TLR7 agonists

Abstract

The present invention relates to TLR7 agonists of Formula I and their use in the treatment of diseases such as cancer and infectious diseases. JPEG2023503996000112.jpg5150

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to TLR7 agonists and prodrugs, pharmaceutical compositions containing them, and their use in therapeutic and prophylactic applications. The present invention provides methods of treating and preventing infectious diseases, immune disorders, and cancer using TLR7 agonists. [Background technology]

[0002] Background of the Invention The ultimate goal of cancer immunotherapy is the eradication of tumor cells by the immune system. Both the innate and adaptive arms of the immune system, primarily natural killer (NK) cells and T cells, respectively, can contribute to tumor cell eradication. Critical to the adaptive immune response against tumor cells is the activation of CD8+ cytotoxic T lymphocytes (CTLs), which can utilize their cytotoxic potential against tumor cells after recognizing tumor-associated antigens (TAAs). Activation of naive CD8+ cells occurs via antigen-presenting cells (APCs), with dendritic cells (DCs) considered to be the most specialized APCs. These cells capture and process TAAs and present epitopes complexed with major histocompatibility complex (MHC) molecules on their membranes. Maturation of APCs by danger signals is essential for epitope presentation in a stimulatory manner to T cells.

[0003] Peripheral T cell tolerance to TAAs prevents effective immune responses against tumors, despite the ability of TAA-specific T cells to eliminate tumor cells. Approaches to overcoming this T cell tolerance to TAAs can be divided into two groups: (a) active specific immunotherapy (also known as cancer vaccines) and (b) passive specific immunotherapy (adoptive transfer of antitumor T cells or monoclonal antibodies). The poor immunogenicity of tumor cells is also a potential challenge in cancer immunotherapy. This low immunogenicity is due to the fact that TAAs are largely self-antigens and also to the downregulation of human leukocyte antigens and costimulatory molecules on the tumor cell membrane. Furthermore, tumor cells actively inhibit the immune system by secreting immunosuppressive factors that interfere with DC and T cell function.

[0004] Toll-like receptors (TLRs) are a group of proteins that play an important role in the innate immune system. TLRs are a type of pattern recognition receptor (PRR) that recognize molecules commonly shared by pathogens but distinct from host molecules, collectively referred to as pathogen-associated molecular patterns (PAMPs). They are typically expressed on sentinel cells such as macrophages and dendritic cells and are single transmembrane, non-catalytic receptors that recognize structurally conserved molecules derived from microorganisms.

[0005] TLRs 3, 7, 8, and 9 form the intracellular TLR family and recognize bacterial or viral nucleic acids. The natural ligands for TLRs 7 and 8 are single-stranded RNAs rich in guanosine and / or uridine. TLRs 7 and 8 can also be activated by certain small molecule synthetic compounds. The imidazoquinoline derivatives imiquimod (R837) and resiquimod (R848) have been described as TLR7 ligands in mice (Hemmi et al., 2002, Nat. Immunol. 3: 196-200). Furthermore, the guanosine analog loxoribine has been identified as a TLR7 ligand (Heil et al., 2003, Eur. J. Immunol. 33: 2987-2997). Additional TLR7 and / or TLR8 ligands include CL097 (3M-001), 852A, and CL075.

[0006] Despite the structural similarity between TLR7 and TLR8, their activation has different effects on innate immune cells and subsequent cytokine production. TLR8 agonists have been reported to be much more effective than TLR7 agonists at inducing proinflammatory cytokines and chemokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-12, and macrophage inflammatory protein (MIP)-1α, in peripheral blood mononuclear cells (PBMCs). In contrast, TLR7 agonists have been reported to activate plasmacytoid dendritic cells and induce interferon (IFN)-α production.

[0007] Since the discovery of the efficacy of the TLR7 / 8 agonist imiquimod in protecting guinea pigs from herpes virus infection, imiquimod has also been shown to be effective against several transplantable mouse tumors. Clinical responses to topical treatment with imiquimod (Aldara® 5% cream) have been reported, proving effective against both primary skin tumors and skin metastases. In these reports, no TAA was added, and the immune-enhancing effects of imiquimod were sufficient to induce antitumor responses. Imiquimod treatment has also been reported to be associated with partial or complete reversal of the aberrant expression of certain genes in premalignant actinic keratoses, thereby demonstrating imiquimod's ability to prevent cancer development. Summary of the Invention [Problem to be solved by the invention]

[0008] Treatment of localized cancers with TLR7 agonists has demonstrated favorable anticancer effects, but oral or systemic administration of these agonists is challenging. In a Phase II clinical trial in patients infected with hepatitis C virus, R848 was administered orally and demonstrated therapeutic effects on plasma hepatitis virus titers, but with dose-limiting toxicity. In another Phase II study, the TLR7 agonist 852A was tested in patients with metastatic melanoma by three weekly intravenous doses. The study demonstrated long-term disease stabilization and increased serum IFNα and IP-10 in some patients, but dose-limiting toxicity in two patients. These studies demonstrate that systemic use of TLR7 agonists in patients can be challenging due to a narrow therapeutic window. [Means for solving the problem]

[0009] Summary of the Invention The present invention relates generally to compounds useful as TLR7 agonists, compositions thereof, methods for making them, and methods for using them.

[0010] In one embodiment, the present invention provides a compound of formula I: [ka] [During the ceremony, R 1 are independently -H, -OH, -OC(O)-R 8 or -F ; R 2 are independently -H, -OH, -OC(O)-R 8 or -F ; R 3 is -OH or -OC(O)-R 8 and ; R 4 -H, -OH, -OC(O)-R 8 or -(C1-C8) alkyl; where R 3 and R 4 may be in the form of a carbonyl oxygen (=O) ; R 5 -H, -OH, -OC(O)-R 8 , -(C1-C8)alkyl, -O-(C1-C8)alkyl, -NH2 or -NHR 8 and ; where R 4 and R 5 can form a 3- to 6-membered cycloalkyl ring ; R 6 is -H, -(C1-C8) alkyl, -C(H)=CH2, -C(H)=C(H)( C1-C8) alkyl), -C(H)= C((C 1 -C 8 )Alkyl) (C1-C8) alkyl), -C(H)=C=CH2, -C(H)=C= C((C 1 -C 8 )Alkyl) H, -CH2C≡CH, -OH or -O(C1-C8)alkyl ; R 7 is -H, -OH, -OCH3, -SH or -Cl ; R 8 are independently -(C1-C8) alkyl, aryl, -(CH2) n(aryl), heteroaryl or -(CH2) n (heteroaryl) ; n is an integer 1, 2, 3, 4, or 5 ; where R 4 or R 5 At least one of them is not -H ;and wherein each alkyl, cycloalkyl, aryl, and heteroaryl is independently CN, NO, halogen, (C-C) alkyl, (C-C) haloalkyl, (C 3 -C 15 )Cycloalkyl , aryl, heteroaryl, OH, alkenyl, alkynyl, O-(C1-C3) alkyl, OC(O)-R 9 , O-(alkylene)aryl, O-(alkylene)heteroaryl, C(O)R 9 , S(C1-C8) alkyl, S(O)(C1-C8) alkyl, SO2(C1-C8) alkyl, C(O)OR 9 , C(O)NR 9 R 9 , C(O)NR 9 SO2(C1-C8) alkyl, NR 9 R 9 , N.R. 9 (CO)OR 9 , NH(CO)R 9 , NH(SO2)(C1-C8) alkyl or NH(SO2)NR 9 R 9 and R 9 are independently —H, —OH, —(C1-C8)alkyl, cycloalkyl, heterocyclyl, or C(O)NR 9 R 9 or N's ​​R 9 R 9 Two R's 9 teeth Together with the nitrogen atom to form a heterocyclyl do.〕 or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof.

[0011] In other embodiments, the TLR7 agonist compounds of the present invention may be used alone or in conjunction with other additional therapeutic agents and methods for the treatment or prevention of cancer or an infection or infectious disease in a subject in need thereof.

[0012] In another embodiment, the present invention provides a pharmaceutical composition comprising: (i) a therapeutically effective amount of at least one compound of Formula I, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof; and (ii) in combination with a pharmaceutically acceptable carrier, diluent, or excipient. Pharmaceutical compositions comprising a pharmaceutically acceptable carrier, diluent, or excipient together with an additional therapeutic agent are also part of the present invention.

[0013] These and other aspects of the present invention will become readily apparent from the following detailed description. To this end, various references detailing certain background information, methods, compounds and / or compositions are set forth herein and are incorporated by reference in their entireties. DETAILED DESCRIPTION OF THE INVENTION

[0014] Detailed Description The present invention provides compounds as TLR7 agonists. The present invention includes TLR7 agonists that activate TLR7 without substantially activating TLR8.

[0015] definition So that the present invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere herein, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0016] In the following description, certain specific details are set forth to provide a thorough understanding of various embodiments of the present invention. However, those skilled in the art will understand that the present invention may be practiced without these details. Unless the context requires otherwise, throughout this specification and claims, the terms "comprises" and variations thereof, such as "including" and "comprising," should be interpreted in an open-ended, inclusive sense (i.e., "including, but not limited to").

[0017] The reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. The appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0018] As used herein, including the appended claims, the singular terms "a," "an," and "the" include the corresponding plural terms unless the context clearly dictates otherwise.

[0019] "Administration" and "treatment," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to the contact of an exogenous pharmaceutical, therapeutic, or diagnostic agent or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell includes contact of the agent with the cell and, when the fluid is in contact with the cell, contact of the agent with the fluid. "Administration" and "treatment" also refer to in vitro and ex vivo treatments, e.g., of cells with pharmaceutical agents, diagnostics, binding compounds, or other cells.

[0020] "Treatment" or "treating" refers to the administration, internally or externally, of a therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments of the present invention, to a subject or patient having or suspected of having one or more disease symptoms for which the agent has therapeutic activity. Generally, the therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, by inducing regression or preventing the progression of such symptoms to some clinically measurable extent. The amount of therapeutic agent that is effective in alleviating any particular disease symptom may vary depending on factors such as the disease state, the age and weight of the patient, and the ability of the drug to elicit a desired response in the subject. Whether a disease symptom has been alleviated can be assessed by any clinical measurement generally used by a physician or other skilled health care provider to assess the severity or progression of a condition.

[0021] "Amino" refers to an -NH2 substituent.

[0022] "Aminocarbonyl" refers to a -C(O)NH2 substituent.

[0023] "Carboxyl" refers to a -CO2H substituent.

[0024] "Carbonyl" refers to the group -C(O)- or -C(=O)-. Both notations are used interchangeably herein.

[0025] "Cyano" refers to a -C≡N substituent.

[0026] "Acetyl" refers to a -C(O)CH3 substituent.

[0027] "Hydroxy" or "hydroxyl" refers to an --OH substituent.

[0028] "Oxo" refers to the =O substituent.

[0029] "Thio" or "thiol" refers to an --SH substituent.

[0030] "Alkyl" refers to an alkyl group containing 1 to 12 carbon atoms (C1-C 12 "Ci-C alkyl" refers to a saturated, straight or branched hydrocarbon chain radical consisting of carbon and hydrogen atoms having 1 to 8 carbon atoms (Ci-C alkyl), or 1 to 6 carbon atoms (Ci-C alkyl), attached to the remainder of the molecule by a single bond. Examples of alkyl groups include methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, and the like. The moieties on which the alkyl group may be substituted are selected from the group consisting of hydroxyl, amino, alkylamino, arylamino, alkoxy, thioalkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, which may be unprotected or protected as needed, as known to those skilled in the art, for example, as taught in Greene, et al., "Protective Groups in Organic Synthesis," John Wiley and Sons, Second Edition, 1991, but are not limited to these.

[0031] "Lower alkyl" has the same meaning as alkyl as defined above, except having one to three carbon atoms (C1-C3 alkyl).

[0032] "Alkenyl" refers to an alkyl group having at least one double bond and 2 to 12 carbon atoms (C2-C 12 C2-C6 alkenyl), 2-8 carbon atoms (C2-C8 alkenyl), or 2-6 carbon atoms (C2-C6 alkenyl) connected to the rest of the molecule by a single bond, e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, etc.

[0033] "Alkynyl" refers to an alkyl group having at least one triple bond and 2 to 12 carbon atoms (C2-C 12 alkynyl), 2 to 10 carbon atoms (C2-C 10C2-C8 alkynyl), or 2-6 carbon atoms (C2-C6 alkynyl) and are connected to the rest of the molecule by a single bond, such as ethynyl, propynyl, butynyl, pentynyl, and hexynyl.

[0034] "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon (alkyl) chain, consisting solely of carbon and hydrogen, respectively, that connects the rest of the molecule to a radical group. Alkylene can have 1 to 12 carbon atoms and is, for example, methylene, ethylene, propylene, n-butylene, and the like. The alkylene chain is attached to the rest of the molecule through a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule are through one carbon or any two carbons within the chain. "Optionally substituted alkylene" refers to alkylene or substituted alkylene.

[0035] "Alkoxy" means a group of the formula -OR a (In the formula, R a is alkyl having the indicated number of carbon atoms as defined above. Examples of alkoxy groups include, but are not limited to, -O-methyl (methoxy), -O-ethyl (ethoxy), -O-propyl (propoxy), -O-isopropyl (isopropoxy), and the like.

[0036] "Aryl" refers to a hydrocarbon ring system radical containing hydrogen, 6 to 18 carbon atoms, and at least one aromatic ring. Examples of aryl are hydrocarbon ring system radicals containing hydrogen, 6 to 9 carbon atoms, and at least one aromatic ring; hydrocarbon ring system radicals containing hydrogen, 9 to 12 carbon atoms, and at least one aromatic ring; hydrocarbon ring system radicals containing hydrogen, 12 to 15 carbon atoms, and at least one aromatic ring; or hydrocarbon ring system radicals containing hydrogen, 15 to 18 carbon atoms, and at least one aromatic ring. For purposes of this invention, aryl groups can be monocyclic, bicyclic, tricyclic, or tetracyclic ring systems, which may include fused or bridged ring systems. Aryl groups include, but are not limited to, aryl groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene and triphenylene. "Optionally substituted aryl" refers to an aryl group or a substituted aryl group. The aryl group may be substituted with one or more moieties selected from the group consisting of hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate or phosphonate, which may be unprotected or protected as needed, as known to those skilled in the art, for example, as taught in Greene, et al., "Protective Groups in Organic Synthesis", John Wiley and Sons, Second Edition, 199.

