composition for use in a method of treatment, said substance or composition comprising a TLR7 ligand prodrug or a pharmaceutically acceptable salt, hydrate, or stereoisomer thereof and said method comprising treat®ng a hepatitis C virus infection im a patient by orally administering said substance or comm position to the patient in need thereof, wherein the oral administration of the TLR7 ligand prodrug achieves a therapeutically effective plasma concentration of the TLR7 ligand while reducing undesirable side effects associated with oral administration of TLR 7 ligands.

   18. A s~ubstance or composition for use in a method of treatmemt, said substance or compositio-n comprising a masked TLR7 ligand prodrug or a pharmaceutically acceptable salt, hydrat-e, or stereoisomer thereof and said method comprising tmreating a hepatitis C virus infection im a patient by orally administering said substance or com position to the patient in need thereof, wherein the oral administration of the masked TLR7 Eigand prodrug achieves a therapeutically effective plasma concentration of the TLR7 ligand while reducing undesirables side effects associated with oral administration of TLR 7 ligands.

   19. A substance or composition for use in a method of treatment according to claim 18, wherein thes masked TLR7 ligand prodrug is selected from analogs and derivatives of guanosine, imidazoquinoline, adenine, and pyrimidine. AMENDED SHEET

   ® 118

   20. A s ubstance or composition for use in a method of treatment according to claim 19, wherein thee prodrug is an (a) amide, carbamate, or amidine moiety after conversion of a TLR7 ligarad amine substituent, (b) ester, carbonate, carbamate, ether, imidate, acetal, aminal, or ketal moiety after conversion of a TLR7 ligand alcoshol substituent, (c) acetal or ketal moiety after conversion of a TLR7 ligand keto substituemt, (d) imidate moiety after conversion. of a TLR7 ligand carbonyl of an amido substituent, (¢) deoxygenated moiety after convesrsion of a TLR7 ligand oxo substituent of pyrimidire or guanosine, or (f) amine.

   21. A substance or composition for use in a method of treattment according to claim 20, wherein the prodrug is selected from an (a) carbamate moiety after conversion of a TLR7 ligand amine substituent, (b) ester, ether, or aminal moiety aftesr conversion of a TLR7 ligand alco hol substituent, or (¢) deoxygenated moiety after co nversion of a TLR7 ligand oxo substituent of pyrimidine or guanosine.

   22. A s-ubstance or composition for use in a method of treatment according to claim 18, wherein the prodrug of the masked TLR7 ligand is selected frosm lo] Rr! R® Rr R | >=x Rr’ HNN N NSN N “NH N= N 1 Ro o} R°<o lo} | a RC ORS RO OR —K R% R IIa , ITb , IIc , Oo rR! [0] N N HN HN LL) at ba HoN An HS NTN Ry 5 O 5 Oo NH R°-o Ro" NESAN $ A <5 KS Js >—R RO ORS RO ©OR® REN Ma 1d lle : uf : AMENDED SHEET

   ® 119 RE CT

   “i . sy O° NZN RNA coco] ‘ RO. ORS Og and 1h i wherein: each R' is H, or a substituted or unsubstituted alkyl, alkenwl, or alkynyl, which may be interrupted by one or more O, S, or N heteroatoms, or a su bstituted or unsubstituted aryl or heteroaryl; R%is H, OH_, SH, halo, or a substituted or unsubstituted alkzyl, alkenyl, or alkynyl, which may be interrupted by one or more O, S, or N heteroatoms , or a substituted or unsubstituted -O-(alkyl), -«O- (aryl), -O-(heteroaryl), -S-(alkyl), -S-(aryl), -S-(heteroaryl), aryl, or heteroaryl; R? is H, OH_ or SH, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, -#O-(alkyl), -O-(aryl), -O-(heteroaryl), -S-(alkyl)), -S-(aryl), -S-(heteroaryl), - NH(alkyl), - INH(aryl), -NH(heteroaryl), -NH(R*)(alkyl), -MNH(R*)(aryl), or -NH(R*)(het.eroaryl); R* is a subst-ituted or unsubstituted alkyl; R’ is indepemdently H, ~C(O)(C;-1salkyl), or a racemic, L- , or D- amino acid group —C(O)CHNH,;R?; R®is H, OR" °, or N(R"); R’ is indeperadently H or a substituted or unsubstituted —CCO)(C,-izalkyl) or —C(0)2(C;- 1salkyl); Ris H, -OH, -O-(alkyl), ~OCO,(C-1salkyl), ~OC(O)(C;- 1salkyl), or a racemic, L-, or D- amino acid ggroup —OC(O)CHNH,R'; R’isH, ora substituted or unsubstituted alkyl, C(O)CH(C x - alkyl)NH,, or -C(O)CH(CHS,- aryl)NH;; R'%is indepe ndently H, Cy. alkyl, Cz alkenyl, C37 alkyny~1, -(CR"*R")(C¢-C1 aryl), ~(CR'?R"%)(C3-Cyg cycloalkyl), -(CR'?R'*)(C4-C)g heterocyclic), (CR'2R"?) OH, -(CR"R")»C0,C 1.18 alkyl, and -(CR’R?)oN(R'*)CO,C 1.15 alkyl, and SOs(aryl), wherein t is an integer from 0 to 6 unless otherwise indicated, and vwherein the alkyl, alkenyl, alkynyl, aryl, cycloalkyl, and heterocyclic moieties of the foregoing groups are optionally substituted with substituents independently selected from halo, cyano, nitro, AMENDED SHEET

   ® 120 trifluoromethyl, trifluoromethoxy, C;-C¢ alkyl, C,-Cg alkenyl, C,-Cg alkynyl, hydroxy, Ci- Ce alkoxy, -NH,, -NH-alkyl, -N(alkyl),, ~-NH-aryl, -N(alkyl)(aryl), -N(aryl),, -NHCHO, -NHC(O)alkyl, -NHC(O)aryl, -N(alkyl)C(O)H, -N(alky)C(O)alkyl, -N(aryl)C(O)H, -N(aryl)C(O)alkyl, -NHCOsalkyl, -N(alkyl)CO»alkyl, -NHC(O)NH,, -N(alkyl)C(O)NH,, -NHC(O)NH-alkyl, -NHC(O) N(alkyl),, -N(alkyl)C(O)NH-alkyl, N(alkyl)C(O) N(alkyl), -NHSOs-alkyl, -N(alkyl)SO,-alkyl, -C(O)alkyl, -C(O)aryl, -OC(O)alkyl, -OC(O)aryl, -CO2- alkyl, -COs-aryl, -CO,H, -C(O)NH,, -C(€QO)NH-alkyl, -C(O)N(alkyl),, -C(O)NH-aryl, -C(O)N(aryl),, -C(O)N(alkyl)(aryl), -S(O)alkyl, -S(O)aryl, -SOsalkyl, -SO,aryl, -SO,NH;, - SO;NH-alkyl, and -SO,N(alkyl); R" is independently H, C,.¢ alkyl, C3-C, g cycloalkyl, or together with nitrogen forms a 5- or 6-membered heterocyclic ring; R'2 and R" are independently H, C,. alkyl, Ca. alkenyl, or Co alkynyl; R" is H, Cy. alkyl, or -CH,-aryl; XisOorS; Y is H, halo, OH, ORY, SH, SRY, NH,, N HRY, N(R, or a substituted or unsubstituted alkyl or aryl; and Z is H, halo, OH, OR", SH, or SR*; or a pharmaceutically acceptable salt, hydrate, metabolite or stereoisomer thereof or a pharmaceutically acceptable salt or hydrate of said stereoisomer.