[0037] "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical, consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, 3 to 9 carbon atoms, 3 to 8 carbon atoms, 3 to 7 carbon atoms, 3 to 6 carbon atoms, or 3 to 5 carbon atoms. Cycloalkyl rings can be saturated or unsaturated and are attached to the remainder of the molecule by a single bond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like.

[0038] "Fused" refers to any ring structure described herein that is fused to an existing ring structure in the compounds of this invention. When the fused ring is a heterocyclyl ring or a heteroaryl ring, any carbon atom of the existing ring structure that becomes part of the fused heterocyclyl ring or fused heteroaryl ring can be replaced with a nitrogen atom.

[0039] "Halo" or "halogen" refers to bromo, chloro, fluoro, or iodo.

[0040] "Haloalkyl" refers to an alkyl group, as defined herein and having the indicated number of carbon atoms, in which one or more of the alkyl group's hydrogen atoms is replaced with a halogen (halo radical), as defined above. The halogen atoms may be the same or different. Examples of haloalkyl include trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and the like.

[0041] "Heterocyclyl," "heterocycle," or "heterocyclic ring" refers to a stable 3- to 18-membered saturated or unsaturated radical consisting of 2 to 12 carbon atoms and 1 to 6 heteroatoms, e.g., 1 to 5 heteroatoms, 1 to 4 heteroatoms, 1 to 3 heteroatoms, or 1 to 2 heteroatoms, selected from the group consisting of nitrogen, oxygen, and sulfur. Examples of heterocycles include, but are not limited to, a stable 3- to 15-membered saturated or unsaturated radical, a stable 3- to 12-membered saturated or unsaturated radical, a stable 3- to 9-membered saturated or unsaturated radical, a stable 8-membered saturated or unsaturated radical, a stable 7-membered saturated or unsaturated radical, a stable 6-membered saturated or unsaturated radical, or a stable 5-membered saturated or unsaturated radical.

[0042] Unless stated otherwise specifically in the specification, a heterocyclyl group can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms of the heterocyclyl group can be optionally oxidized; the nitrogen atom can be optionally quaternized; and the heterocyclyl group can be partially saturated or fully saturated. Examples of non-aromatic heterocyclyl groups include, but are not limited to, azetidinyl, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, thietanyl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Heterocyclyl includes heteroaryl as defined herein, and examples of aromatic heterocyclyl are listed below in the definition of heteroaryl.

[0043] "Heteroaryl" refers to a 5- to 14-membered ring system radical containing hydrogen, 1 to 13 carbon atoms, 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, and at least one aromatic ring. For purposes of this invention, a heteroaryl group can be a stable 5- to 12-membered ring, a stable 5- to 10-membered ring, a stable 5- to 9-membered ring, a stable 5- to 8-membered ring, a stable 5- to 7-membered ring, or a stable 6-membered ring containing at least one heteroatom, at least two heteroatoms, at least three heteroatoms, at least four heteroatoms, at least five heteroatoms, or at least six heteroatoms. Heteroaryl can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can include fused or bridged ring systems; and the nitrogen, two carbon, or sulfur atoms of the heteroaryl group can be optionally oxidized; and the nitrogen atom can be optionally quaternized. The heteroatom can be a member of an aromatic or non-aromatic ring, so long as at least one ring of the heteroaryl is aromatic.Examples include azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indophenyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridin ... including, but not limited to, dolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl).

[0044] The compounds of the present invention may exhibit the phenomenon of tautomerism. While Formula I cannot specifically describe all possible tautomeric forms, it is understood that Formula I is intended to represent all tautomeric forms of the compounds described and is not limited to only the specific compounds depicted by the formula drawings. For example, R 7 When is OH, it is understood that for Formula I, whether the substituent is shown in the enol or keto form, as shown below, represents the same compound. Compound 1: [ka] Compounds such as compounds 1A and 1B: [ka] It will be apparent to one skilled in the art that tautomeric forms such as , , and .

[0045] It will also be apparent to those skilled in the art that compounds having trifluoromethyl ketones may also exist in the form of their corresponding hydrates. [ka]

[0046] An "isolated nucleic acid molecule" or "isolated polynucleotide" means DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof, where the isolated polynucleotide is not associated with all or a portion of polynucleotides found in nature or is associated with polynucleotides with which it is not naturally associated. For the purposes of this disclosure, a "nucleic acid molecule comprising" a particular nucleotide sequence should be interpreted as not including intact chromosomes. An isolated nucleic acid molecule "comprising" a particular nucleic acid sequence may, in addition to the particular sequence, include coding sequences for up to 10 or up to 20 or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the described nucleic acid sequence and / or may include vector sequences.

[0047] The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

[0048] A nucleic acid or polynucleotide is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expected to be expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. Generally, but not always, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[0049] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the terms "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that not all progeny will have precisely the same DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. When different designations are intended, they will be clear from the context.

[0050] As used herein, "germline sequence" refers to the sequence of an unrearranged immunoglobulin DNA sequence. Any suitable source of unrearranged immunoglobulin sequences can be used. Human germline sequences can be obtained, for example, from the JOINSOLVER germline database on the National Institutes of Health website. Mouse germline sequences can be obtained, for example, as described in Giudicelli et al. (2005) Nucleic Acids Res. 33: D256-D261.

[0051] The term "together" indicates that the components administered in the methods of the present invention can be formulated into a single composition for simultaneous delivery or formulated into two or more compositions (e.g., a kit). Each component can be administered to a subject at a different time than the other components are administered; for example, each administration can be administered at several intervals over a period of time rather than simultaneously (e.g., separately or sequentially). Furthermore, the separate components can be administered to a subject by the same or different routes.

[0052] As used herein, the term "effective amount" refers to an amount of a TLR7 agonist compound of the present invention that, when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to cause a measurable improvement in one or more symptoms of a disease, e.g., cancer, or cancer progression. An effective dose also refers to the amount of the compound or pharmaceutical composition thereof sufficient to cause at least partial improvement of a symptom, e.g., tumor shrinkage or disappearance, lack of tumor growth, or prolonged survival. When applied to an individual active ingredient administered alone, an effective dose refers to the ingredient alone. When applied to a combination, an effective dose refers to the combined amount of the active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent produces an improvement in a diagnostic measure or parameter by at least 10%, usually at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%. An effective amount also produces an improvement in a subjective measure when a subjective measure is used to assess disease severity.

[0053] A "subject" is a mammal, such as a human, dog, cat, horse, cow, mouse, rat, monkey (e.g., cynomolgus monkey, e.g., Macaca fascicularis), or rabbit. In a preferred embodiment of the invention, the subject is a human subject.

[0054] TLR7 agonists In certain embodiments of formula I, R 1 is -H or -OH.

[0055] In some embodiments, R 1 is -H.

[0056] In some embodiments, R 2 is -H or -OH.

[0057] In some embodiments, R 2 is -H.

[0058] In some embodiments, R 2 is -F.

[0059] In some embodiments, R 3 is -OH or -OC(O)-CH3.

[0060] In some embodiments, R 3 is -OH.

[0061] In some embodiments, R 4 is —H or —(C1-C8)alkyl.

[0062] In some embodiments, R 4 is -H or -CH2CH3.

[0063] In some embodiments, R 5 is —H or —(C1-C8)alkyl.

[0064] In some embodiments, R 5 is -H or -CH2CH3.

[0065] In some embodiments, R 6 is -CH2C≡CH, CH2CH2CH3 or CH2CH2CH2CH3.

[0066] In some embodiments, R 6 is -CH2C≡CH.

[0067] In some embodiments, R 7is -H or -OH.

[0068] In some embodiments, R 7 is -OH.

[0069] In some embodiments, R 8 is -(C1-C8) alkyl.

[0070] In some embodiments, R 8 is -CH3.

[0071] General implementation method Compounds of formula I can be prepared by the following general methods.

[0072] In one method (Scheme 1), the chlorine atom of the symmetrical 4,6-dichloropyrimidine-2,5-diamine [55583-59-0] can be replaced with a benzylic amine, such as 4-methoxybenzylamine, to form II. Intermediate II can be exposed to phosgene or a phosgene equivalent, such as carbonyldiimidazole, to form the corresponding cyclic urea III. The chlorine atom of III can be replaced with an appropriate alcohol, preferably a benzylic alcohol, to form 2-amino-6,9-dibenzyl-7,9-dihydro-8H-purin-8-one IV. The N-7- of purin-8-one IV can be converted to R under basic conditions. 6 Alkylation with -Lv can give the N-7 alkyl compound V. 6is a C1-C8 alkyl group, and Lv is defined as a leaving group such as a halogen atom, OSO2CH3 (mesylate), OSO2CF3 (triflate), or OSO2Ar (where Ar is 4-methylphenyl) (tosylate). After N-7 alkylation, O-6 and N-9 of V can be deprotected under appropriate conditions. For example, under acidic conditions such as trifluoroacetic acid in combination with trifluoromethanesulfonic acid, O-6 and N-9 of V are simultaneously removed to give 2-amino-7-alkyl-7,9-dihydro-1H-purine-6,8-dione VI. The primary 2-amino group can then be protected to form VII (where P is a protecting group such as acyl or carbamyl). The synthesis of several furanose sugar intermediates VIII is known in the art and can be obtained in several steps from the corresponding carboxaldehyde VIIIa or epoxide VIIIb. Generally, purine intermediates VI and VII can then be exposed to sugar derivative VIII under a variety of nucleoside-forming reaction conditions, followed by hydroxyl and amine deprotection, if necessary, to afford the 9-β-furano-purine nucleoside analogs of Formula I. For a comprehensive review of synthetic conditions for forming nucleosides, see Romeo, et al., Chem. Rev. 2010, 110, pp. 3337-3370. [ka]

[0073] In another method (Scheme 2), the preparation begins with intermediate III (above), where 6-chloropurine can be hydro-dehalogenated under acidic conditions with hydrogen and catalytic Pd or Pt metal or activated Zn to form IX. N-7 alkylation of intermediate IX can be carried out under basic conditions, R 6 -Level 1 can be achieved to obtain intermediate X. 6and Lv are defined above in the general method used in Scheme 1. Following N-7 alkylation, N-9 of X can be deprotected under appropriate conditions. For example, the N-9 4-methoxybenzyl group of X can be removed under acidic conditions, such as trifluoroacetic acid in combination with trifluoromethanesulfonic acid, to give 2-amino-7-alkyl-7,9-dihydro-8H-purin-8-one XI. The primary 2-amino group of XI can then be protected to form XII (where P is a protecting group such as acyl or carbamyl). Generally, purine intermediates XI and XII are then exposed to a sugar derivative VIII under a variety of nucleoside-forming reaction conditions, followed by hydroxyl and amine deprotection, if necessary, to give the 9-β-furano-purine nucleoside analogs of Formula I. [ka]

[0074] Alternatively, as shown in Scheme 3, 2-amino-9-benzylic-7,9-dihydro-8H-purin-8-one IX can be obtained by first hydro-dehalogenating chloro-pyrimidine II with hydrogen and catalytic Pd or Pt metal or activated Zn under acidic conditions to give N- 4 The imidazolone ring can then be formed by exposure of XIII to phosgene or a phosgene equivalent such as carbonyldiimidazole to give intermediate IX. [ka]

[0075] Using an alternative method (Scheme 4), the preparation of compounds of formula I can be carried out by the synthesis of guanosine nucleoside analogs XIV (wherein R 1 and R 2 can be H, OH, F, and R 3 is OH and R 4 and R 5The synthesis of modified sugar guanosine analogs can begin with the bromine S of XV (where S can be H or alkyl). The synthesis of modified sugar guanosine analogs can be achieved using the methods described in Zou, et al., Can. J. Chem., 1987, p. 1436 and Robins et al., JOC, 1996, p. 9207. C-8 of the guanine base can be brominated under the conditions described in Holmes, et al., JACS, 1964, p. 1242 and Sheu et al., JACS, 1995, p. 6439 to give the 8-bromo-guanosine derivative XV. The oxygen can be brominated with the bromine S of XV using the alkoxide of benzyl alcohol described in Holmes, et al., JACS, 1965, p. 1772 and Sheu et al., JACS, 1995, p. 6439. N Ar substitution at C-8 provides the 8-benzyloxo-guanosine derivative XVI. Prior to debenzylation of the C-8 oxygen, the nitrogen at the N-1 position must be protected to achieve selective N-7 alkylation. Broom et al., JOC, 1969, p. 1025, describes the amination of the N-1 of guanosine, essentially acting as a protecting group. Thus, intermediate XVI can be exposed to hydroxylamine-O-sulfonic acid under basic conditions to provide the N-1 amino-guanosine derivative XVII. The benzyl group can be removed from the C-8 oxygen under a variety of deetherification conditions, preferably by catalytic hydrogenation under palladium metal, to provide the cyclic urea intermediate XVIII. N-7 alkylation of intermediate VI can be achieved under basic conditions by R 4 -Lv to give the N-7 alkyl intermediate XVIII. 6 is a C1-C8 alkyl group, and Lv is defined as a leaving group such as a halogen atom, OSO2CH3 (mesylate), OSO2CF3 (triflate), or OSO2Ar (where Ar is 4-methylphenyl) (tosylate). The final step in the synthesis is the N-1 deamination of XVIII, achieved by forming the diazonium salt with sodium nitrite under aqueous acidic conditions as described in U.S. Pat. No. 5,093,318, to give the desired compound of formula I (where R 7 is OH). [ka]

[0076] In another method using modified guanosines (Scheme 5), the amide oxygen of intermediate XVI (above) can be converted to the 6-chlorine purine intermediate XX, typically with phosphorus oxychloride. The benzyl ether at C-8 can then be selectively removed with catalytic hydrogenation or a boron trihalide such as BCl to afford intermediate XXI. N-7 alkylation of intermediate XXI can be carried out under basic conditions with R 6 -Lv to give N-7 alkyl compounds of formula I. 6 and Lv is defined above.

[0077] R 7 Compounds of formula I where is chlorine may further comprise R 7 can be converted to other compounds of formula I where R is H, OH, or OCH. 7 To obtain compounds of formula I where R is H, a hydro-dehalogenation reaction can be utilized. This transformation can be achieved under hydrogenation conditions, usually Pd or Pt or activated zinc in acetic acid. Substitution of the C-6 chlorine with a hydroxyl group can be achieved under aqueous basic or acidic conditions. Displacement of this chlorine with methoxide anion gives R 7 Compounds of formula I in which is OCH3 can be obtained. [ka]

[0078] In another method (Scheme 6), intermediate XVI (above) can be utilized and the amide oxygen converted to the 6-thiopurine intermediate XXII, typically with diphosphorus pentasulfide, Lawesson's reagent, or an equivalent. The thio group can be reduced with Raney nickel to afford intermediate XXIII. The benzyl ether at C-8 can then be selectively removed with catalytic hydrogenation or a boron trihalide such as BCl3 to afford compounds of formula I. R 6 N-7 alkylation with -Lv under basic conditions gives compounds of formula I.6 and Lv is defined above. [ka]

[0079] Therapeutic Use of TLR7 Agonists TLR7 activation of innate immunity is primarily mediated by plasmacytoid dendritic cells (pDCs). These cells are the major physiological producers of type I interferons—up to 1,000 times more than any other cell type. TLR7 activation is therefore a critical "gatekeeper" for the global induction of innate immune responses. TLR7 has significant advantages as a therapeutic target compared to other TLRs; for example, it can be activated with orally available small molecules. Unlike some other TLRs, systemic activation of TLR7 avoids stimulating excessive TNF production.

[0080] Administration of TLR7 agonists directly and indirectly engages in a variety of antitumor mechanisms, including the production of cytokines and chemokines that have direct antitumor activity; activation of natural killer (NK) cells, the primary effector cells of the innate immune system for the control of cancer, which can subsequently lyse tumor cells by both antibody-dependent (antibody-dependent cellular cytotoxicity, or ADCC) and -independent mechanisms; T cell activation and reversal of T cell exhaustion through antigen presentation by direct cell-cell interaction with pDCs and the subsequent production of cytokines and chemokines, all of which augment T cell-mediated attack against tumor cells; increased proliferation and maturation of normal B cells and their precursors, which can enhance the endogenous production of antibodies with antitumor activity; and direct activity against abnormal B cells via activation of TLR7 on these cells, which can induce apoptosis and supersensitivity to chemotherapy.

[0081] In certain embodiments, the TLR7 agonist compounds of the present invention may be used alone or in conjunction with other additional therapeutic agents and methods for the treatment or prevention of cancer or an infection or infectious disease in a subject in need thereof.

[0082] In certain embodiments, the TLR7 agonist compounds of the present invention may be used alone or in conjunction with a tumor vaccine.

[0083] In certain embodiments, the TLR7 agonist compounds of the present invention may be used alone or in combination with chemotherapeutic agents.

[0084] In certain embodiments, the TLR7 agonist compounds of the present invention may be used alone or in conjunction with radiation therapy.

[0085] In certain embodiments, the TLR7 agonist compounds of the present invention can be used alone or in conjunction with targeted therapies. Examples of targeted therapies include hormone therapy, signal transduction inhibitors (e.g., EGFR inhibitors, such as cetuximab (Erbitux) and erlotinib (Tarceva)); HER2 inhibitors (e.g., trastuzumab (Herceptin) and pertuzumab (Perjeta)); BCR-ABL inhibitors (e.g., imatinib (Gleevec) and dasatinib (Sprycel)); ALK inhibitors (e.g., crizotinib (Xalkori) and ceritinib (Zycadia)); BRAF inhibitors (e.g., vemurafenib (Zelboraf) and dabrafenib (Tafinlar)), gene expression modulators, apoptosis inducers (e.g., bortezomib (Velcade) and carfilzomib (Kyprolis)), angiogenesis inhibitors (e.g., bevacizumab (Avastin) and ramucirumab (Cyramza)), monoclonal antibodies conjugated to toxins (e.g., brentuximab These include vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla).

[0086] In certain embodiments, the TLR7 agonist compounds of the present invention may be used in combination with anti-cancer therapeutics or immunomodulatory drugs, such as immunomodulatory receptor inhibitors or antibodies or antigen-binding fragments thereof that specifically bind to the receptor.

[0087] In certain embodiments, the TLR7 agonist compounds of the present invention may be used in combination with an immune checkpoint inhibitor, an OX40 agonist, a 4-1BB agonist, an ICOS agonist, a GITR agonist, or an IL2-receptor agonist.

[0088] In certain embodiments, the TLR7 agonist compounds of the present invention may be used in combination with inhibitors or antagonists of PD-1, PD-L1, CTLA4, TIM3, LAG3, SIRPα, CD47, VISTA, BTLA, or TIGIT.

[0089] In certain embodiments, the TLR7 agonist compounds of the present invention may be used in combination with a therapeutic antibody whose anti-tumor activity is mediated, at least in part, by ADCC.

[0090] In certain embodiments, the TLR7 agonist compounds of the present invention may be used in combination with therapeutic antibodies with anti-tumor activity mediated at least in part via ADCC, including rituximab, trastuzumab, and alemtuzumab.

[0091] In certain embodiments of the invention, the TLR7 agonist compounds of the invention are in combination with anti-OX40 antibodies or other pathway agonists, including MOXR0916 and GSK3174998.

[0092] In certain embodiments of the invention, the TLR7 agonist compounds of the invention are in combination with anti-4-1BB antibodies, including urelumab, utomilumab, or other pathway agonists.

[0093] In certain embodiments of the invention, a TLR7 agonist compound of the invention is in combination with an anti-ICOS antibody or other pathway agonist.

[0094] In certain embodiments of the invention, the TLR7 agonist compounds of the invention are in combination with anti-GITR antibodies or other pathway agonists.

[0095] In certain embodiments of the invention, the TLR7 agonist compounds of the invention are in combination with IL-2-receptor or other pathway agonists.

[0096] In certain embodiments of the invention, the TLR7 agonist compound of the invention is in combination with an anti-PD1 or anti-PDL1 antibody, including nivolumab (Opdivo), pembrolizumab (Keytruda), atezolizumab (Tecentriq), durvalumab (Imfinzi), or avelumab (Bavencio).

[0097] In certain embodiments of the invention, the TLR7 agonist compounds of the invention are in combination with a CTLA-4 / CD80-CD86 antagonist, including ipilimumab (Yervoy).

[0098] In certain embodiments of the invention, the TLR7 agonist compounds of the invention are in combination with Tim-3 pathway antagonists, including MBG453 and TSR-022.

[0099] In certain embodiments of the invention, the TLR7 agonist compounds of the invention are in combination with LAG-3 pathway antagonists, including BMS-986016, GSK2831781 and IMP321.

[0100] In one embodiment of the invention, a TLR7 agonist compound of the invention is in combination with an anti-SIRPα antibody.

[0101] In one embodiment of the invention, a TLR7 agonist compound of the invention is in combination with an anti-CD47 antibody.

[0102] In certain embodiments of the invention, a TLR7 agonist compound of the invention is in combination with a Vista pathway antagonist.

[0103] In certain embodiments of the invention, a TLR7 agonist compound of the invention is in combination with a BTLA pathway antagonist.

[0104] In certain embodiments of the present invention, a TLR7 agonist compound of the present invention is in combination with a TIGIT pathway antagonist.

[0105] In other embodiments, the TLR7 agonist compounds of the present invention increase the activity of immune cells. The increase in immune cell activity can be detected using any method known in the art. In some embodiments, the increase in immune cell activity can be detected by measuring immune cell proliferation. For example, the increase in T cell activity can be detected by measuring T cell proliferation or signaling events such as tyrosine phosphorylation of immune receptors or downstream kinases that signal transcription factors. In other embodiments, the increase in immune cell activity can be detected by measuring CTL or NK cell cytotoxicity function against specific target cells or IFNγ cytokine responses associated with the stimulation of anti-tumor immunity. In yet other embodiments, the increase in immune cell activity can be detected by measuring ex vivo T cell activation in a sample from a subject.

[0106] Additional agents useful in eliciting cytolytic T cell responses may be used in combination with the TLR7 agonist compounds of the present invention, including, but not limited to, B7 costimulatory molecules, interleukin-2 (e.g., NKTR-214), interferon-γ, GM-CSF, CTLA-4 antagonists, OX-40 / OX-40 ligand, CD40 / CD40 ligand, sargramostim, levamisole, vaccinia virus, Bacillus Calmette-Guerin (BCG), liposomes, alum, Freund's complete or incomplete adjuvant, detoxified endotoxin, mineral oil, lipolecithin, pluronic polyols, polyanions, peptides, and surfactants such as oils or hydrocarbon emulsions.

[0107] In certain embodiments of the present invention, the TLR7 agonist compounds of the present invention are selected from the group consisting of casopitant (GlaxoSmithKline), netupitant (MGI-Helsinn) and other NK-1 receptor antagonists, palonosetron (sold as Aloxi by MGI Pharma), aprepitant (sold as Emend by Merck and Co.; Rahway, NJ), diphenhydramine (sold as Benadryl® by Pfizer; New York, NY), hydroxyzine (sold as Atarax® by Pfizer; New York, NY), metoclopramide (sold as Reglan® by A.H. Robins Co,; Richmond, VA), lorazepam (sold as Ativan® by Wyeth; Madison, NJ), alprazolam (sold as Xanax® by Pfizer; New York, NY), haloperidol (Ortho-McNeil; Raritan, NJ as Haldol®), droperidol (Inapsyn®), dronabinol (Sold as Marinol® by Solvay Pharmaceuticals, Inc.; Marietta, GA), dexamethasone (Sold as Decadron® by Merck and Co.; Rahway, NJ), methylprednisolone (Sold as Medrol® by Pfizer; New York, NY), prochlorperazine (Sold as Compazine® by GlaxoSmithKline; Research Triangle Park, NC), granisetron (Sold as Compazine® by Hoffmann-La Roche Inc.; sold as Kytril® by Nutley, NJ), ondansetron (sold as Zofran® by GlaxoSmithKline; Research Triangle Park, NC), dolasetron (sold as Anzemet® by Sanofi-Aventis; New York, NY), and tropisetron (sold as Navoban® by Novartis; East Hanover, NJ).

[0108] Other side effects of cancer treatment include red blood cell and white blood cell deficiencies. Thus, in some embodiments of the invention, the TLR7 agonist compound is combined with an agent that treats or prevents such deficiencies, including filgrastim, PEG-filgrastim, erythropoietin, epoetin alfa, and darbepoetin alfa.

[0109] In another embodiment, the present invention relates to a composition comprising one or more TLR7 agonist compounds of the present invention and a pharmaceutically acceptable carrier or diluent. Such a composition may further comprise one or more other therapeutically active ingredients, such as a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation molecule (SLAM protein), OX40, 4-1BB (CD137), ICOS (CD278), GITR, an IL2R beta (CD122), and / or an IL2R gamma agonist antibody or an antigen-binding fragment or soluble fusion thereof.

[0110] The present invention includes compositions comprising a TLR7 agonist compound of the present invention in combination with one or more antibodies that target the PD-1 / PD-L1 interaction or the CTLA-4 / CD80-CD86 interaction. Non-limiting examples of such antibodies include pembrolizumab, nivolumab, avelumab, REGN2810, MEDI-0680, PDR-001, SHR-1210, BGB-A317, PF-06801591, TSR-042, atezolizumab, durvalumab, BMS-936559, ipilimumab, and tremelimumab.

[0111] Compositions that induce a T cell immune response that preferentially stimulates a cytolytic T cell response over an antibody response are preferred, although those that stimulate both types of responses can be used as well.

[0112] In other embodiments, compositions comprising one or more TLR7 agonist compounds may further comprise one or more other therapeutically active ingredients that are immune checkpoint inhibitors, OX40 agonists, 4-1BB agonists, ICOS agonists, GITR agonists, or IL2-receptor agonists.

[0113] In other embodiments, compositions comprising one or more TLR7 agonist compounds may further comprise one or more other therapeutically active ingredients that are inhibitors or antagonists of PD-1, PD-L1, CTLA4, TIM3, LAG3, SIRPα, CD47, VISTA, BTLA, or TIGIT.

[0114] In other embodiments, compositions comprising one or more TLR7 agonist compounds may further comprise one or more other therapeutically active ingredients that are therapeutic antibodies with anti-tumor activity mediated at least in part by ADCC.

[0115] In other embodiments, compositions comprising one or more TLR7 agonist compounds may further comprise one or more other therapeutically active ingredients that are therapeutic antibodies with anti-tumor activity mediated at least in part by ADCC, including rituximab, trastuzumab, and alemtuzumab.

[0116] Further provided herein are methods for treating or preventing cancer or an infection or infectious disease in a subject, including a human subject, in need thereof, using the TLR7 agonist compounds disclosed herein. In some embodiments of the present invention, the subject has cancer or a precancerous condition. In other embodiments of the present invention, the subject has an infection or infectious disease.

[0117] In other embodiments, the present invention also relates to a method for treating or preventing cancer in a human subject, comprising administering to the subject an effective amount of one or more TLR7 agonist compounds of the present invention, optionally together with an additional therapeutic agent or method; and a method for treating an infection or infectious disease in a human subject, comprising administering to the subject an effective amount of one or more TLR7 agonist compounds of the present invention, optionally together with an additional therapeutic agent or method.

[0118] In yet other embodiments, the present invention provides compounds for the treatment or prevention of cancer; for treating infections or infectious diseases; for acting as vaccine adjuvants; or for increasing immune cell activation. Ruta To this end, the present invention relates to a method for increasing the activity of immune cells, comprising administering to a subject in need thereof an effective amount of one or more TLR7 agonist compounds of the present invention.