   23. A substance or composition for use in a method of treatment according to claim 22, wherein R' is H or a substituted or unsubstituted alkyl, alkenyl, or alkynyl; R?is H, OH, halo, or a substituted or unsubstituted alkyl, alkenyl, or alkynyl, or -CH,-O-(alkyl); R’ is H, OH, or SH, or a substituted or unisubstituted -O-(alkyl), -S-(alkyl), or -NH(alkyl); R’ is independently H, —C(0)(C;- alkyl), or a racemic, L-, or D- amino acid group — C(O)CHNH,R®, wherein R® is an unsubstituted alkyl; R® is H or OR'’, wherein R'’ is independently C.¢ alkyl, C37 alkenyl, C3 _; alkynyl, (CR'"?R")(C¢-C}0 aryl), - (CR'’R")(C4-Cyg heterocyclic), and -(CR"?R'?),.oN(R'*)CO,C 1.15 alkyl, wherein t is an integer from 0 to 4 unless otherwise indicated, and wherein the alkyl, alkenyl, aryl, and heterocyclic moieties of the foregoing groups are optionally substituted with 1 to 3 substituents independently selected from halo, cyano, nitro, trifluoromethyl, trifluoromethoxy, C1-Cs alkyl, C,-C¢ alkenyl, C,-Cs alkynyl, hydroxy, C,-Cs alkoxy, -CO»- alkyl, -CO,-aryl, -OC(O)alkyl, and -OC(Q)aryl, and wherein R'? and R" are independently H, C4 alkyl, or Ca. alkenyl; and R'is HI, ~CHj, or -CH,CH3; R is independently H or a substituted or unsubstituted —C(O)(C,;-salkyl) or —C(O),(C,;-1salkyl); R%is H, -OH, -O- AMENDED SHEET

   ® 121 (alkyl), -OCO%(C,-1salkyl), or a racemic, L-, or D- amino acid group —OC(O)CHNH,R'; X is O or S; Y is H, halo, OH, OR", SH, or SR*; and Z is H, halo, OH, OR", SH, or SR".

   24. A substance or composition for use in a method of treatment according to claim 18, wherein the masked TLR7 ligand prodrug is selected from J eg CH3CH,0 >=o “x =o nS N EN N HO HO HO ~~ OH HO OH 0} No (ON PG ay 0 o} AN No wt NZ SN NZ —N [=o r =o 0 =o EN N EN N Sn N 0} ran ~) ~ ~~] PS N “ 5 , < = HO OH HO OH , HO OH NH; NH, wn N OCH,CH3 N Br Br i 7 NH 2 ANN INE NNN a. EN Po J = 0] = o lo} 0] Br lo} Br Br A 0} fo}

   NT. NN C 5 Ly AMENDED SHEET

   ® 122 NH, NH, NH, OR NC NZ SN > T S—ocH, Qu S— OCH, CH, ia N 5 Ny Ia N OCH, | , OCH , OCH; | , fo) NH, fo: NH» 0 oN NN“ | Ne “Hocker; On Hoch, os | Y»>—o = N OCH,CH oS AA A Sa So TO Ly OCHj , OCH3 , OH , NH; «© NH, 0 NH, o I Voc ORS Voces I Soci © | “0 | S>—o 7 N Ny N EN N OCH, | , OcH, | c NH, o NH fo) — N ANN OCHS RE N N eT - PNY a OCHj3 , OCH; , N H, lo) NH, fe] ~~ a. Mo NES Mo [| Ho | Ho B® Be OCH; , OCHs , NZ AN = NZ SN >= ony PS | >= J N N J N N Cl OCH; , OCH | , 0 lo) CHACH,0 S S = S = S HN HN N N ETS Te Fe °N N N HN ON N HN" “NT TN HN ON N AcO AcO HO HO AQ OAC ACO OAc HO oH HO oH AMENDED SHEET

   ® 123 lo Ng Ge o 0 0 oto wr S NZ SS NS s [ =o r pa “r pa AS N oS N PN N o A $ - s ~ So “, HO OH HO OH HO OH |, 0 CH30 Ao an NT Ss NPN —S [=o TU =o 9 S—o HoN sy N nS N nS N HO» HO HO HO OH AO OAc HO OH , and fo Vo

   [0] PP T NG Tr y= HN NT TN ~ AcO AO OAc ,

   25. A sub stance or composition for use in a method of treatment accordEng to claim 18, wherein the o ral administration of the masked TLR7 ligand prodrug achieves an in vivo effective plasama concentration of the TLR7 ligand that is 10% to 500% of the effective in vivo exposures obtained upon oral administration of the TLR7 ligand alone.

   26. A substance or composition for use in a method of treatment according to claim 18, wherein the oral administration of the masked TLR 7 ligand prodrug achievess an in vivo effective plasma concentration of the TLR7 ligand that is 50% to 200% of the effective in vivo exposure obtained upon oral administration of the TLR7 ligand alone.

   27. A subsstance or composition for use in a method of treatment accordi ng to claim 25, wherein the owal administration of the masked TLR7 ligand prodrug achievess a AMENDED SHEET

   ® 124 “therapeutically effective plasma concentration of the corresponding TLR7 ligand without «causing gastrointestinal irritation.

   228. A substance or composition for use in a method of tre atment according to claim 18, “wherein the oral administration of the masked TLR7 ligand p rodrug reduces undesirable ssjde effects in a patient relative to the side effects upon oral a.dministration of the TLR7 ligand alone.

   29. A substance or composition for use in a method of tre atment according to claim 18, “wherein the oral administration of the masked TLR7 ligand p rodrug reduces undesirable side effects by 50% in a patient relative to the side effects upon oral administration of the “TLR7 ligand alone.

   30. A substance or composition for use in a method of tre atment according to claim 18, —wherein the side effect is gastrointestinal irritancy.

   31. A substance or composition for use in a method of tre atment according to claim 30, =wherein the irritancy is hemorrhage.

   32. A substance or composition for use in a method of tre atment according to claim 30, ~wherein the irritancy is lesions.

   33. A substance or composition for use in a method of treatment according to claim 30, =wherein the irritancy is emesis.

   34. A substance or composition for use in a method of treatment according to claim 18, wnherein the patient is human.

   3S. A substance or composition for use in a method of treatment according to claim 18, ssaid method further comprising administering a pharmaceuticzally acceptable excipient, carrier, or vehicle.

   36. A substance or composition for use in a method of treatment according to claim 18, ssaid method further comprising administering an additional thherapeutic agent. AMENDED SHEET

   ® 125

   37. A substance or composition for use in a method of tmreatment according to claim 36, wherein the additional therapeutic agent is an antiviral agen_t.

   38. A substance or composition for use in a method of tmreatment according to claim 18, wherein the therapeutically or prophylactically effective dosse is 0.001 to 100 mg/kg per day.

   39. A substance or composition for use in a method of tareatment according to claim 38, wherein the therapeutically or prophylactically effective dosse is about 0.1 to 25 mg/kg per day.

   40. A substance or composition for use in a method of te-eatment according to claim 39, wherein the therapeutically or prophylactically effective dosse is about 1 to 20 mg/kg per day.

   41. A substance or composition for use in a method of treatment according to claim 18, wherein the masked TLR?7 ligand prodrug is administered p.arenterally.

   42. A substance or composition for use in a method of tr-eatment according to claim 41, wherein the masked TLR7 ligand prodrug is administered iratravenously.

   43. A substance or composition for use in a method of tr-eatment according to claim 18, wherein the masked TLR?7 ligand prodrug is administered omrally.

   44. A substance or composition for use in a method of treatment according to claim 18, wherein the masked TLR7 ligand prodrug is administered mmucosally.

   45. A substance or composition for use in a method of tr-eatment, said substance or composition comprising a masked TLR7 ligand prodrug andl said method comprising treating diseases responsive to immuno therapy while reducing undesirable side effects associated with immunologic agents, by orally administering said substance or composition to a patient in need of immuno therapy, wherein the masked TLR?7 ligand prodrug achieves a therapeutically effective plasma concentration of a TLR7 | igand in the patient. AMENDED SHEET

   ® 126

   46. A substance or composition for use in a rmethod of treatment according to claim 45, “wherein the masked TLR7 ligand prodrug is selected from analogs and derivatives of seuanosine, imidazoquinoline, adenine, and pyrimidine.