[0119] In some embodiments, the present invention relates to a method for treating a subject using the TLR7 agonist compound of the present invention, wherein the subject has cancer or a precancerous condition. In some embodiments, the cancer is, for example, osteosarcoma, rhabdomyosarcoma, neuroblastoma, kidney cancer, leukemia, renal transitional cell carcinoma, bladder cancer, Wilms' carcinoma, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, bone cancer, lung cancer (e.g., non-small cell lung cancer), gastric cancer, colorectal cancer, cervical cancer, synovial sarcoma, head and neck cancer, squamous cell carcinoma, multiple myeloma, renal cell carcinoma, retinoblastoma, hepatoblastoma, hepatocellular carcinoma, melanoma, renal cell carcinoma, leukemia, renal ...leukemia, renal cell carcinoma, leukemia, renal cell carcinoma, leukemia, renal cell carcinoma, leukemia, renal cell carcinoma, leukemia, renal cell carcinoma, leukemia, renal cell carcinoma, leukemia, renal cell carcinoma, leukemia, renal cell carcinoma, leukemia, renal cell carcinoma, leukemia, renal cell carcinoma, leukemia, renal cell carcinoma, leukemia, renal cell carcinoma, leuk The cancer is selected from the group consisting of rhabdoid tumor, Ewing's sarcoma, chondrosarcoma, brain cancer, glioblastoma, meningioma, pituitary adenoma, vestibular schwannoma, primitive neuroectodermal tumor, medulloblastoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, ependymoma, choroid plexus papilloma, polycythemia vera, thrombocythemia, idiopathic myelofibrosis, soft tissue sarcoma, thyroid cancer, endometrial cancer, carcinoid cancer, or liver cancer, breast cancer, and gastric cancer. In some embodiments of the present invention, the cancer is, for example, any of the various metastatic cancers described above.

[0120] In certain embodiments, the present invention provides a method of treating or preventing a viral infection in a subject using a TLR7 agonist of Formula I. In certain embodiments, the viral infection is an infection with a virus selected from the group consisting of human immunodeficiency virus (HIV), hepatitis virus (A, B, C, or D), herpesvirus (e.g., VZV, HSV-I, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus (e.g., SARS-CoV, MERS, and SARS-CoV-2), respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.

[0121] The present invention includes methods for treating and preventing viral infections in a subject, comprising administering an effective amount of a TLR7 agonist compound of Formula I and one or more additional treatment modalities (e.g., small molecule therapeutics, protein or peptide therapeutics, antibodies, serum from individuals who have recovered from a viral infection, and therapeutic or prophylactic vaccines).

[0122] In certain embodiments, the present invention provides methods of treating a subject using a TLR7 agonist compound of the present invention, wherein the subject has a bacterial infection. In some embodiments, the bacterial infection is caused by Chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Corynebacterium diphtheriae, Salmonella, bacilli, Vibrio cholerae, Clostridium tetani, Clostridium botulinum, or the like. The infection is caused by a bacterium selected from the group consisting of Bacillus botulinum, Bacillus anthricis, Yersinia pestis, Mycobacterium leprae, Mycobacterium lepromatosis, and Borrelia.

[0123] In certain embodiments, the present invention provides methods of treating a subject using a TLR7 agonist compound of the present invention, wherein the subject has a fungal infection. In some embodiments, the fungal infection is caused by a fungal species selected from the group consisting of Candida (such as albicans, krusei, glabrata, and tropicalis), Cryptococcus neoformans, Aspergillus (such as fumigatus and niger), Genus Mucorales (such as mucor, absidia, and rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, and Coccidioides immitis. The infection is caused by a fungus selected from the group consisting of Histoplasma immitis and Histoplasma capsulatum.

[0124] In certain embodiments, the present invention provides methods of treating a subject using a TLR7 agonist compound of the present invention, wherein the subject has a parasitic infection. In certain embodiments, the parasitic infection is an infection with a parasite selected from the group consisting of Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba, Giardia lambia, Cryptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Nippostrongylus brasiliensis.

[0125] The present invention includes a method of treating or preventing cancer in a subject, comprising administering to the subject an effective amount of a TLR7 agonist compound of the present invention and one or more antibodies targeting the PD-1 / PD-L1 or CTLA-4 / CD80-CD86 interaction. In one embodiment of the present invention, the TLR7 agonist compound of the present invention is in combination with an anti-PD1 or anti-PDL1 antibody.

[0126] The present invention includes a method of treating or preventing cancer in a subject, comprising administering an effective amount of a TLR7 agonist compound of the invention in combination with an immune checkpoint inhibitor, an OX40 agonist, a 4-1BB agonist, an ICOS agonist, a GITR agonist, or an IL2-receptor agonist.

[0127] The present invention includes a method of treating or preventing cancer in a subject, comprising administering an effective amount of a TLR7 agonist compound of the invention in combination with an inhibitor or antagonist of PD-1, PD-L1, CTLA4, TIM3, LAG3, SIRPα, CD47, VISTA, BTLA, or TIGIT.

[0128] The present invention includes a method of treating or preventing cancer in a subject, comprising administering an effective amount of a TLR7 agonist compound of the invention in combination with a therapeutic antibody having anti-tumor activity mediated at least in part through ADCC.

[0129] The present invention includes a method of treating or preventing cancer in a subject, comprising administering an effective amount of a TLR7 agonist compound of the invention in combination with a therapeutic antibody having anti-tumor activity mediated at least in part through ADCC, including rituximab, trastuzumab, and alemtuzumab.

[0130] The present invention includes a method of treating or preventing cancer in a subject, comprising administering an effective amount of a TLR7 agonist compound of the invention in combination with an anti-OX40 antibody or other pathway agonist, including MOXR0916 and GSK3174998.

[0131] The present invention includes a method of treating or preventing cancer in a subject, comprising administering an effective amount of a TLR7 agonist compound of the invention in combination with an anti-4-1BB antibody, including urelumab, utomilumab, or other pathway agonist.

[0132] The present invention includes a method of treating or preventing cancer in a subject, comprising administering an effective amount of a TLR7 agonist compound of the invention in combination with an anti-ICOS antibody or other pathway agonist.

[0133] The present invention includes a method of treating or preventing cancer in a subject, comprising administering an effective amount of a TLR7 agonist compound of the invention in combination with an anti-GITR antibody or other pathway agonist.

[0134] The present invention includes a method of treating or preventing cancer in a subject, comprising administering an effective amount of a TLR7 agonist compound of the present invention in combination with an IL-2-receptor or other pathway agonist.

[0135] The present invention includes methods of treating or preventing cancer in a subject, comprising administering an effective amount of a TLR7 agonist compound of the invention in combination with an anti-PD1 or anti-PDL1 antibody, including nivolumab (Opdivo), pembrolizumab (Keytruda), atezolizumab (Tecentriq), durvalumab (Imfinzi), or avelumab (Bavencio).

[0136] The present invention includes a method of treating or preventing cancer in a subject, comprising administering an effective amount of a TLR7 agonist compound of the invention in combination with a CTLA-4 / CD80-CD86 antagonist, including ipilimumab (Yervoy).

[0137] The present invention includes a method of treating or preventing cancer in a subject, comprising administering an effective amount of a TLR7 agonist compound of the present invention in combination with a Tim-3 pathway antagonist, including MBG453 and TSR-022.

[0138] The present invention includes a method of treating or preventing cancer in a subject, comprising administering an effective amount of a TLR7 agonist compound of the present invention in combination with a LAG-3 pathway antagonist, including BMS-986016, GSK2831781, and IMP321.

[0139] The present invention includes a method of treating or preventing cancer in a subject, comprising administering an effective amount of a TLR7 agonist compound of the present invention in combination with an anti-SIRPα antibody.

[0140] The present invention includes a method of treating or preventing cancer in a subject, comprising administering an effective amount of a TLR7 agonist compound of the present invention in combination with an anti-CD47 antibody.

[0141] The present invention includes a method of treating or preventing cancer in a subject, comprising administering an effective amount of a TLR7 agonist compound of the present invention in combination with a Vista pathway antagonist.

[0142] The present invention includes a method of treating or preventing cancer in a subject, comprising administering an effective amount of a TLR7 agonist compound of the invention in combination with a BTLA pathway antagonist.

[0143] The present invention includes a method of treating or preventing cancer in a subject, comprising administering an effective amount of a TLR7 agonist compound of the present invention in combination with a TIGIT pathway antagonist.

[0144] Pharmaceutical Compositions and Administration The present invention also provides pharmaceutical compositions comprising (i) a therapeutically effective amount of at least one compound of Formula I, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, in combination with (ii) a pharmaceutically acceptable carrier, diluent, or excipient. Pharmaceutical compositions comprising a pharmaceutically acceptable carrier, diluent, or excipient together with an additional therapeutic agent are also part of the present invention.

[0145] As used herein, the term "drug" refers to a chemical substance intended for use in the cure, treatment, or prevention of disease and subject to approval by the U.S. Food and Drug Administration (or its foreign equivalent) as a prescription or over-the-counter drug. Details of the techniques for formulation and administration of such compositions are provided in the Remington, The Science and Practice of Pharmacy 21 st Edition (Mack Publishing Co., Easton, PA) and Nielloud and Marti-Mestres, Pharmaceutical Emulsions and Suspensions: 2 ndThe TLR7 agonist compounds of the present invention are described in the "Pharmaceutical and Pharmaceutical Sciences" and "US Pharmacopeia: National Formulary," Mack Publishing Company, Easton, PA (1984). To prepare pharmaceutical compositions or sterile compositions of the TLR7 agonist compounds of the present invention, the compounds are mixed with pharmaceutically acceptable carriers or additives. For example, see Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984).

[0146] Formulations of therapeutic and diagnostic agents can be prepared, for example, by mixing them with acceptable carriers, additives, or stabilizers in the form of a lyophilized powder, a slurry, an aqueous solution, or a suspension (see, e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; see Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0147] Toxicity and therapeutic efficacy of the compounds or compositions of the invention administered alone or in combination with other therapeutic agents may be measured, for example, by the LD 50 (lethal dose to 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is known as the therapeutic index (LD ). 50 / ED 50 The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds is preferably administered with little or no toxicity and with an ED 50 The dosage may vary within this range depending upon the dosage form and route of administration used.

[0148] In further embodiments, the additional therapeutic agent administered to a subject along with the TLR7 agonist compound of the present invention is in accordance with the Physicians' Desk Reference 2003 (Thomson Healthcare; 57th edition (November 1, 2002)).

[0149] Methods of administration may vary. For purposes of the present invention, pharmaceutical compositions may be administered by a variety of means, including non-parenteral, parenteral, inhalation spray, topical, or rectal administration in formulations containing pharmaceutically acceptable carriers, adjuvants, and vehicles. "Non-parenteral administration" includes oral, buccal, sublingual, topical, transdermal, ocular, otic, nasal, rectal, cervical, pulmonary, mucosal, and vaginal routes. As used herein, the term parenteral includes, but is not limited to, subcutaneous, intravenous, intramuscular, intraarterial, intradermal, intrathecal, and epidural injections and various infusion techniques. As used herein, intraarterial and intravenous injections include administration via a catheter. Intratumoral (directly into the tumor mass) or peritumoral (around the tumor mass) administration of the compounds of the present invention is also contemplated. As used herein, the term oral includes, but is not limited to, oral ingestion or delivery by sublingual or buccal routes. Oral administration includes liquid drinks, energy bars, and pill formulations.

[0150] Pharmaceutical compositions may be in any form suitable for the intended method of administration. For oral use, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups, or elixirs may be prepared. Compositions for oral use may be prepared by any method known in the art for the preparation of pharmaceutical compositions, and such compositions may contain one or more additives, including sweeteners, flavoring agents, coloring agents, and preservatives, to provide an easy-to-swallow formulation. Tablets containing drug compounds mixed with non-toxic pharmaceutically acceptable additives suitable for tablet manufacture are acceptable. These additives may be, for example, inert diluents, such as calcium or sodium carbonate, lactose, calcium or sodium phosphate; granulating and disintegrating agents, such as maize starch or alginic acid; binders, such as starch, gelatin, or acacia; and lubricants, such as magnesium stearate, stearic acid, or talc. Tablets may be uncoated or may be coated by known techniques including enteric-coating, colonic-coating or microencapsulation to delay disintegration and absorption in the gastrointestinal tract and / or provide a sustained action over a longer period, for example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.

[0151] The present invention provides a container (e.g., a plastic or glass vial, e.g., with a cap or a chromatography column, a hollow needle, or a syringe cylinder) comprising one or more compounds of the present invention or pharmaceutical compositions thereof. The present invention also provides an injection device comprising one or more compounds of the present invention or pharmaceutical compositions thereof. An injection device is a device for introducing a substance into a patient's body via a parenteral route, e.g., intramuscularly, subcutaneously, or intravenously. For example, the injection device is a syringe (e.g., pre-filled with a pharmaceutical composition, such as an auto-injector), and includes, for example, a cylinder or barrel that holds the liquid to be injected (e.g., one or more compounds of the present invention or pharmaceutical compositions thereof), a needle that pierces the skin and / or a blood vessel to inject the liquid; and a plunger that forces the liquid through the cylinder and needle bore. In certain embodiments of the present invention, the injection device comprising one or more compounds of the present invention or pharmaceutical compositions thereof is an intravenous (IV) injection device. Such devices contain one or more compounds of the present invention or pharmaceutical compositions thereof in a cannula or trocar / needle, which may be connected to a bag or reservoir for holding a liquid (e.g., saline; or lactated Ringer's solution containing NaCl, sodium lactate, KCl, CaCl, and optionally glucose) to be introduced into the patient's body via the cannula or trocar / needle, which may be connected to tubing. In some embodiments of the present invention, one or more compounds of the present invention or pharmaceutical compositions thereof can be introduced into the device once the trocar and cannula are inserted into a subject and the trocar is removed from the inserted cannula. IV devices can be introduced, for example, into a peripheral vein (e.g., the hand or arm); into the superior vena cava, inferior vena cava, or right atrium (e.g., central IV); or into the subclavian artery, internal jugular, or femoral vein and advanced toward the heart until, for example, the superior vena cava or right atrium (e.g., central venous line). In some embodiments of the present invention, the injection device is an autoinjector; a jet injector, or an external infusion pump. Jet injectors use a high-pressure, narrow jet of liquid to penetrate the epidermis to introduce one or more compounds of the invention, or pharmaceutical compositions thereof, into the patient's body.An external infusion pump is a medical device that introduces pharmaceutical compositions into a patient's body in controlled amounts. External infusion pumps can be electrically or mechanically powered. Various pumps operate in different ways, for example, syringe pumps hold liquid in a syringe reservoir and a movable piston controls liquid delivery, while elastomeric pumps hold liquid in a stretchable balloon reservoir and pressure from the balloon's elastic walls drives liquid delivery. In peristaltic pumps, a series of rollers depress a length of flexible tubing, pushing the liquid forward. In multi-channel pumps, liquid can be delivered from multiple reservoirs at multiple rates.