   =47. A substance or composition for use in a rmethod of treatment according to claim 45, “wherein the prodrug is an (a) amide, carbamate, «or amidine moiety after conversion of a "TLR?7 ligand amine substituent, (b) ester, carbon ate, carbamate, ether, imidate, acetal, zminal, or ketal moiety after conversion of a TLIRR7 ligand alcohol substituent, (c) acetal or ketal moiety after conversion of a TLR7 ligand keto substituent, (d) imidate moiety after conversion of a TLR7 ligand carbonyl of an amiclo substituent, (¢) deoxygenated moiety after conversion of a TLR7 ligand oxo substituert of pyrimidine or guanosine, or (f) amine.

   =}8. A substance or composition for use in a rmethod of treatment according to claim 47, =wherein the prodrug is selected from an (a) carbamate moiety after conversion of a TLR7 Rigand amine substituent, (b) ester, ether, or animal moiety after conversion of a TLR7 NMigand alcohol substituent, or (¢) deoxygenated moiety after conversion of a TLR7 ligand oxo substituent of pyrimidine or guanosine.

   =49. A substance or composition for use in a method of treatment according to claim 45, wvherein the masked TLR7 ligand prodrug is selected from 0 rR! <8 R' wy ; NZ N R os =X Rr’ HN N HS N “NH RS 0 5 o NT Ng ie R™~0 N RSG ORS RG ORS D> R? : IIa , IIb , IIc , (¢] rR! O i HN N HN N oY Nr S—y HN A HN As N N

   R'. Oo 5 oO NH Ro" RP~0"™\ NSN p— pu Js >—R® 5,8 © 5 [PS “55 3 N RO OR RO OR RTENTIN 1d , Ile , If , AMENDED SHEET

   ® 127 RE ON ps HN SN Ra NZ N RNAs cot] ’ RO" ORS Hg nd 11h i wherein: each R' is H, or a substituted or unsubstitiated alkyl, alkenyl, or alkynyl, which may be interrupted by one or more O, S, or N hetesroatoms, or a substituted or unsubstituted aryl or heteroaryl, Ris H, OH, SH, halo, or a substituted or wminsubstituted alkyl, alkenyl, or alkynyl, which may be interrupted by one or more O, S, o rN heteroatoms, or a substituted or unsuabstituted -O-(alkyl), -O-(aryl), -O-(heteroaryl), -S-(zalkyl), -S-(aryl), -S-(heteroaryl), aryl, or heteroaryl; R? is H, OH, or SH, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, -O-(alkyl), -O-(aryl), -O-(heter-oaryl), -S-(alkyl), -S-(aryl), -S-(heteroaryl), - NH(alkyl), -NH(aryl), -NH(heteroaryl), -NJH(R*)(alkyl), -NH(R*(aryl), or -NH(R*)(heteroaryl); R* is a substituted or unsubstituted alkyl; R’ is independently H, ~C(O)(C;-1salkyl), or a racemic, L-, or D- amino acid group —C(O)CHNH,R?; R®is H, OR", or N(R'")y; R’ is independently H or a substituted or u_nsubstituted ~C(O)(C,-13alkyl) or —C(OW(C;- 1salkyl); R%is H, -OH, -O-(alkyl), -OCO2(C;-1galk v1), -OC(O)(C,-1salkyl), or a racemic, L—, or D- amino acid group ~OC(O)CHNH:R'; R’ is H, or a substituted or unsubstituted allkyl, C(O)CH(C, alkyl)NH,, or —C(O)CCH(CH,- aryl)NH,; R!%is independently H, Cy alkyl, C3.; alkenyl, C3.7 alkynyl, -(CR'?R"*)(C¢-C1q arwl), -(CR"R"?)(C3-Cyg cycloalkyl), -(CR'*R "Ww ((C4-C1g heterocyclic), -(CR'?R'?)- OH, ~(CR" R")4C0O,C).15 alkyl, and -(CR"?R'Z)s(N(R'*)CO,C,. 5 alkyl, and SO, (aryl) , wherein t is an integer from 0 to 6 unless otherwise indicated, and wherein the alkyl, alkenyl, alkynyl, aryl, cycloalkyl, and heterocyclic -moieties of the foregoing groups are optionally substituted with substituents independently selected from halo, cyano, nitro, AMENIDED SHEET trifluoromethyl, trifluoromethoxy, C,-Cg alkyl, C,-C¢ alkenyl, Co-C¢ alkynwl, hydroxy, Ci- Ce alkoxy, -NIH,, -NH-alkyl, -N(alkyl),, -NH-aryl, -N(alkyl)(aryl), -N(ary]»,, -NHCHO, -NHC(O)alky1, -NHC(O)aryl, -N(alkyl)C(O)H, -N(alkyl)C(O)alkyl, -N(ary’1)C(O)H, -N(aryl)C(O)alkyl, -NHCOsalkyl, -N(alkyl)CO,alkyl, -NHC(O)NH,, -N(al kyl)C(O)NH,, -NHC(O)NH-alkyl, -NHC(O) N(alkyl),, -N(alkyl)C(O)NH-alkyl, N(alky])C(O) N(alkyl)2, -NHSO,-alky1, -N(alkyl)SO,-alkyl, -C(O)alkyl, -C(O)aryl, -OC(O)alkyl, -OC(O)aryl, -CO,- alkyl, -COs-axyl, -CO,H, -C(O)NH,;, -C(O)NH-alkyl, -C(O)N(alkyl),, -C(O)NH-aryl, -C(O)N(aryl)o, -C(O)N(alkyl)(aryl), -S(O)alkyl, -S(O)aryl, -SO,alkyl, -SOzaryl, -SO,NH>, - SO,NH-alkyl , and -SO,N(alkyl),; R'!is independently H, C,. alkyl, C5-C), cycloalkyl, or together with nitro gen forms a 5- or 6-membered heterocyclic ring; R'? and R" ame independently H, C, alkyl, C, alkenyl, or Cy. alkynyl; R'is H, C16 alkyl, or -CHy-aryl; XisOorS; Y is H, halo, ©OH, OR*, SH, SRY, NH,, NHR? N(R%),, or a substituted or urasubstituted alkyl or aryl; and Z is H, halo, OH, OR, SH, or SR*; or a pharmaceutically acceptable salt, hydrate, metabolite or stereoisomer thereof or a pharmaceutically acceptable salt or hydrate of said stereoisomer.

   50. A substance or composition for use in a method of treatment according to claim 49, whesrein R' is H or a substituted or unsubstituted alkyl, alkenyl, ox alkynyl; R? is H, OH, halo, or a substituted or unsubstituted alkyl, alkenyl, or alkynyl, or —CH,-O-(alkyl); R? is H, OH, or SH, or a substituted or unsubstituted -O-(alkyl), -S-(alkyl), or -NH(alkyl); R’ is independently H, —~C(O)(C,-1salkyl), or a racemic, L-, or D- amino aci d group — C(O)CHNH,R’, wherein R’ is an unsubstituted alkyl; R®is H or OR'®, wherein R'is independently Cy. alkyl, C.7 alkenyl, C3.; alkynyl, (CR"*R'3)(C¢-C)q aryl), - (CR'’R')(C4-Cio heterocyclic), and -(CR'*R'?)oN(R')CO,C 1.15 alkyl, wherein t is an integer from O to 4 unless otherwise indicated, and wherein the alkyl, alkenyl, aryl, and heterocyclic moieties of the foregoing groups are optionally substituted with 1 to 3 substituents independently selected from halo, cyano, nitro, trifluoromethyl trifluoromethoxy, Ci-Cg alkyl, C,-Cg alkenyl, C,-Cs alkynyl, hydroxy, C-C alkoxy, -CO,- alkyl, -CO»-ar-yl, -OC(O)alkyl, and -OC(O)aryl, and wherein R'? and R"* ar e independently H, Cy alkyl, or C,. alkenyl; and R'is H, —CHs, or —-CH,CH3; R'is indepe=ndently H or a substituted or unsubstituted —C(O)(C,-jgalkyl) or —C(O)2(Ci-1salkyl); R%is ©, -OH, -O- AMENDED SHEET

   (alkyl), ~OCO,(C)-1galkyl), or a racemic, L-, or D- amino acid group —OC(0)CHNH,R'; X is O or S; Y is H, halo, OH, OR", SH, or SR*; and 2 is H, halo, OH, OR", SH, or SR".