[0152] The compounds and pharmaceutical compositions disclosed herein can be delivered by needleless hypodermic injection devices, such as those disclosed in U.S. Patents 6,620,135; 6,096,002; 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556. Such needleless devices containing pharmaceutical compositions are also part of the present invention. The pharmaceutical compositions disclosed herein can also be administered by infusion. A number of well-known implants and modules for administering pharmaceutical compositions include those disclosed in U.S. Patent 4,487,603, which discloses an implantable microinfusion pump for dosing at a controlled rate; U.S. Patent 4,447,233, which discloses a drug infusion pump that delivers drugs at precise infusion rates; U.S. Patent 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; and U.S. Patent 4,439,196, which discloses an osmotic drug delivery system with multi-chamber compartments. Many other such implants, delivery systems, and modules are known to those skilled in the art, and those incorporating the pharmaceutical compositions of the present invention are within the scope of the present invention.

[0153] Alternatively, the TLR7 agonist compounds of the present invention can be administered locally, rather than systemically, for example, by injecting the compound into a tumor.Furthermore, the TLR7 agonist compounds of the present invention can be administered in a targeted drug delivery system, for example, in liposomes coated with tissue-specific antibodies that target tumors characterized by immunopathology.The liposomes are directed to and selectively taken up by the corresponding tissue.Such methods and liposomes are part of the present invention.

[0154] When the disclosed compounds or salts thereof are named or represented by structure, it is understood that the compounds or salts, including solvates (particularly hydrates), may exist in crystalline form, amorphous form, or mixtures thereof. The compounds or salts thereof or solvates (particularly hydrates) may also exhibit polymorphism (i.e., the ability to occur in different crystalline forms). These various crystalline forms are generally known as "polymorphs." When named or represented by structure, the disclosed compounds or solvates (particularly hydrates) are also understood to include all polymorphs thereof. Polymorphs have the same chemical composition but differ in packing, geometric arrangement, and other descriptive properties of the crystalline solid state. Polymorphs may differ in physical properties such as density, shape, hardness, stability, and dissolution properties. Polymorphs generally have different melting points, IR spectra, and X-ray powder diffraction patterns, which can be used for identification. Those skilled in the art will recognize that different polymorphs can be produced, for example, by changing or adjusting the conditions used during crystallization or recrystallization of the compound.

[0155] For the solvates of the compounds of the present invention or their salts in crystalline form, those skilled in the art will recognize that solvent molecules are incorporated into the crystal lattice during crystallization to form pharmaceutically acceptable solvates.Solvates can include non-aqueous solvents such as ethanol, isopropanol, dimethyl sulfoxide, acetic acid, ethanolamine and ethyl acetate, or can include water as the solvent incorporated into the crystal lattice.Solvates in which water is the solvent incorporated into the crystal lattice are generally referred to as "hydrates".Hydrates include stoichiometric hydrates and compositions containing various amounts of water.The present invention includes all such solvates.

[0156] For potential use as pharmaceuticals, the salts of the compounds of the invention are preferably pharmaceutically acceptable. Suitable pharmaceutically acceptable salts are described by P. Heinrich Stahl and Camille G. Wermuth in Handbook of Pharmaceutical Salts: Properties, Selection, and Use, 2004. nd ed. (Wiley-VCH: 2011) and also Remington's Pharmaceutical Sciences, 18 th ed. (Mack Publishing, Easton PA: 1990) and also Remington: The Science and Practice of Pharmacy, 19 th ed. (Mack Publishing, Easton PA: 1995).

[0157] Representative pharmaceutically acceptable salts include, for example, alkali metal salts, alkaline earth salts, ammonium salts, water soluble and water insoluble salts, such as acetate, amsonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fiunarate, gluceptate, gluconate, glutamate, glycolyl arsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, Includes lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methyl bromide, methyl nitrate, methyl sulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate (1,1-methene-bis-2-hydroxy-3-naphthoate, eimbonate), pantothenate, phosphate / diphosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosaliculate, suramate, tannate, tartrate, teoclate, tosylate, triethiodide, trifluoroacetate, and valerate salts. A pharmaceutically acceptable salt may have more than one charged atom in its structure. In this case, the pharmaceutically acceptable salt may have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0158] Salts of the compounds of the invention may be prepared by any suitable method known in the art, including treatment of the free base with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or an organic acid such as acetic acid, trifluoroacetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, formic acid, alginic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidyl 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 such as benzoic acid or cinnamic acid, a sulfonic acid such as p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, and the like.

[0159] Pharmaceutically acceptable salts can be prepared with bases which provide pharmaceutically acceptable cations, such as alkali metal salts (especially sodium and potassium), alkaline earth metal salts (especially calcium and magnesium), aluminum and ammonium salts, zinc, and physiologically acceptable organic bases such as diethylamine, isopropylamine, oramine, benzathine, benethamine, tromethamine (2-amino-2-(hydroxymethyl)propane-1,3-diol), morpholine, epolamine, piperidin, salts formed from benzophenone, piperazine, picoline, dicyclohexylamine, N,N'-dibenzylethylenediamine, 2-hydroxyethylamine, tri-(2-hydroxyethyl)amine, chloroprocaine, choline, deanol, imidazole, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), procaine, dibenzylpiperidine, dehydroabietylamine, glucamine, collidine, quinine, quinolone, erbumine, and basic amino acids, such as lysine and arginine.

[0160] If a compound containing a basic amine or other basic functional group is isolated as a salt, the corresponding free base form of the compound will have a higher pK than the inorganic or organic base of the salt, preferably the free base form of the compound. aSimilarly, if a compound containing a phosphate diester, phosphorothioate diester, or other acidic functional group is isolated as a salt, the corresponding free acid form of the compound will have a lower pK than the inorganic or organic acid of the salt, preferably the free acid form of the compound. a The compounds may be prepared by any suitable method known in the art, including treatment with an inorganic or organic acid having the formula:

[0161] The effective amount of a compound described herein, or a pharmaceutically acceptable salt, pharmaceutically acceptable solvate, or pharmaceutically acceptable hydrate thereof, for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the route and dose of administration, and the severity of side effects.

[0162] A "pharmaceutically acceptable prodrug" is a compound that can be converted under physiological conditions or by solvolysis to a specified compound or a pharmaceutically acceptable salt of such compound before exerting its pharmacological effect. Generally, prodrugs are formulated for the purposes of improved chemical stability, improved patient acceptance and compliance, improved bioavailability, extended duration of action, improved organ selectivity, improved formulation (e.g., aqueous solubility), and / or reduced side effects (e.g., toxicity). Prodrugs are commonly referred to as Burger's Medicinal Chemistry and Drug Chemistry, 1, 172-178, 949-982 (1995). See also Bertolini et al., J. Med. Chem., 40, 2011-2016 (1997); Shan, et al., J. Pharm. Sci., 86 (7), 765-767; Bagshawe, Drug Dev. Res., 34, 220-230 (1995); Bodor, Advances in Drug Res., 13, 224-331 (1984); Bundgaard, Design of Prodrugs (Elsevier Press 1985); Larsen, Design and Application of Prodrugs, Drug Design and Development (Krogsgaard-Larsen et al., eds., Harwood Academic Publishers, 1991); Dear et al., J. Chromatogr. B, 748, 281-293 (2000); Spraul et al., J. Pharmaceutical & Biomedical Analysis, 10, 601-605 (1992); and Prox et al., Xenobiol., 3, 103-112 (1992).

[0163] Methods for co-administration with additional therapeutic agents are well known in the art (Hardman, et al. (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, NY; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila., PA; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., PA). Generally, co-administration or co-administration refers to treating a subject with two or more agents, and these agents can be administered simultaneously or at different times. For example, these agents can be delivered to a single subject as separate administrations, which can be essentially simultaneous or at different times, and can be by the same route or different routes of administration. The agents may be delivered in the same dosage (eg, in the same formulation) so that they are administered to a single subject at the same time and by the same route of administration.

[0164] Generally, each dose of a compound of the invention individually contains from about 10 mg to about 2000 mg, e.g., from about 10 mg to about 50 mg, from about 50 mg to about 100 mg, from about 100 mg to about 250 mg, from about 250 mg to about 500 mg, from about 500 mg to about 1000 mg, or from about 1000 mg to about 2000 mg.

[0165] General method All synthetic chemistry was performed in standard laboratory glassware unless otherwise noted in the examples. Commercially available reagents were used as received. Microwave reactions were performed in a Biotage Initiator using the instrument software to control heating time and pressure. Analytical LC / MS was performed on an Agilent 1290 infinity, Mass: 6150 SQD (ESI / APCI) or Agilent 1200 SERIES, Mass: 6130 SQD (ESI / APCI); variable wavelength detector and an Agilent 6130 single quadrupole mass spectrometer, alternating positive and negative ion scan using Chemistation software. Retention times were determined from extracted 220 nm UV chromatograms. HPLC was performed on a Waters 2695 system equipped with a variable wavelength detector using Empower software. Retention times were determined from extracted 210 nm and 300 nm UV chromatograms. 1 H NMR was performed at 400 MHz on a Bruker Avance 400 or 500 MHz on a Bruker Avance DRX-500 using Topspin software. For complex splitting patterns, apparent splittings were tabulated. Analytical thin-layer chromatography was performed on silica (Macherey-Nagel ALUGRAM Xtra SIL G, 0.2 mm, UV 254 Indicator) and visualized with UV light. Silica gel chromatography was performed manually or on a Grace automated chromatograph with gradient elution. Melting points were collected on a Büchi B-540 melting point apparatus. [Example]

[0166] Example 1 Compounds 1, 2 and 3

[0167] 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, Compound 1 [ka]

[0168] 2-Amino-9-((2R,3R,5S)-3-hydroxy-5-((R)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 2 [ka]

[0169] 2-Amino-9-((2R,3R,5S)-3-hydroxy-5-((R)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(propa-1,2-dien-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 3 [ka]

[0170] Compounds 1, 2 and 3 were prepared by the following multi-step method. [ka]

[0171] (3R,5S)-5-(1-acetoxypropyl)tetrahydrofuran-2,3-diyl diacetate; Preparation of intermediate (7S) [ka]

[0172] Process 1 :O-((3aR,5R,6S,6aR)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-6-yl)S-methylcarbonodithioate(1S) To a suspension of NaH (60% in mineral oil, 36 g, 0.961 mol) in THF (500 mL) was added dropwise a solution of commercially available (3aR,5S,6S,6aR)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-6-ol [582-52-5] (100 g, 0.384 mol) and imidazole (1.3 g, 1.92 mmol) in THF (500 mL) at 0 °C. The reaction mixture was stirred at 10 °C for 15 min. Carbon disulfide (121 mL, 1.92 mol) was added to this mixture, and the reaction mixture was stirred at RT for 1 h. Subsequently, iodomethane (118 mL, 1.92 mol) was added at 0 °C. Stirring was continued at RT for 2 h, after which the reaction mixture was quenched with saturated NH4Cl solution (1 L) and extracted with EtOAc (2 × 1.5 L). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (100–200 mesh, eluted with 5% EtOAc in petroleum ether) to afford 120 g (89%) of O-((3aR,5R,6S,6aR)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-6-yl)S-methylcarbonodithioate (1S) [1667-96-2] as a colorless oil. 1 H NMR (500MHz, CDCl3): δ 5.92-5.91 (m, 2H), 4.68-4.67 (m, 1H), 4.34-4.29 (m, 2H), 4.12-4.04 (m, 2H), 2.59 (s, 3H), 1.61 (s, 3H), 1.54 (s, 3H), 1.33-1.32 (m, 6H)

[0173] Process 2 (3aR,5S,6aR)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole(2S) A solution of O-((3aR,5R,6S,6aR)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-6-yl)S-methylcarbonodithioate (1S) (120 g, 342 mmol) in toluene (800 mL) was degassed with argon for 45 minutes. A solution of AIBN (39 g, 239 mmol) and nBu3SnH (150 mL, 411 mmol) in toluene (400 mL) was degassed with argon in a separate flask for 30 minutes. This solution was added dropwise to the toluene solution of (1S). The reaction mixture was stirred at 120 °C for 6 hours and then cooled to RT. The toluene was removed under reduced pressure, and the residue was partitioned between acetonitrile and hexane (1:1, 1000 mL). The acetonitrile layer was washed with hexane (3 × 500 mL) and concentrated to give a residue, which was purified by column silica gel chromatography (100–200 mesh, eluted with 10% EtOAc in petroleum ether) to give 70 g (83.7%) of (3aR,5S,6aR)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole (2S) [4613-62-1] as a colorless oil. 1 H NMR (400MHz, CDCl3): δ 5.82 (d, J = 3.6 Hz, 1H), 4.76-4.74 (m, 1H), 4.17-4.10 (m, 3H), 3.84-3.81(m, 1H), 2.21-2.16 (m, 1H), 1.80-1.73 (m, 1H), 1.54 (s, 3H), 1.51 (s, 3H), 1.36 (s, 3H), 1.32 (s, 3H)

[0174] Process 3 (R)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethane-1,2-diol (3S) A solution of (3aR,5S,6aR)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole (2S) (70 g, 286 mmol) in 60% AcOH in water (350 mL, 5 volumes) was stirred at RT for 16 h. The AcOH was removed under reduced pressure, and the residue was partitioned between acetonitrile and hexane (1:1, 500 mL). The acetonitrile layer was washed with hexane (3 × 300 mL), and the acetonitrile layer was concentrated. The residue was purified by column silica gel chromatography (100–200 mesh, eluted with 50% EtOAc in petroleum ether) to give 35 g (60%) of (R)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethane-1,2-diol (3S) [4005-46-3] as a colorless oil. 1 H NMR (500MHz, DMSO-d6): δ 5.81 (d, J = 3.5 Hz, 1H), 4.75 (t, J = 4 Hz, 1H), 4.25-4.21 (m, 1H), 3.94-3.91 (m, 1H), 3.74-3.71 (m, 1H), 3.62-3.58 (m, 1H), 2.08-2.00 (m, 1H), 1.88-1.82 (m, 1H), 1.54 (s, 3H), 1.32 (s, 3H)

[0175] Process 4 (3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole-5-carbaldehyde (4S) To an ice-cold solution of (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethane-1,2-diol (3S) (3.3 g, 16.17 mmol) in methanol (35 mL) was added sodium periodate (4.15 g, 19.411 mmol). The reaction mixture was stirred at room temperature for 16 h, and the resulting suspension was filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluted with 1:2 EtOAc in petroleum ether) to give 2.5 g of (3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole-5-carbaldehyde (4S) [4613-68-7] as a colorless oil, which was used immediately in the next step.