   51. A substance or composition for use in a method of treatment according to claim 45, wherein the masked TLR7 ligand prodrug is selected from J eg CH 5CHL0 =o “I »=0 oS N HoN As N HO HO HO OH , HO OH 0} No 0 ~ SP 0 0 EN wo AN py [ =o 9 =o “ =o nS N EN N nS N o) HO OH HO OH , HO oH NH, NH, > Wn NX 0CH,CH, Br Br fo} 0} NH 2 I PNY He A gr Ay Pw Nu YY Xe x o Oo Oo Br 0} TY ory” ~ 0} 0 PN NENG Lr > Lr y AMENDED SHESET

   7 ® 130 NH, NH, NH; > oom | S—och,ch ba y Ia y ba Ny OCHs3 N OCHj N OCH, 5 fo} NH, 0 NH ) aN PI 0 A CI H—ocaH, © NH fo Z N_ OCH,CH Ny N EN N N 1 oe SD J Ly OCH; , OCH, , OH , NH; fo NH, o NH 0 Ed TY H—o0CsHs; nS —0CeHi o o ba N 7 y 7 N OCH; | , OCH; , OCH, NH, o NH, o ( NF N M—ocrHis NZ ~N Mo jen A OCH5 , OCHs NH, o { NH o / 8 = NZ N »o [ do [| Sd y 7 y OCH; , OCH; , NH, o - NH, o NZ SN yo NZ SN > Ys PN a 07 °N N 0” °N N Cl

   NX. I P OCHa , OCH; , 0) fo) CHaCH,0 s Js s s " HN N= N=" [ =o Ju =o “x =o gs =o A ~ © Sn N nS N nS N nS N AcO AcO HO HO AG OAc , AO OAc HO oH | HO OH , AMENDED SHEET

   @® 131 Q No o a i 0 Io Ao S s S HN NT NE yg yo Y ro [gs y= HN ONT TN H,NT ONT ON HoNT NT TN o HN 0 o 0 z NE HO HO HO OH HO OH HO OH , 0 CH;0 a Co CT “ oS N EN N on N HO HO HO HO oH AO OAc #0 oH and 0 No ON PN S NZ NS ry 7° H,NT NT TN AcO AO OAc R

   52. A substance or composition for use in a method of treatmert according to claim 45, wherein the disease is a viral infection.

   53. As substance or composition for use in a method of treatmert according to claim 52, wherein the viral infection is selected from an RNA or DNA virus including adenovirus, cytomegalovirus, hepatitis A virus (HAV), hepadnaviruses includirmg hepatitis B virus (HBV), flaviviruses including Yellow Fever virus, hepaciviruses iracluding hepatitis C virus (HCV), lmerpes simplex type 1 and 2, herpes zoster, human herpesv-irus 6, human immunodeficiency virus (HIV), human papilloma virus (HPV), influenza A virus, influenza B virus, rneasles, parainfluenza virus, pestivirus, poliovirus, poxvir-us (including smallpox and monlkeypox virus), rhinovirus, coronovirus, respiratory syncyti al virus (RSV), multiple AMENDED SHEET

   ® 132 families of viruses that cause hemorrhagic fevers, imcluding the Arenaviruses, the Bunyaviruses and Filoviruses, and a range of viral encephalitides caused by RNA and DNA viruses.

   54. A substance or composition for use in a met hod of treatment according to claim 45, wherein the disease is a cancer.

   SS. A substance or composition for use in a method of treatment according to claim 45, wherein the oral administration of the masked TLR7 ligand prodrug achieves an in vivo effective plasma concentration of the TLR7 ligand t hat is 10% to 500% of the effective in vivo exposure obtained upon oral administration of €he TLR7 ligand alone.

   56. A substance or composition for use in a method of treatment according to claim 55, wherein the oral administration of the masked TLR7 ligand prodrug achieves an in vivo effective plasma concentration of the TLR7 ligand tkat is 50% to 200% of the effective in vivo exposure obtained upon oral administration of tthe TLR7 ligand alone.

   57. A substance or composition for use in a methnod of treatment according to claim 56, wherein the oral administration of the masked TLR7 ligand prodrug achieves a therapeutically effective plasma concentration of the corresponding TLR7 ligand without causing gastrointestinal irritation.

   58. A substance or composition for use in a meth«od of treatment according to claim 45, wherein the oral administration of the masked TLR7 ligand prodrug reduces undesirable side effects in a patient relative to the side effects upon oral administration of the TLR7 ligand alone.

   59. A substance or composition for use in a method of treatment according to claim 45, wherein the oral administration of the masked TLR7 Aigand prodrug reduces undesirable side effects by 50% in a patient relative to the side effects upon oral administration of the TLR7 ligand alone.

   60. A substance or composition for use in a methoed of treatment according to claim 45, wherein the side effect is gastrointestinal irritancy. AMENDED SHEET

   ® 133

   61. A substance or composition for use in a rmethod of treatment according to claim 60, wherein the irritancy is hemorrhage.

   62. A substance or composition for use in a rmethod of treatment according to claim 60, wherein the irritancy is lesions.

   63. A substance or composition for use in a rnethod of treatment according to claim 60, wherein the irritancy is emesis.

   64. A substance or composition for use in a method of treatment according to claim 45, wherein the patient is human.

   65. A substance or composition for use in a method of treatment according to claim 45, further comprising administering a pharmaceutically acceptable excipient, carrier, or vehicle.

   66. A substance or composition for use in a meethod of treatment according to claim 45, further comprising administering an additional therapeutic agent.

   67. A substance or composition for use in a m ethod of treatment according to claim 66, wherein the additional therapeutic agent is an anti viral agent.

   68. A substance or composition for use in a me=thod of treatment according to claim 45, wherein the therapeutically or prophylactically eff ective dose is 0.001 to 100 mg/kg per day.

   69. A substance or composition for use in a method of treatment according to claim 68, wherein the therapeutically or prophylactically effective dose is about 0.01 to SO mg/kg per day.

   70. A substance or composition for use in a me=thod of treatment according to claim 69, wherein the therapeutically or prophylactically effective dose is about 0.1 to 20 mg/kg per day.

   71. A substance or composition for use in a method of treatment according to claim 45, wherein the masked TLR7 ligand prodrug is admin_istered parenterally. AMENDED SHEET

   ® 3a

   72. A substance or composition for use in a amethod of treatment according to claim 71, wherein the masked TLR7 ligand prodrug is administered intravenously.

   73. A substance or composition for use in a rnethod of treatment according to claim 45, wherein the masked TLR7 ligand prodrug is administered orally.

   74. A substance or composition for use in a rnethod of treatment according to claim 45, wherein the masked TLR7 ligand prodrug is administered mucosally.

   75. Use of a TLR7 ligand or a pharmaceutically acceptable salt, hydrate, or stereoisomer thereof in the manufacture of a preparation for treating a hepatitis C virus infection in a patient by administering to the patient in need the=reof a therapeutically or prophylactically effective amount of said preparation.

   76. Use of a TLR7 ligand or a pharmaceutically acceptable salt, hydrate, or stereoisomer thereof in the manufacture of a preparation for treating a hepatitis C virus infection in a patient by administering to the patient in need thereof a therapeutically or prophylactically effective amount of said preparation, wherein the TLR7 ligand is selected from analogs and derivatives of guanosine, imidazoquinoline, adenine, and pyrimidine. AMENDED SHEET

   ® 135

   77. Use according to claim 76, wherein the TLR7 ligand is selected from PF HN oe NH; Rens HNN al a 1 HO ~) , oe] Lg R WS on HO OH Ia Ib Ie 0 Rg Q wr RR NH NS 1 HaN” NT pt el eel eS HO oH? HO OM , ) Id Ie If o} [A NZ NH Je , and oe] HO OH °° Ig Ih wherein: cach R' is H, or a substituted or unsubstituted alkyl, alkenyl, or alkynyl, which may be interrupted by one or more O, S, or IN heteroatoms, or a substituted or unsubstituted aryl oer heteroaryl; R*isH, OH, SH, halo, or a substituted or unsubstituted alkyl, alkenyl, or alkynyl, which may be interrupted by one or more OO, S, or N heteroatoms, or a substituted or unsubstituted -O-(alkyl), -O-(aryl), -O- (heteroaryl), -S-(alkyl), -S-(aryl), -S-(heteroaryl), aryl, or heteroaryl; R’ is H, OH, or SH, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, -O-(alkyl), -O-(aryl), -O-Cheteroaryl), -S-(alkyl), -S-(aryl), -S-(heteroaryl), -NH(alkyl), -NH(aryl), -NH(heteroaryl), -NH(R4)(alkyl), -NH(R*)(aryl), or -NH(R*)(heteroaryl), wherein R* is a substituted or unsubstituted alkyl; XisQorS; Y is H, halo, OH, OR*, SH, SR*, or a substituted or unsubstituted alkyl or aryl, Z is H, halo, OH, ORY, SH, or SR*; A MENDED SHEET

   ® 136 or a pharmaceutically acceptable salt, hydrate, metabolite or stereoisomer thereof or a pharmaceutically acceptable salt or hydrate of said stereoisomer.

   78. Use according to claim 76, wherein the TLR7 ligand is selected from Formula Ia, Ib, Ic, Id, Ie, If, Ig, and Ih, wherein R'is H or a substituted or unsubstituted alkyl, alkenyl, or alkynyl; R? is H, OH, halo, or a substituted ©r unsubstituted alkyl, alkenyl, or alkynyl, or —CH,-O-(alkyl); R? is H, OH, or SH, or a substituted or unsubstituted -O-(alkyl), -S-(alkyl), or -NH(alkyl); X is O or S; Y is H, halo, OH, OR", SH, or SR*; and Z is H, halo, OH, OR", SH, or SR*.

   79. Use according to claim 76, wherein the TL.R7 ligand is selected from ) 0] o 0] wT no ORE nS N HN" OS NTN HN N o HO OH, HO OH HO OH NH, NH, an N PN | S—oH NZ NH HCO Ny Na x 0 oT © NH, NH» N N NT IB N= [| D—ch0cH,ch N Ly ~ , and OH .

   80. Use according to claim 76, wherein the patient is human.

   81. Use according to claim 76, further comprising administering a pharmaceutically acceptable excipient, carrier, or vehicle. AMENDED SHEET

   ® 137

   82. Use according to claim 76, further comprising administering an additional therapeutic agent.

   83. Use according to claim 82, wherein the additional therapeutic agent is an antiviral agent.

   84. Use according to claim 76, wherein the therapeutically or prophylactically effective dose 1s 0.001 to 100 mg/kg per day.

   85. Use according to claim 84, wherein the therapeutically or prophylactically effective dose is about 0.01 to 50 mg/kg; per day.

   86. Use according to claim 85, wherein the therapeutically or prophylactically effectives dose is about 0.1 to 20 mg/kg per day.

   87. Use according to claim 76, wherein the TLR7 ligand is administered parenterally.

   88. Use according to claim 76, wherein the TLR7 ligand is administered intravenously.

   89. Use according to claim 76, wherein the TLR7 ligand is administered orally.

   90. Use according to claim “76, wherein the TLR7 ligand is administered mucosally.

   91. Use of a TLR7 ligand prodrug or a pharmaceutically acceptable salt, hydrate, or stereoisomer thereof in the manufacture of a preparation for treating a hepatitis C virus infection in a patient by orally administering said prepatration to the patient in need thereof, wherein the oral administration of the TLR7 ligand prodrug achieves a therapeutically effective plasma concentration of the TLR7 ligand while reducing undesirable side effects associated with oral administration of TLR7 ligands.

   92. Use of amasked TLR7 ligand prodrug or a pharmaceutically acceptable salt, hydrate, or stereoisomer thereof in the manufacture of a medicament for treating a hepatitis C virus infection in a patient by orally administering said preparation to the patient in need thereof, wherein the oral administration of the masked TLR7 ligand prodrug achieves a AMENDED SHEET

   ® 138 therapeutically effective plasma concentration of the TLR7 ligand while reducing undesirable side effects associated with oral administration of TLR7 ligands.

   93. Use according to claim 92, wherein the masked TLR7 ligand prodrug is selected from analogs and derivatives of guanosine, imiidazoquinoline, adenine, and pyrimidine.

   94. Use according to claim 93, wherein the prodrug is an (a) amide, carbamate, or amidine moiety after conversion of a TLR7 lig and amine substituent, (b) ester, carbonate, carbamate, ether, imidate, acetal, aminal, or ketal moiety after conversion of a TLR7 ligand alcohol substituent, (c) acetal or ketal moiety a fter conversion of a TLR7 ligand keto substituent, (d) imidate moiety after conversior of a TLR7 ligand carbonyl of an amido substituent, (e) deoxygenated moiety after conwersion of a TLR7 ligand oxo substituent of pyrimidine or guanosine, or (f) amine.

   95. Use according to claim 94, wherein the prodrug is selected from an (a) carbamate moiety after conversion of a TLR7 ligand amime substituent, (b) ester, ether, or aminal moiety after conversion of a TLR7 ligand alcolol substituent, or (¢) deoxygenated moiety after conversion of a TLR7 ligand oxo substituesnt of pyrimidine or guanosine.

   96. Use according to claim 92, wherein the prodrug of the masked TLR7 ligand is selected from 0] Rr! RE Rr! on NPN R Ir =X RY. SN N HNN N “NH 0 5 o NV Ng Ro R*~o N RO OR® RO OR —K Rr? R Ila , Ib , IIc , oO Rr? O HN N HN N oY r Dan HS HN N , 5 0} 5 0 " ha R ~0 R ~0" NE N \ RO ORS 50 ORS A ba R%O \_g IId , Ile , If , AMENDED» SHEET

   RE or “ HS O° NZN RIN Ng cot] ’ RO ORS Hg and ITh , wherein: eachR'is H, or a substituted or unsubstituted alkyl, alkenyl, or alkynyl, which may be interrupted by one or more O, S, or N heteroatoms, or a substituted or unsubstituted aryl or heteroaryl; R? is H, OH, SH, halo, or a substituted or unsubstituted alkyl, alkenyl, or alkynyl, which may be interrupted by one or more O, S, or N heeteroatoms, or a substituted or unsubstituted -O-(alky!), -O-(aryl), -O-(heteroaryl), -S-(alkyl®, -S-(aryl), -S-(heteroaryl), aryl, or heteroaryl; R’ is H, OH, or SH, or a substituted or unsubstiztuted alkyl, alkenyl, alkynyl, aryl, heteroaryl, -O-(alkyl), -O-(aryl), -O-(heteroaryl)), -S-(alkyl), -S-(aryl), -S-(heteroaryl), - NH(alkyl), -NH(aryl), -NH(heteroaryl), -NH(R* (alkyl), -NH(R*)(aryl), or -NH(R*)(heteroaryl); R* is a substituted or unsubstituted alkyl; R’ is independently H, -C(O)(C,-1salkyl), or a racemic, L-, or D- amino acid group —C(O)CHNH,R; R®is H, OR, or N(R"); R’ is independently H or a substituted or unsubs tituted —C(O)(C,-salkyl) or —C(0)5(C;- 1salkyl); R? is H, —OH, -O-(alkyl), —OCO,(C- alkyl), —OC(O)(C,-isalkyl), or a racemic, L-, or D- amino acid group ~OC(O)CHNH,R'; R® is H, or a substituted or unsubstituted alkyl, CC(O)CH(C,.¢ alkyl)NH,, or —-C(O)CH(CH,- aryl)NHj; R'%is independently H, Cy. alkyl, C3.7 alkenyl, Ci.; alkynyl, (CR"*R")(C¢-C1p aryl), -(CR'R")(C;-C 0 cycloalkyl), -(CR'’R'*)(C4-C 1p heterocyclic), -(CR?R"?)» OH, -(CR"R'?)14CO,C}.15 alkyl, and -(CR"’R™);»(NCR')CO,C). 15 alkyl, and SO4(aryl), wherein t is an integer from 0 to 6 unless otherwise indicaated, and wherein the alkyl, alkenyl, alkynyl, aryl, cycloalkyl, and heterocyclic moieti_es of the foregoing groups are optionally substituted with substituents independently selected from halo, cyano, nitro, AMENDED SHEET