[0176] Process 5 : 1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-1-ol (5S) To a solution of (3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole-5-carbaldehyde (4S) (2.6 g (crude), 15.11 mmol) in THF (30 mL) was added ethylmagnesium bromide (1 M in THF, 15.1 mL, 15.11 mmol) at −20° C. under argon. After stirring at room temperature for 16 h, the reaction mixture was quenched with saturated NH4Cl solution and extracted with EtOAc (3 × 100 mL). The combined organic layers were concentrated under reduced pressure, and the resulting crude compound was purified by column silica gel chromatography (eluted with 1:4 EtOAc in petroleum ether) to give 2.1 g of 1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-1-ol (5S) (diastereomeric mixture) as a colorless oil: 1H NMR (400MHz, CDCl3): δ 5.80 (t, J = 3.2 Hz, 1H), 4.75 (t, J = 4.2 Hz, 1H), 4.23-4.13 (m, 1H), 3.86-3.83 (m, 1H), 3.41-3.38 (m, 1H), 2.07-2.00 (m, 2H), 1.84-1.75 (m, 1H), 1.59-1.47 (m, 4H), 1.32 (s, 3H), 1.01 (t, J = 7.4 Hz, 3H)

[0177] Process 6 : 1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propyl acetate (6S) To a solution of 1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-1-ol (5S) (2.1 g, 10.39 mmol) in CHCl (20 mL) was added EtN (2.9 mL, 20.79 mmol) and DMAP (253 mg, 2.07 mmol). The stirred solution was cooled to 0 °C for 10 min, during which time AcO (1.48 mL, 15.59 mmol) was added. The reaction mixture was allowed to warm to room temperature and stirred for 16 h. The reaction was quenched with aqueous NaHCO and extracted with CHCl (3 × 100 mL). The combined organic layers were dried over NaSO and concentrated under reduced pressure to give 2.4 g of 1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propyl acetate (6S) (diastereomeric mixture) as a colorless oil. The product was used directly in the next step.

[0178] Process 7 (3R,5S)-5-(1-acetoxypropyl)tetrahydrofuran-2,3-diyl diacetate (7S) To a solution of 1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propyl acetate (6S) (2.4 g, 9.836 mmol) in dichloromethane (30 mL) was added AcOH (5.62 mL, 98.36 mmol), AcO (4.68 mL, 49.18 mmol), and concentrated HSO (0.5 mL) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 3 h. The reaction mixture was quenched with aqueous NaHCO and extracted with dichloromethane (3 × 100 mL). The combined organic layers were dried over NaSO and concentrated under reduced pressure. The resulting residue was purified by flash chromatography (SiO, 0-15% EtOAc petroleum ether) to give 1.5 g of (3R,5S)-5-(1-acetoxypropyl)tetrahydrofuran-2,3-diyl diacetate (7S) (diastereomeric mixture) as a colorless oil: 1 H NMR (400MHz, CDCl3): δ 6.12 (m, 1H), 5.19 (m, 1H), 4.91-4.83 (m, 1H), 4.44-4.34 (m, 1H), 2.19-2.09 (m, 9H), 1.74-1.51 (m, 2H), 1.48 (s, 2H), 0.94 (t, J = 7.4 Hz, 3H)

[0179] Process 8 :6-chloro-N 4 -(4-Methoxybenzyl)pyrimidine-2,4,5-triamine (1) 4-Methoxybenzylamine (114.8 g, 83.728 mol) was added to a stirred mixture of 4,6-dichloropyrimidine-2,5-diamine [55583-59-0] (100 g, 55.81 mol) and TEA (169 mL, 167.45 mol) in ethanol (1.0 L) at 0 °C, and the resulting reaction mixture was stirred at reflux temperature for 18 h. The solvent was evaporated under reduced pressure, and the concentrated mass was poured into ice-cold water and stirred for 30 min. The precipitated solid was collected by filtration, washed with water, and dried under reduced pressure to give 6-chloro-N 4 -(4-Methoxybenzyl)pyrimidine-2,4,5-triamine (1) (100 g, 64%) was obtained as a brown solid. ES+, m / z 280.1 [M+H]+ ; C 12 H 14 ClNO; 1 H NMR (500MHz, DMSO-d6): δ 8.25 (d, J =8.5 Hz, 2H), 6.92 (t, J =6.0 Hz, 1H), 7.87 (d, J =8.5 Hz, 2H), 5.63 (s, 2H), 4.47 (d, J =5.5 Hz, 2H), 3.91 (s, 2H), 3.72 (s, 3H)

[0180] Process 9 :2-amino-6-chloro-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (2) 6-Chloro-N 4 A mixture of 1-(4-methoxybenzyl)pyrimidine-2,4,5-triamine (1) (50 g, 17.92 mol) and 1,1'-carbonyldiimidazole (100 g, 61.64 mol) in acetonitrile (500 mL) was stirred at reflux for 18 hours. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. Ice-cold water was added to the resulting residue, and the mixture was stirred at room temperature for 30 minutes. The precipitated solid was collected by filtration, washed with water, and dried to give 2-amino-6-chloro-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (2) (50 g, 91%) as a brown solid; C 13 H 12 ClN5O2; 1 H NMR (500MHz, DMSO-d6): δ 11.31 (s, 1H), 7.23 (d, J = 9.0 Hz, 2H), 6.88 (d, J = 5.0 Hz, 2H), 6.62 (s, 2H), 4.80 (s, 2H), 3.71 (s, 3H)

[0181] Step 10 :2-amino-6-(benzyloxy)-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (3) Sodium hydroxide (9.967 g, 262.29 mmol) was added to a suspension of 2-amino-6-chloro-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (2) (40 g, 131.14 mmol) in benzyl alcohol (45.0 mL). The resulting reaction mixture was stirred at 120° C. for 18 hours. The reaction mixture was quenched with ice water (200 mL), and diethyl ether (150 mL) was added and stirred for 15 minutes. The resulting precipitated solid was collected by filtration, washed with water, and dried to give 2-amino-6-(benzyloxy)-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (3) (30 g, 60%) as a brown solid. ES+, m / z 378.1 [M+H] + ; C 20 H 19 N5O3; 1 H NMR (400MHz, DMSO-d6): δ 10.81 (s, 1H), 7.48 (d, J = 7.2 Hz, 2H), 7.40-7.36 (t, J = 7.6 Hz, 2H), 7.34-7.31 (t, J = 5.2 Hz, 1H), 7.21 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.4 Hz, 2H), 6.27 (s, 2H), 5.41 (s, 2H), 4.78 (s, 2H), 3.71 (s, 3H)

[0182] Step 11 2-Amino-6-(benzyloxy)-9-(4-methoxybenzyl)-7-(prop-2-yn-1-yl)-7,9-dihydro-8H-purin-8-one (4) Propargyl bromide (7.1 mL, 63.66 mmol) was added to a suspension of 2-amino-6-(benzyloxy)-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (3) (20 g, 53.05 mmol) and KCO (10.98 g, 79.57 mmol) in DMF (100 mL) at 0 °C and stirred at room temperature for 18 h. The reaction mixture was quenched with ice water (200 mL), and diethyl ether (150 mL) was added and stirred for 15 min. The resulting precipitated solid was collected by filtration, washed with water, and dried to give 2-amino-6-(benzyloxy)-9-(4-methoxybenzyl)-7-(prop-2-yn-1-yl)-7,9-dihydro-8H-purin-8-one (4) (20 g, 91%) as a brown solid. ES+, m / z 416.1 [M+H] + ; C 23 H 21 N5O3; 1 H NMR (400MHz, DMSO-d6): δ 7.52 (d, J = 7.2 Hz, 2H), 7.38 (t, J = 7.6 Hz, 2H), 7.32 (t, J = 5.2 Hz, 1H), 7.22 (d, J = 8.4 Hz, 2H), 6.81 (d, J = 8.4 Hz, 2H), 6.27 (s, 2H), 5.44 (s, 2H), 4.82 (s, 2H), 4.56 (s, 2H), 3.71 (s, 3H), 3.32-3.26 (m, 1H)

[0183] Step 12 :2-amino-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione (5) Trifluoromethanesulfonic acid (27 g, 180.72 mmol) was added to a suspension of 2-amino-6-(benzyloxy)-9-(4-methoxybenzyl)-7-(prop-2-yn-1-yl)-7,9-dihydro-8H-purin-8-one (4) (25 g, 60.24 mmol) in trifluoroacetic acid (21 mL, 180.72 mmol) at 0 °C under argon, and the resulting reaction mixture was stirred at room temperature for 18 h under argon. The reaction mixture was quenched with ice-cold water, made basic with saturated NaHCO solution under vigorous stirring, and collected by filtration. The residual solid was dissolved in ethyl acetate, stirred for 30 min, collected by filtration, and dried to give 2-amino-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione (5) (12 g, 36%) as a brown solid. ES+, m / z 206.1 [M+H] + ; C8H7N5O2; 1 H NMR (400MHz, DMSO-d6): δ 11.32 (s, 1H), 6.74 (s, 2H), 4.53 (s, 2H), 3.14 (s, 1H)

[0184] Step 13 N-(6,8-dioxo-7-(prop-2-yn-1-yl)-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (6) Acetic anhydride (4.85 mL, 47.56 mmol) was added to a solution of 2-amino-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione (5) (6.5 g, 31.70 mmol) in DMF (65 mL) at room temperature under argon, and the resulting reaction mixture was stirred at room temperature for 18 hours under argon. The reaction mixture was cooled to 0° C. (a solid formed upon stirring) and stirred for 30 minutes. The product was collected by filtration, washed with ethanol, and dried under reduced pressure to give N-(6,8-dioxo-7-(prop-2-yn-1-yl)-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (6) (4 g, 51.2%) as a brown solid. ES+, m / z 248.1 [M+H] + ; C 10 H9N5O3; 1H NMR (400MHz, DMSO-d6): δ 11.83 (s, 2H), 4.61 (s, 2H), 3.23 (s, 1H), 2.16 (s, 3H)

[0185] Step 14 (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (7A) and (R)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (7B) To N-(6,8-dioxo-7-(prop-2-yn-1-yl)-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (6) (700 mg, 2.834 mmol) and (3R,5S)-5-(1-acetoxypropyl)tetrahydrofuran-2,3-diyl diacetate (7S) (979 mg, 3.40 mmol) dissolved in 1,2-dichloroethane (10 mL) was added bis(trimethylsilyl)acetamide (2.14 mL, 8.502 mmol). The reaction mixture was stirred at 80° C. for 30 minutes under argon and then cooled to room temperature. The 1,2-dichloroethane was removed under reduced pressure, and the residue was dissolved in MeCN (10 mL), followed by the addition of trimethylsilyl trifluoromethanesulfonate (0.08 mL, 0.437 mmol). The reaction mixture was heated to 80° C. and stirred for 16 h, cooled to room temperature, diluted with water, and extracted with EtOAc (3×100 mL). The combined organic layers were washed with water (100 mL), brine (100 mL), dried over Na2SO4, and concentrated. The crude compound was purified by reverse phase preparative HPLC (Waters Preparative HPLC (Empower-3 software) (Column: X-SELECT-C18 (250*19), 5μ Mobile phase: 10 mM ammonium bicarbonate in HO:MeCN Gradient: (T%B): -0 / 25, 8 / 45, 12 / 45, 12.1 / 98, 14 / 98, 14.1 / 25, 17 / 25; Flow rate: 15 ml / min; Diluent: MeCN+HO+THF+MeOH) to give (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (7A) (90 mg) (ES+, m / z 476.2 [M+H] + ; C 21 H 25 N5O8) and (R)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (7B) (90 mg) (ES+, m / z 476.2 [M+H] + ; C21 H 25 N5O8) were both obtained as off-white solids.