   @® 140 trifluoromethyl, trifluoromethoxy, C,-Cs alkyl, C>-Cg alkenyl, C>-Cg alkynyl, hydroxy, Ci- Cs alkoxy, -NH,, -NH-alkyl, -N(alkyl), -NH-aryl, -N( alkyl)(aryl), -N(aryl),, -NHCHO, -NHC(O)alkyl, -NHC(O)aryl, -N(alkyl)C(O)H, -N(alk_y)C(O)alkyl, -N(aryl)C(O)H, -N(aryl)C(O)alkyl, -NHCOQsalkyl, -N(alkyl)CO,alkyl, ~NHC(O)NH,, -N(alkyl)C(O)NHa, -NHC(O)NH-alkyl, -NHC(O) N(alkyl),, -N(alky)C(O )NH-alkyl, N(alkyl)C(O) N(alkyl)s, -NHSO,-alkyl, -N(alkyl)SO;-alkyl, -C(O)alkyl, -C(O)aryl, -OC(O)alkyl, -OC(O)aryl, -CO,- alkyl, -COs-aryl, -CO,H, -C(O)NH,, -C(O)NH-alkyl, - C(O)N(alkyl),, -C(O)NH-aryl, -C(O)N(aryl),, -C(O)N(alkyl)(aryl), -S(O)alkyl, -S(O)aryl, -SOsalkyl, -SO»aryl, -SO,NH; - SO;NH-alkyl, and -SO,;N(alkyl),; R' is independently H, C, alkyl, C5-Cyo cycloalkyl, o=r together with nitrogen forms a 5- or 6-membered heterocyclic ring; : R'? and R" are independently H, Cg alkyl, Cy.¢ alkenw|1, or C,.¢ alkynyl; RM isH, Cie alkyl, or -CH,-aryl; XisOQorS; Y is H, halo, OH, OR, SH, SR*, NH», NHR*, N(R"), Or a substituted or unsubstituted alkyl or aryl; and Z is H, halo, OH, OR", SH, or SRY; or a pharmaceutically acceptable salt, hydrate, metabolste or stereoisomer thereof or a pharmaceutically acceptable salt or hydrate of said stereoisomer.

   97. Use according to claim 96, wherein R' iss H or a substituted or unsubstituted alkyl, alkenyl, or alkynyl; R? is H, OH, halo, or a substituted or unsubstituted alkyl, alkenyl, or alkynyl, or —CH,-O-(alkyl); R}is H, OH, or SH, or a substituted or unsubstituted -O- (alkyl), -S-(alkyl!), or -NH(alkyl); Ris independently H_, —C(O)(C,-zalkyl), or a racemic, L- , or D- amino acid group ~C(0)CHNH,R®, wherein R® i_s an unsubstituted alkyl; RéisH or OR! wherein R'Y is independently C,. alkyl, C3.7 alkemyl, Ca.7 alkynyl, -(CR"?R"*)(C¢-Co aryl), (CR'*R"?),(C4-C) heterocyclic), and -(CR'R")—(N(R'"*)CO,C,. 5 alkyl, wherein t is an integer from 0 to 4 unless otherwise indicated, and wherein the alkyl, alkenyl, aryl, and heterocyclic moieties of the foregoing groups are option_ally substituted with 1 to 3 substituents independently selected from halo, cyano, nitro, trifluoromethyl, trifluoromethoxy, C,-Cs alkyl, C,-C¢ alkenyl, C,-Cg alkynyl, hydroxy, C,-Cg alkoxy, -CO,- alkyl, -CO,-aryl, -OC(O)alkyl, and -OC(O)aryl, and whezrein R'? and R" are independently H, Ci. alkyl, or Co. alkenyl; and RMis H, —CHj3, or -CH,CH;; R’ is independently H or a substituted or unsubstituted —C(O)(C,-3alkyl) or —C(O)=(C,-salkyl); R%is H, —OH, -O- AMENDED SHEET

   ® 141 (alkyl), “OCO4(C;-zalkyl), or a racemic, L-, or D- &amino acid group ~OC(O)CHNHzR'; X is O or S; Y is H, halo, OH, OR?, SH, or SR*; and Z_ is H, halo, OH, OR", SH, or SR".

   98. Use according to claim 92, wherein the mask<ed TLR7 ligand prodrug is selected from ny CHasCH,0 GN NZ N =o yr =o nS N nS N HO HO* Ho oH , HO OH 0} No oz RR Su 0 0 “So” No Sa NZ N NZ N [=o gs =o r =o oS N Sy N oS N o} man AL © HO © HO © we om , Ho OH , HO OH NH, NH, Wn wo NOCH, CH; = | N Br x Br o} 0} " . 2 Ho Ao Ho gen Ay EN Pn YL x x fo) lo} O Br Oo 0 0 NEN NZ SN RSS RSS Ly AMENDED SH_EET

   NH, NH, NH; dS 9 S—ocH, [| D—ocHacn, ba \ 7 N ba \ OCH4 , OCH3 N OCH; Py 0) NH 0 NH; fo) PN. NZ | No M—ock;cH, eS Soca of | »>—0 Zz N_ OCH,CH “ N 2CH3 Su N oS N > N LC I. = Ly OCHj3 , OCHs , OH N NH» 0 NH, 0 NH; 0 NZ N oct NT N Noch, NZ N H—ocetss PNY a PN a SNE Sas J NT ON 0” NT TN 0” NT TN OCH; C C 4 kd ? NH, fe) NH, [0] ~ NZ N Mochi or Mo 0 N\ \ Yr >= ING va 0” NTN o” "NTN o oO c © OCH, i: OCH, > NH, 0 Z NH, 0 pi NF NN 0 NZ SN 0 D \ rr )—d BY Sa

   J . ® J CO ) OCH3 . OCH3 > NH, 0 > N H, Ie) NE = NZ N bas A\ A\ oY =e BN Wha : SO 2 TO = OCH; , OCH . lo} 0 CH3CH,O S S = S = S HN HN N N We Mme Te XL oN NT TN HN" NT ON HN TN N HN” TNT ON AcO AcO HO HO AG OAc , AG OAc HO oH HO OH AMENDED SHEET"

   LJ 143 0 No Ge o} 0 Io oP wr NZ SS NE Co 0 ny HN N EN N nS N 0 0 IN RR ~~ HO OH HO oH HO OH ol CH30 oP =o “TI =o 98 =o EN N EN N EN N 0 lo} 0 HO oH AO OAc HO OH , and 0 No OA L NZ SS YY =o HN" N N ~] AcO AO OAc o

   99. Use according to claim 92, wherein the oral administration of the masked TLR7 ligand prodrug achieves an in vivo effective plasma coracentration of the TLR7 ligand that is 10% to 500% of the effective in vivo exposure obtained upon oral administration of the TLR7 ligand alone.

   100. Use according to claim 92, wherein the oral administration of the masked TLR7 ligand prodrug achieves an in vivo effective plasma comcentration of the TLR7 ligand that is 50% to 200% of the effective in vivo exposure obtained upon oral administration of the TLR7 ligand alone. AMENDED SHEET

   ® 144

   101. Use according to claim 99, wherein the oral administration of the masked TLR7 ligand p rodrug achieves a therapeutically effective plasma concertration of the corresponding TLR7 ligand without causing gastrointestinal irritation.

   102. Use according to claim 92, wherein the oral administratiom of the masked TLR7 ligand prodrug reduces undesirable side effects in a patient relative to the side effects upon oral administration of the TLR7 ligand alone.

   103. UJse according to claim 92, wherein the oral administratiora of the masked TLR7 ligand prodrug reduces undesirable side effects by 50% in a patiemt relative to the side effects mpon oral administration of the TLR7 ligand alone.

   104. Use according to claim 92, wherein the side effect is gastreointestinal irritancy.

   105. Use according to claim 104, wherein the irritancy is hemo-rrhage.

   106. Use according to claim 104, wherein the irritancy is lesiorms.

   107. Use according to of claim 104, wherein the irritancy is em- esis.

   108. UJse according to claim 92, wherein the patient is human.

   109. Use according to claim 92, further comprising administerimg a pharmaceutically acceptab le excipient, carrier, or vehicle.

   110. Use according to claim 92, further comprising administerirg an additional therapeutic agent.

   111. UFse according to claim 110, wherein the additional therapesutic agent is an antiviral agent.

   112. Use according to claim 92, wherein the therapeutically or porophylactically effective dose is 0.001 to 100 mg/kg per day. AMENDED SHEET

   ® 145

   113. Use according to claim 112, wherein the therapeutically or prophylactically effective dose isabout 0.1 to 25 mg/kg per day.

   114. Use according to claim 113, wherein the therapeutically «or prophylactically effective dose as about 1 to 20 mg/kg per day.

   115. Use according to claim 92, wherein the masked TLR7 ligand prodrug is administered parenterally.

   116. Use according to claim 115, wherein the masked TLR 7 l&gand prodrug is administered intravenously.

   117. Use according to claim 92, wherein the masked TLR7 ligzand prodrug is admimistered orally.

   118. Use according to claim 92, wherein the masked TLR7 lig and prodrug is admin istered mucosally.

   119. Use of a masked TLR7 ligand prodrug in the manufacture of a preparation for treating diseases responsive to immuno therapy while reducing umdesirable side effects associated with immunologic agents, by orally administering said preparation to a patient in need o-f immuno therapy, wherein the masked TLR7 ligand prodrug achieves a therapesutically effective plasma concentration of a TLR7 ligand 1 n the patient.

   120. Use according to claim 119, wherein the masked TLR7 ligzand prodrug is selected from analogs and derivatives of guanosine, imidazoquinoline, adesnine, and pyrimidine.

   121. Use according to claim 119, wherein the prodrug is an (a) amide, carbamate, or amidin_ e moiety after conversion of a TLR7 ligand amine substituent, (b) ester, carbonate, carbamate, ether, imidate, acetal, aminal, or ketal moiety after coraversion of a TLR7 ligand alcohol substituent, (c) acetal or ketal moiety after conversion of &2 TLR7 ligand keto substituent, (d) imidate moiety after conversion of a TLR7 ligand carbonyl of an amido substituent, (¢) deoxygenated moiety after conversion of a TLR7 1 igand oxo substituent of pyrimicline or guanosine, or (f) amine. AMENDED SHEET

   @® 146

   122. Use accordi ng to claim 121, wherein the prodrug is selected from an (a) carbamate moiety after conversion of a TLR7 ligand amine substituent, (b) ester, eth er, or animal . moiety after conver-sion of a TLR7 ligand alcohol substituent, or (€) deox=ygenated moiety after conversion of a TLR7 ligand oxo substituent of pyrimidine or guano sine.

   123. Use accordimg to claim 119, wherein the masked TLR7 ligand proedrug is selected from 0] R! R® rR! Yd rT R | =x rR HN" NTN NSN N “NH NZ 1 Ro 0} Ro~o o) | Naa RO ORS RO ORS = Rr? Ila , IIb , Ile , oO Rg! 0) H 0 y wr HoN ~n HN A NTN , 5 0) (0) " hu R™-o0 Ro" NPN gS E> < ~ PN | FR’ RO OR® RO ©R® R®7N N= 11d , Ile , If , RE - 7 0} ®t HaN A NTN oy ] rR! ANA RO z SN PP RO OR® Mg and Th i wherein: each R! is H, or a subbstituted or unsubstituted alkyl, alkenyl, or alkynyl, which may be interrupted by one or- more O, S, or N heteroatoms, or a substituted or unsubstituted aryl or heteroaryl; R?is H, OH, SH, hale, or a substituted or unsubstituted alkyl, alkenyl, or al-kynyl, which may be interrupted bsy one or more O, S, or N heteroatoms, or a substituted or unsubstituted AMENDED SHEET

   ® 147 -O-(alkyl), -O-(azryl), -O-(heteroaryl), -S-(alkyl), -S-(aryl), -S-(heteroamyl), aryl, or heteroaryl; R? is H, OH, or SH, or a substituted or unsubstituted alkyl, alkenyl, alkzynyl, aryl, heteroaryl, -O-(alkyl), -O-(aryl), -O-(heteroaryl), -S-(alkyl), -S-(aryl), —S-(heteroaryl), - NH(alkyl!), -NH(aryl), -NH(heteroaryl), -NH(R*)(alkyl), -NH(R*)(aryl)-, or -NH(R*)(heteroar-yl); R* is a substitute] or unsubstituted alkyl; RSs independently H, —C(O)(C;-1galkyl), or a racemic, L-, or D- amine acid group —-C(O)CHNH,R?; R®is H, OR", or N(R"); R'is independently H or a substituted or unsubstituted —-C(O)(C,-galkys1) or —C(0)x(C;- isalkyl); R®is H, —OH, -O—(alkyl), -OCO(C,-1salkyl), -OC(O)(C;-isalkyl), or a racemic, L-, or D- amino acid group —-OC(O)CHNH.R'; R’ is H, or a substituted or unsubstituted alkyl, C(O)CH(C.6 alkyl)NH> , or -C(O)CH(CH- aryl)NHs; R'is independen€ly H, C, alkyl, Cs.7 alkenyl, Ca.7 alkynyl, -(CR"?R">(C¢-C 1 aryl), A(CR"RP)(C3-C, ¢ cycloalkyl), -(CR'?R'?)(C4-C\y heterocyclic), (CR' ZR") O11, ~(CRR"™)20C0,C 1.15 alkyl, and -(CRR"*).oN(R'*)CO,C,_15 alkyl, ancl SO, (aryl), wherein t is an integer from 0 to 6 unless otherwise indicated, and wherein the al kyl, alkenyl, alkynyl, aryl, cycleoalkyl, and heterocyclic moieties of the foregoing groups are optionally substituted with stabstituents independently selected from halo, cyano, n-tro, trifluoromethyl, tra fluoromethoxy, C;-C¢ alkyl, C,-C¢ alkenyl, C,-C¢ alk-ynyl, hydroxy, C;- Ce alkoxy, -NH,, - NH-alkyl, -N(alky!),, -NH-aryl, -N(alkyl)(ary!), -N(ar-yl),, -NHCHO, -NHC(O)alkyl, -N-HC(O)aryl, -N(alkyl)C(O)H, -N(alkyl)C(O)alkyl, -N( aryl)C(O)H, -N(aryD)C(O)alkyl, -NHCO»alkyl, -N(alky)COaalkyl, -NHC(O)NH,, -N (alky)C(O)NH,, -NHC(O)NH-alkyR, -NHC(O) N(alkyl),, -N(alkyl)C(O)NH-alkyl, N(alks/)C(O) N(alkyl),, -NHSO,-alkyl, -N(alkyl)SO,-alkyl, -C(O)alkyl, -C(O)aryl, -OC(O)alkyl,., -OC(O)aryl, -CO,- alkyl, -COs-aryl, -CCO,H, -C(O)NH,, -C(O)NH-alkyl, -C(O)N(alkyl);, -C(O)NH-aryl, -C(O)N(aryl),, -C(®O)N(alkyl)(aryl), -S(O)alkyl, -S(O)aryl, -SOzalkyl, -S-Osaryl, -SO;NH, - SO,NH-alkyl, and ~SO,N(alkyl),; R'! is independent] y H, C,_ alkyl, C3-C)o cycloalkyl, or together with ni€rogen forms a 5- or 6-membered heterocyclic ring; R'? and R" are independently H, C,. alkyl, Co. alkenyl, or Cy. alkynyl; Ris H, Ci alkyl, or -CHj-aryl,

   AMENDED SHEET

   @® 148 XisQorS; Y is H, halo, OH, OR*, SF, SR*, NH,, NHR*, N(R%),, or a substituted or unstubstituted alkyl or aryl; and Z is H, halo, OH, OR", SH, or SR; or a pharmaceutically acceptable salt, hydrate, metabolite or stereoisomer thereof or a pharmaceutically acceptable salt or hydrate of said stercoisomer.