[0186] Step 15 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 1 To a solution of (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (7A) (90 mg, 0.189 mmol) in methanol (10 mL) was added KCO (104 mg, 0.757 mmol) at 0° C. The reaction mixture was stirred at room temperature for 16 hours and then concentrated under reduced pressure. The resulting residue was purified by GRACE flash chromatography (reverse phase using 0.01% formic acid in acetonitrile as eluent) to afford 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 1 (25 mg, 21%) as a white solid. - , m / z 348.1 [MH]; C 15 H 19 N5O5, 1H NMR (500MHz, DMSO-d6): δ 11.38 (brs, 1H), 6.66 (s, 2H), 5.51 (d, J = 3.2 Hz, 1H), 5.38 (d, J = 4.4 Hz, 1H), 4.76-4.72 (m, 1H), 4.68-4.66 (m, 1H), 4.59 (m, 2H), 4.02-3.98 (m, 1H), 3.27-3.25 (m, 1H), 3.22-3.20 (m, 1H), 2.40-2.33 (m, 1H), 1.80-1.75 (m, 1H), 1.40-1.38 (m, 1H), 1.31-1.25 (m, 1H), 0.95 (t, J = 8.0 Hz, 3H)

[0187] Process 16 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((R)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 2 and 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((R)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(propa-1,2-dien-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 3 To a solution of (R)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (7B) (90 mg, 0.189 mmol) in methanol (10 mL) was added KCO (104 mg, 0.757 mmol) at 0° C. The reaction mixture was stirred at room temperature for 16 h and then concentrated under reduced pressure. The resulting residue was purified by GRACE flash chromatography (reverse phase using 0.01% formic acid in acetonitrile as eluent) to give 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((R)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 2 (15 mg) as a white solid. - , m / z 348.1 [MH]; C 15 H 19 N5O5, 1 H NMR (500MHz, DMSO-d6): δ 10.99 (brs, 1H), 6.54 (s, 2H), 5.49 (d, J = 3.6 Hz, 1H), 5.37 (d, J = 4.8 Hz, 1H), 4.82-4.78 (m, 1H), 4.63-4.58 (m, 3H), 3.96-3.91(m, 1H), 3.41-3.36 (m, 1H), 3.23-3.22 (m, 1H), 2.45-2.39 (m, 1H), 1.87-1.81 (m, 1H), 1.45-1.41 (m, 1H), 1.25-1.20 (m, 1H), 0.88 (t, J = 8.0 Hz, 3H). The isomerized propargylic by-product, 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((R)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(propa-1,2-dien-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 3 (10 mg), was also isolated as an off-white solid. ES + , m / z 350.1 [M+H]; C 15 H 19 N5O5,1 H NMR (500MHz, DMSO-d6): δ 11.79 (brs, 1H), 7.31 (t, J = 6.5 Hz, 1H), 6.58 (s, 1H), 5.48 (d, J = 4 Hz, 1H), 5.43 (d, J = 6.5 Hz, 2H), 5.34 (d, J = 5 Hz, 1H), 4.79(s, 1H), 4.78-4.77 (m, 1H), 3.96-3.93 (m, 1H), 3.41-3.39 (m, 1H), 2.40-2.36 (m, 1H), 1.85-1.81 (m, 1H), 1.42-1.40 (m, 1H), 1.25-1.21 (m, 2H), 0.87 (t, J = 7.5 Hz, 3H)

[0188] Alternative Preparation of Compound 1. Example 2 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 1 [ka]

[0189] Compound 1 was also prepared by the following alternative multi-step process. [ka]

[0190] Process 1 (R)-2-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2-hydroxyethyl 4-methylbenzenesulfonate (8S) To a solution of (R)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethane-1,2-diol (3S) (25 g, 122 mmol) in dry pyridine (250 mL) was added p-toluenesulfonyl chloride (34.7 g, 183 mmol) at 0 °C. After stirring at room temperature for 12 h, pyridine was removed under reduced pressure. The residue was diluted with water (500 mL) and extracted with EtOAc (2 × 600 mL). The combined EtOAc layers were dried over NaSO and concentrated. The concentrate was purified by column silica gel chromatography (100–200 mesh, eluted with 25% EtOAc in petroleum ether) to give 26.4 g (60%) of (R)-2-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2-hydroxyethyl 4-methylbenzenesulfonate (8S) [5875-16-4] as a light yellow oil. 1 H NMR (500MHz, CDC13): δ 7.80 (d, J = 6.5 Hz, 2H), 7.36 (d, J = 8.00 Hz, 2H), 5.76 (d, J = 4 Hz, 1H), 4.72 (t, J = 4.00 Hz, 1H), 4.17-4.14 (m, 2H), 4.01-3.97 (m, 2H), 2.45 (s, 3H), 2.41 (d, J = 4 Hz, 1H), 2.10-2.04 (m, 1 H), 1.80-1.75 (m, 1 H), 1.51 (s,3 H), 1.33 (s, 3 H)

[0191] Process 2 (3aR,5S,6aR)-2,2-dimethyl-5-((R)-oxiran-2-yl)tetrahydrofuro[2,3-d][1,3]dioxole (9S) To a solution of (R)-2-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2-hydroxyethyl 4-methylbenzenesulfonate (3S) (26 g, 72 mmol) in anhydrous THF (260 mL) cooled to −78 °C was added potassium bis(trimethylsilyl)amide (108 mL, 108 mmol, 1 M in THF) under a N atmosphere. After stirring at −78 °C for 1 h, the reaction mixture was poured into saturated NHCl solution. The organic layer was separated, and the aqueous phase was extracted with EtOAc (2 × 200 mL). The combined organic layers were dried over NaSO and concentrated under reduced pressure. The crude product was purified by column silica gel chromatography (100–200 mesh, eluted with 25% EtOAc in petroleum ether) to afford 10.5 g (78%) of (3aR,5S,6aR)-2,2-dimethyl-5-((R)-oxiran-2-yl)tetrahydrofuro[2,3-d][1,3]dioxole (9S) [2457-93-4] as a light yellow oil. 1 H NMR: (400MHz, CDC13): δ 5.84 (d, J = 5.4 Hz, 1H), 4.75 (t, J = 4.4 Hz, 1H), 4.23-4.18 (m, 1H), 3.15-3.13 (m, 1H), 2.83-2.80 (m, 1H), 2.61 (m, 1H), 2.07 (dd, J = 13.2, 4.8 Hz, 1H), 1.75-1.68 (m, 1H), 1.50 (s, 3H), 1.32 (s, 3H)

[0192] Process 3 (R)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-1-ol (10S) To a suspension of CuI (2 g, 10.7 mmol) in dry THF (300 mL) was added methylmagnesium bromide (3 M in diethyl ether, 53 mL, 159 mmol) at −78 °C under a N atmosphere. After stirring at −78 °C for 1 h, a solution of (3aR,5S,6aR)-2,2-dimethyl-5-((R)-oxiran-2-yl)tetrahydrofuro[2,3-d][1,3]dioxole (9S) (10 g, 53 mmol) in THF (40 mL) was added and stirred at −78 °C for an additional 2 h. The reaction mixture was poured into saturated NHCl solution (200 mL) and the organic layer was separated. The aqueous phase was extracted with EtOAc (2 × 200 mL). The combined organic layers were dried over NaSO and concentrated under reduced pressure. The crude product was purified by column silica gel chromatography (100–200 mesh, eluted with 30% EtOAc in petroleum ether) to give 8.2 g (75%) of (R)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-1-ol (10S) as a light yellow oil. 1 H NMR (500MHz, CDC13): δ 5.81 (d, J = 3.5 Hz, 1H), 4.75-4.73 (m, 1H), 4.23-4.19 (m, 1H), 3.85-3.84 (m, 1H), 2.04-2.02 (m, 1H), 1.91-1.90 (m, 2H), 1.51 (s, 3H), 1.45-1.39 (m, 2H), 1.32 (s, 3H), 1.01 (t, J = 7.5 Hz, 3H)

[0193] Process 4 (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propyl 4-nitrobenzoate (11S) To a stirred solution of (R)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-1-ol (10S) (8 g, 39 mmol), triphenylphosphine (31 g, 118 mmol), and 4-nitrobenzoic acid (20 g, 118 mmol) in THF (240 mL) was added diethyl azodicarboxylate (18.6 mL, 118 mmol) dropwise at 0 °C under a N atmosphere. After stirring at RT for 10 h, the mixture was quenched with saturated NaHCO solution (200 mL) and extracted with EtOAc (2 × 200 mL). The combined organic layers were dried over NaSO and concentrated under reduced pressure. The residue was purified by column silica gel chromatography (100–200 mesh, eluted with 15% EtOAc in petroleum ether) to give 9 g (64.7%) of (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propyl 4-nitrobenzoate (11S) as a light yellow solid. 1 H NMR (400MHz, CDC13): δ 8.34-8.27 (m, 2H), 8.25-8.21 (m, 2H), 5.83 (d, J = 3.6Hz, 1H), 5.20-5.15 (m, 1H), 4.73 (t, J = 4.0 Hz, 1H), 4.40-4.38 (m, 1H), 2.12 (dd, J = 13.2, 3.6 Hz, 1H), 1.88-1.78 (m, 2H), 1.68-1.62 (m, 1H), 1.55 (s, 3H), 1.32 (s, 3H), 1.01 (t, J =7.2Hz, 3H)

[0194] Process 5 (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-1-ol (12S) To a stirred solution of (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propyl 4-nitrobenzoate (11S) (9 g, 26 mmol) in methanol (180 mL) was added KCO (7.5 g, 55 mmol). After stirring at room temperature for 30 min, the resulting mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude material was purified by column chromatography on silica gel (100-200 mesh, eluted with 30% EtOAc in petroleum ether) to give 4.5 g (87%) of (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-1-ol (12S) as a light yellow oil. 1 H NMR (400MHz, CDC13): δ 5.81 (d, J = 3.6 Hz, 1H), 4.74 (t, J = 4.4 Hz, 1H), 4.17-4.13 (m, 1H), 3.42-3.38 (m, 1H), 2.05-2.00 (m, 2H), 1.80-1.71 (m, 1H), 1.57-1.50 (m, 5H), 1.32 (s, 3H), 1.01 (t, J= 7.6 Hz, 3H)

[0195] Process 6 (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propyl acetate (13S) To a stirred solution of (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-1-ol (12S) (4.5 g, 22 mmol), TEA (31 mL, 267 mmol), and DMAP (0.538 g, 4.4 mmol) in anhydrous DCM (90 mL) was added acetic anhydride (20 g, 198 mmol). After stirring at room temperature for 10 h, the reaction mixture was quenched with saturated aqueous NaHCO3 (100 mL). The organic layer was separated, and the aqueous phase was extracted with DCM (2 × 100 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column silica gel chromatography (100–200 mesh, eluted with 20% EtOAc in petroleum ether) to give 4.4 g (80.5%) of (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propyl acetate (13S) as a colorless oil. 1 H NMR (400MHz, CDC13): δ 5.80 (d, J = 3.6 Hz, 1H), 4.9-4.87 (m, 1H), 4.72 (t, J = 4.4 Hz, 1H), 4.30-4.25 (m, 1H), 2.10 (s, 3H), 2.03-1.99 (m, 1H), 1.69-1.55 (m, 3H), 1.51 (s, 3H), 1.32 (s, 3H), 0.91 (t, J = 4.0 Hz, 3H)

[0196] Process 7 (3R,5S)-5-((S)-1-acetoxypropyl)tetrahydrofuran-2,3-diyl diacetate (14S) To a solution of (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propyl acetate (13S) (4.4 g, 18 mmol), acetic acid (10.4 mL, 180 mmol), and acetic anhydride (8.8 mL, 90 mmol) in anhydrous DCM (90 mL) was added concentrated H2SO4 (0.44 mL) at 0 °C. After stirring at RT for 3 h, the reaction mixture was quenched by the addition of saturated aqueous NaHCO3 solution (100 mL). The organic layer was separated, and the aqueous phase was extracted with DCM (2 × 100 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column silica gel chromatography (100–200 mesh, eluted with 30% EtOAc in petroleum ether) to give 2.7 g (52%) of (3R,5S)-5-((S)-1-acetoxypropyl)tetrahydrofuran-2,3-diyl diacetate (14S) as a colorless oil. 1 H NMR (400MHz, CDC13): δ 6.10 (s, 1H), 5.18 (d, J = 4.4 Hz, 1H), 4.87-4.82 (m, 1H), 4.44-4.34 (m, 1H), 2.12-2.10 (m, 9H), 1.61-1.54 (m, 4H), 0.94 (m, 3H)

[0197] Process 8 (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (7A) To a solution of N-(6,8-dioxo-7-(prop-2-yn-1-yl)-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (6) (300 mg, 1.21 mmol), (3R,5S)-5-((S)-1-acetoxypropyl)tetrahydrofuran-2,3-diyl diacetate (14S) (384 mg, 1.33 mmol) in 1,2-dichloroethane (20 mL) was added bis(trimethylsilyl)acetamide (0.91 mL, 3.63 mmol). The resulting reaction mixture was stirred at 80° C. for 30 minutes under argon. The reaction mixture was warmed to RT, and the 1,2-dichloroethane was removed under reduced pressure. The residue was dissolved in ACN (20 mL), and trimethylsilyl trifluoromethanesulfonate (0.335 mL, 1.815 mmol) was added. The reaction mixture was heated at 80° C. for 16 hours. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was diluted with aqueous NaHCO (50 mL) and extracted with EtOAc (3×50 mL). The combined EtOAc layers were washed with water (30 mL), brine (30 mL), dried over NaSO, and concentrated under reduced pressure. The crude compound was purified by GRACE flash chromatography (using 80% EtOAc in petroleum ether as eluent) to afford 200 mg (34.6%) of (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (7A) as an off-white solid. ES+, m / z 476.2 [M+H] + ; C 21 H 25 N5O8

[0198] Process 9 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 1 To a solution of (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (7A) (200 mg, 0.421 mmol) in MeOH (20 mL) was added KCO (232 mg, 1.684 mmol) at 0 °C. The reaction mixture was stirred at RT for 16 h. Methanol was removed under reduced pressure at 30 °C. The residue was purified by normal-phase GRACE flash chromatography (using 7% MeOH in 7% MeOH as eluent) followed by reverse-phase GRACE flash chromatography (using 0.01% aqueous HCOH and ACN as eluent). Lyophilization of the purified fractions afforded 32 mg (22%) of 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 1, as an off-white solid. + , m / z 350.2 [M+H]; C 15 H 19 N5O 5: 1 H NMR (400MHz, DMSO-d6): δ 11.00 (brs, 1H), 6.55 (brs, 2H), 5.51 (d, J = 4.0 Hz, 1H), 5.39 (d, J = 5.2 Hz, 1H), 4.76-4.72 (m, 1H), 4.58-4.55 (m, 3H), 4.02-3.98 (m, 1H), 3.31-3.30 (m, 1H), 3.23-3.22 (m, 1H), 2.39-2.33 (m, 1H), 1.78-1.75 (m, 1H), 1.45-1.38 (m, 1H), 1.31-1.25 (m, 1H), 0.87 (t, J = 7.2 Hz, 3H)