   124. Use accord ing to claim 123, wherein R' is H or a substituted o=r unsubstituted alkyl, alkenyl, or alkynyl; R%is H, OH, halo, or a substituted or unsubstituted alkyl, alkenyl, or alkynyl, or ~CH,-O-(alkyl); R? is H, OH, or SH, or a substituted or unsubstituted -O- (alkyl), -S-(alkyl), or -NHqalky!); R’ is independently H, ~-C(OXC,-salkyl), or a racemic, L- , or D- amino acid group —C(O)CHNH-R®, wherein R® is an unsubstituted alk yl; R® is H or OR’, wherein R'* is independently C,.¢ alkyl, Cs.7 alkenyl, C3. alkynyl, (CIR"*R"?)(Ce-Cio aryl), (CR"R"*)(C4-C}o heterocyclic), and -(CR'ZR").oN(R')CO,C).15 alky~1, wherein t is an integer from 0 to 4 unless otherwise indicated, and wherein the alkyl, alkerayl, aryl, and heterocyclic moieties of th e foregoing groups are optionally substituted with 1 to 3 substituents independently selected from halo, cyano, nitro, trifluoromethyl, trifluoromethoxy, C;-Cs alkyl, C5-Cg alkenyl, C,-Cs alkynyl, hydroxy, C,-C¢ alkoxy, -CO»- alkyl, -CO;-aryl, -OC(O)alL kyl, and -OC(O)aryl, and wherein R'* and R"? are i_ndependently H, Cy alkyl, or C,.¢ alkenwl; and R'is H, —CHa;, or -CH,CHs; R'is independently H or a substituted or unsubstituted —-C(O)(C,-1salkyl) or —C(0),(C,-1salkyl); R® is H, —OH, -O- (alkyl), -OCO,(C,-1salkyl) , or a racemic, L-, or D- amino acid group ~OC(O)CCHNH:R'; X is O or S; Y is H, halo, OH , ORY, SH, or SR? and Z is H, halo, OH, OR*, SH, or SR*

   125. Use according to claim 199, wherein the masked TLR7 ligand prodrug is selected from g ; CH3CH,0 Fe Tee H>=N" "N N HN" TN N ~~ ~~] HO OH Ho oH , AMENDED SHEET

   C 149 (e] No (OP i o 0 Sot J =o Tr =o “I 0 NS N nS N on a (e] HO OH HO OH , HO OH NH» NH; on on Xx OCH,CH, N Br Br 0 2 NH 2 Ho Ag INP NNN A - EN YY ~ A § N 0 0 [e] Br Qo

   [0] (@] PEN NN NZ | A) N= | A) NH, NH, NH, OR RE To HC CH C H C , OCH; | , C , 1 NH; fo) NH, 0 I S—ocHcH, \ a8 Soca, “07 NH o \ oS N oS | a LR cD JO OCHa , OCH3 , ~~ , NH, > NH, Yo NH; 3 on OC4Hq 8 OCsH44 rt OCgH43 rr So AN N»—0 A phe AJ ~~ A CI GRY OCH, OCHs3 , OCH; R AMENDED SHEET

   ® 150 NH, o NH, 0 { GS MH—0C Hes nS M0 0] 0 AN =n ENA C N C ° NH; o { NH, o / NS Mo I M0 Io] 0 EN AN C s . = NH, o O NH, 0 ONS Mo SS 0 > 0] lo] EN AN EN " C 5 C > 0 [e] CH3CH,0 RS ORE OL Y= 0 lo) lo) —0 eS N nS N oS N nS N AcO AO HO HO AO OAc , aC onc , HO OH , HO oH, lo} No 0 o oo we NZ SNS NZ 5S [ =o “ =o “Tr =o HN N nS N nS NJ lo) HO oH HO oH HO OH, AMENDED SHEET

   0 i" A on NZ S N ’ S [ =o “TC =o r =o HN N EN N HN sy N HO HO HO HO oH, AG OAc He om , and 0 No a o NF SS fe =o HN ONT ON ~] AcO AO ‘OAc ,

   126. Use according to claim 119, wherein the disease is a viral infection. 12 7. Use according to claim 126, wherein the viral infection i s selected from an RNA or DMNA virus including adenovirus, cytomegalovirus, hepatitis A wirus (HAV), hegpadnaviruses including hepatitis B virus (HBV), flaviviruses Lncluding Yellow Fever virus, hepaciviruses including hepatitis C virus (HCV), herpes smmplex type 1 and 2, herpes zosster, human herpesvirus 6, human immunodeficiency virus (H_IV), human papilloma virus (HPV), influenza A virus, influenza B virus, measles, parainflue nza virus, pestivirus, poliovirus, poxvirus (including smallpox and monkeypox virus), rhinovirus, coronovirus, res piratory syncytial virus (RSV), multiple families of viruses th_at cause hemorrhagic fev=ers, including the Arenaviruses, the Bunyaviruses and Filoviruses, and a range of viral encephalitides caused by RNA and DNA viruses.

   128. Use according to claim 119, wherein the disease is a cancer.

   129. Use according to claim 119, wherein the oral administrataon of the masked TLR7 ligand prodrug achieves an in vivo effective plasma concentratiom of the TLR7 ligand that is 10% to 500% of the effective in vivo exposure obtained upon oraml administration of the TLIR?7 ligand alone. AMENDED SHEET

   @® 152

   130. Use according to claim 129, wherein the oral administration of the masked TLR7 Ligand prodrug achieves an in vivo effective plasma concermtration of the TLR7 ligand that is 50% to 200% of the effective in vivo exposure obtained up«on oral administration of the "T'LR7 ligand alone. L 31. Use according to claim 130, wherein the oral admimistration of the masked TLR7 ligand prodrug achieves a therapeutically effective plasma «concentration of the corresponding TLR7 ligand without causing gastrointestinal irritation. 1 32. Use according to claim 119, wherein the oral admimmistration of the masked TLR7 13 gand prodrug reduces undesirable side effects in a patient relative to the side effects upon o»ral administration of the TLR7 ligand alone. 1 33. Use according to claim 119, wherein the oral admin_istration of the masked TLR7 11 gand prodrug reduces undesirable side effects by 50% in am patient relative to the side effects upon oral administration of the TLR7 ligand alone.

   134. Use according to claim 119, wherein the side effect is gastrointestinal irritancy.

   135. Use according to claim 134, wherein the irritancy is hemorrhage.

   136. Use according to claim 134, wherein the irritancy is lesions.

   137. Use according to claim 134, wherein the irritancy is emesis.

   138. Use according to claim 119, wherein the patient is human.

   139. Use according to claim 119, further comprising admmnistering a pharmaceutically acceptable excipient, carrier, or vehicle. 14-0. Use according to claim 119, further comprising administering an additional therapeutic agent. AMENDED SHEET i

   141. Use according to claim 140), wherein the additional therapeutic agent is an anti viral agent.

   142. Use according to claim 119, wherein the therapeutically or prophylactically effective dose is 0.001 to 100 mg/kg per day.

   143. Use according to claim 142, wherein the therapeutically or prophylactically effective dose is about 0.01 to 50 mg/kg per day.

   144. Use according to claim 143, wherein the therapeutically or prophylactically effective dose is about 0.1 to 20 mg/kg per day.

   145. Use according to claim 119, wherein the masked TLR7 ligand prodrug is administered parenterally.

   146. Use according to claim 145 _ wherein the masked TLR7 ligand prodrug is administered intravenously.

   147. Use according to claim 119_ wherein the masked TLR7 ligand prodrug is ; administered orally.

   148. Use according to claim 119, wherein the masked TLR7 ligand prodrug is administered mucosally.

   149. A substance or composition for use in a method of treatment, substantially as described herein.

   150. Use ofa TLR7 ligand, a TLIR7 ligand prodrug, a masked TLR7 ligand prodrug, org pharmaceutically acceptable salt, hydrate, or stereoisomer thereof in the manufacture of™ apreparation, substantially as described herein. AMENDED SHEET