[0199] Example 32-Amino-7-(cyclopropylmethyl)-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 4 [ka]

[0200] Preparation of intermediates (12) and (14) Intermediate compounds (12) and (14) were prepared in the following multiple steps. [ka]

[0201] Process 1 2-Amino-6-chloro-7-(cyclopropylmethyl)-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (8) Cyclopropylmethyl bromide (32.9 g, 24.39 mol) was added to a suspension of 2-amino-6-chloro-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (3) (62 g, 20.32 mol) and K2CO3 (42 g, 30.49 mol) in DMF (500 mL) at 0 °C, followed by stirring at room temperature for 18 h. The reaction mixture was poured into ice-cold water and stirred at room temperature for 30 min. The precipitated solid product was collected by filtration and dried under reduced pressure to give 2-amino-6-chloro-7-(cyclopropylmethyl)-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (8) (60 g, 82%) as a brown solid. TLC: 30% ethyl acetate in hexane; R f : 0.4; ES+, m / z 360.1 [M+H] + ; C 17 H 18 ClN5O2; 1H NMR (400MHz, DMSO-d6): δ 7.23 (d, J = 8.4 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 6.75 (s, 2H), 4.86 (s, 2H), 3.80 (d, J = 6.8 Hz, 2H), 3.71 (s, 3H), 1.21-1.17 (m, 1H), 0.50-0.46 (m, 2H), 0.37-0.34 (m, 2H)

[0202] Process 2 2-Amino-6-chloro-7-(cyclopropylmethyl)-7,9-dihydro-8H-purin-8-one (9) Anhydrous AlCl powder (25 g, 187 mmol) was added to a stirred solution of 2-amino-6-chloro-7-(cyclopropylmethyl)-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (8) (50 g, 139.2 mmol) in 1,2-dichlorobenzene (250 mL) at room temperature under argon, and the resulting reaction mixture was stirred at 100 °C for 18 h under argon. The reaction mixture was cooled to room temperature, quenched with ice-cold water, basified with saturated NaHCO solution under vigorous stirring, and filtered. The collected solid was dissolved in 10% MeOH in dichloromethane, stirred for 30 min, and filtered (repeated three times with 1.0 L solvent volume). The filtrate was passed through a pad of Celite and concentrated under reduced pressure. The solid thus obtained was washed twice with 10% methanol in dichloromethane and collected by filtration. The filtered solid was washed twice again with acetonitrile, collected by filtration, and dried to give 2-amino-6-chloro-7-(cyclopropylmethyl)-7,9-dihydro-8H-purin-8-one (9) (12 g, 36%) as a brown solid. TLC: 50% ethyl acetate in hexane; R f : 0.3; ES+, m / z 240.1 [M+H] + ; C9H 10 ClN5O. 1H NMR (500MHz, DMSO-d6): δ 11.81 (s, 1H), 6.58 (s, 2H), 3.73-3.72 (d, J = 7.0 Hz, 2H), 1.21-1.17 (m, 1H), 0.50-0.46 (m, 2H), 0.37-0.34 (m, 2H).mp 286-290℃ Process 3 :2-Amino-6-(benzyloxy)-7-(cyclopropylmethyl)-7,9-dihydro-8H-purin-8-one (10) Sodium metal (1.43 g, 62.6 mmol) was added to benzyl alcohol (25.0 mL) and the sodium was allowed to dissolve over 1 h. The resulting viscous liquid was cooled to rt, and 2-amino-6-chloro-7-(cyclopropylmethyl)-7,9-dihydro-8H-purin-8-one (9) (5.0 g, 20.86 mmol) was added. The resulting reaction mixture was stirred at 100 °C for 1 h and quenched with ice water (200 mL). Diethyl ether (150 mL) was added and stirred for 15 min. The resulting precipitated solid was filtered, washed with water, and dried. The solid was dissolved in 15% methanol-dichloromethane (500 mL), filtered through a pad of Celite, washed with brine, and concentrated. The resulting solid was washed with diethyl ether and filtered to give 2-amino-6-(benzyloxy)-7-(cyclopropylmethyl)-7,9-dihydro-8H-purin-8-one (10) (3.5 g, 54%) as an off-white solid. TLC: 70% ethyl acetate in petroleum ether; R f : 0.6; ES+, m / z 312.2 [M+H] + ; C 16 H 17 N5O2. 1 H NMR (500MHz, DMSO-d6): δ 11.30 (brs, 1H), 7.48-7.46 (m, 2H), 7.41-7.38 (m, 2H), 7.35-7.30 (m, 1H), 6.15 (s, 2H), 5.41 (s, 2H), 3.56 (d, J = 7.0 Hz, 2H), 1.14-1.10 (m, 1H), 0.36-0.32 (m, 2H), 0.23-

[0203] Process 4 2-Amino-7-(cyclopropylmethyl)-7,9-dihydro-1H-purine-6,8-dione (11) A suspension of 2-amino-6-(benzyloxy)-7-(cyclopropylmethyl)-7,9-dihydro-8H-purin-8-one (10) (10.0 g, 32.15 mmol), anhydrous LiBr (3.34 g, 38.58 mmol), and chlorotrimethylsilane (4.53 g, 108.6 mmol) in acetonitrile (700 mL) was stirred at room temperature for 16 hours. Methanol (60 mL) was added to the reaction mixture and stirred for 30 minutes. The solvent was evaporated to a volume of approximately 100 mL and collected by filtration. The filter cake was washed with acetonitrile and dried. The solid was dissolved in saturated NaHCO3 solution (200 mL), stirred for 1 hour, filtered, washed with water, and dried. The solid was dissolved in 10% methanol / dichloromethane (50 mL), filtered, and dried to give 2-amino-7-(cyclopropylmethyl)-7,9-dihydro-1H-purine-6,8-dione (11) (8.5 g, 80%) as a light brown solid. TLC: 10% methanol in dichloromethane; R f : 0.4; ES+, m / z 222.1 [M+H] + ; C9H 11 N5O2. 1 H NMR (500MHz, DMSO-d6): δ 11.06 (s, 1H), 10.68 (s, 1H), 6.36 (s, 2H), 3.58 (d, J = 7.0 Hz, 2H), 1.19-1.14 (m, 1H), 0.40-0.36 (m, 2H), 0.33-0.30 (m, 2H).mp 364-368℃

[0204] Process 5 N-(7-(cyclopropylmethyl)-6,8-dioxo-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (12) Acetic anhydride (0.13 mL, 1.3574 mmol) was added to a solution of 2-amino-7-(cyclopropylmethyl)-7,9-dihydro-1H-purine-6,8-dione (11) (200 mg, 0.9049 mmol) in acetic acid (5 mL) at ambient temperature under an argon atmosphere, and the resulting reaction mixture was heated at 140° C. for 6 h. The reaction mixture was cooled to 0° C. (a solid formed with stirring) and stirred for 30 min. The product was filtered and dried under reduced pressure to give N-(7-(cyclopropylmethyl)-6,8-dioxo-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (12) (180 mg, 75.6%) as a brown solid: C 11 H 13 N5O3: LC-MS showed 98% of the desired m / z (M+H; 264.1). 1 H NMR (400MHz, DMSO-d6): δ 12.01 (s, 1H), 11.68 (s, 1H), 11.65 (s, 1H), 3.66 (d, J =7.2 Hz, 2H), 2.15 (s, 3H), 2.21-2.15 (m, 1H), 043-0.38 (m, 2H), 0.36-0.32 (m, 2H)

[0205] Process 6 2-Amino-7-(cyclopropylmethyl)-7,9-dihydro-8H-purin-8-one (13) 10% Pd-C (5.0 g) was added to a solution of 2-amino-6-chloro-7-(cyclopropylmethyl)-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (8) (20 g, mmol) in methanol (500 mL). The reaction mixture was hydrogenated at room temperature for 24 hours under 80 psi H gas pressure. The reaction mixture was filtered through a pad of Celite, and the filtrate was evaporated to give the crude product. Acetonitrile was added to the crude compound with stirring for 15 minutes and filtered. This process was repeated, and the solid was dried under reduced pressure to give 2-amino-7-(cyclopropylmethyl)-7,9-dihydro-8H-purin-8-one (13) (11 g, 64%) as an off-white solid. TLC: 70% ethyl acetate in hexane; R f : 0.2; ES+, m / z 206.2 [M+H]+ ; C9H 11 N5O. 1 H NMR (500MHz, DMSO-d6): δ 12.68 (s, 1H), 8.11 (s, 1H), 7.87 (s, 2H), 3.60-3.59 (d, J = 7.5 Hz, 2H), 1.18-1.12 (m, 1H), 0.50-0.46 (m, 2H), 0.37-0.34 (m, 2H).mp 245-249℃

[0206] Process 7 N-(7-(cyclopropylmethyl)-8-oxo-8,9-dihydro-7H-purin-2-yl)acetamide (14) To a stirring solution of 2-amino-7-(cyclopropylmethyl)-7,9-dihydro-8H-purin-8-one (13) (2 g, 9.75 mmol) in AcOH (50 mL) was added AcO (3.68 mL, 39.02 mmol). The resulting solution was heated at 120 °C for 12 h. The reaction mixture was cooled to RT, and the resulting solid was collected by filtration, washed with diethyl ether (100 mL), and dried to give 1.6 g (51%) of N-(7-(cyclopropylmethyl)-8-oxo-8,9-dihydro-7H-purin-2-yl)acetamide (14) as an orange solid. ES+, m / z 248.2 [M+H] + ; C 11 H 13 N5O2: 1 H NMR (400MHz, DMSO-d6): δ 12.97 (s, 1H), 11.71 (s, 1H), 8.31 (s, 1H), 3.74, (d, J = 7.2 Hz, 2H), 2.22 (s, 3H), 1.45-1.13 (m, 1H), 0.53 (t, J = 3.6 Hz, 2H), 0.48 (t, J = 3.6 Hz, 2H)

[0207] Process 8(S)-1-((2S,4R,5R)-5-(2-acetamido-7-(cyclopropylmethyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (15) [ka] N-(7-(cyclopropylmethyl)-6,8-dioxo-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (12) (200 mg, 0.7604 mmol), (3R,5S)-5-((S)-1-acetoxypropyl)tetrahydrofuran-2,3-diyl diacetate (14S) (219 mg, 0.7604 mmol), and bis(trimethylsilyl)acetamide (0.58 mL, 2.2812 mmol) were dissolved in 1,2-dichloroethane (10 mL). The reaction mixture was stirred at 80 °C for 30 min under argon and then concentrated under reduced pressure. The resulting residue was diluted with ACN (50 mL), TMSOTf (0.212 mL, 1.1406 mmol) was added, and the mixture was placed in a preheated oil bath at 80 °C and stirred for 16 h. The reaction mixture was cooled to room temperature, and the solvent was removed by rotary evaporation. The resulting solid was dissolved in ethyl acetate (100 mL) and extracted with saturated aqueous NaHCO (2 × 30 mL). The organic phase was dried over NaSO and concentrated. The crude product was purified by flash chromatography (SiO, 0–80% EtOAc-petroleum ether) to give (S)-1-((2S,4R,5R)-5-(2-acetamido-7-(cyclopropylmethyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (15) (175 mg, 46.9%) as a light yellow solid: C 22 H 29 N5O 8: ES + , m / z 492.2 [M+H] + This compound was used further without any further purification.

[0208] Process 92-Amino-7-(cyclopropylmethyl)-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 4 To a solution of (S)-1-((2S,4R,5R)-5-(2-acetamido-7-(cyclopropylmethyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (15) (175 mg, 0.3564 mmol) in methanol (10 mL) was added KCO (20 mg, 0.1424 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 36 h, concentrated, and purified by preparative HPLC (column: X-SELECT-C18 (250*19), 5 μL mobile phase: 0.1% TFA:ACN in HO gradient: (T%B): -0 / 5, 1 / 5, 8 / 40, 9 / 40, 9.1 / 98, 11 / 98, 11.1 / 5, 14 / 5, flow rate: 20 mL). The purified fractions were collected and lyophilized to give 2-amino-7-(cyclopropylmethyl)-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 4 (60 mg, 47.6%) as a white solid. 16 H 23 N5O 5: ES + , m / z 366.2 [M+H] + . 1 H NMR (500MHz, DMSO-d6): δ 10.87 (s, 1H), 6.45 (s, 2H), 5.52 (s, 1H), 4.76-4.75 (m, 1H), 4.01-3.97 (m, 1H), 3.64 (d, J = 7 Hz, 2H), 3.27-3.25 (m, 1H), 2.39-2.35 (m, 1H), 1.79-1.74 (m, 1H), 1.41-1.37 (m, 1H), 1.28-1.17 (m, 2H), 0.88-0.85 (t, J = 7 Hz, 3H), 0.40 (d, J = 7.5 Hz, 2H), 0.34 (d, J = 3.5 Hz, 2H)

[0209] Example 4 2-Amino-7-(cyclopropylmethyl)-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7,9-dihydro-8H-purin-8-one, compound 5 [ka]

[0210] Compound 5 was prepared in two steps. [ka]

[0211] Process 1 (S)-1-((2S,4R,5R)-5-(2-acetamido-7-(cyclopropylmethyl)-8-oxo-7,8-dihydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (16) A stirred solution of N-(7-(cyclopropylmethyl)-8-oxo-8,9-dihydro-7H-purin-2-yl)acetamide (14) (300 mg, 1.21 mmol), (3R,5S)-5-((S)-1-acetoxypropyl)tetrahydrofuran-2,3-diyl diacetate (14S) (384.7 mg, 1.33 mmol), and bis(trimethylsilyl)acetamide (0.73 mL, 3.64 mmol) in 1,2-dichloroethane (25 mL) was heated at 80° C. for 30 min under argon. The reaction mixture was cooled to RT, and the 1,2-dichloroethane was removed under reduced pressure. The residue was dissolved in ACN (30 mL), TMSOTf (0.40 mL, 1.82 mmol) was added, and the reaction mixture was maintained at 80° C. for 16 h. The mixture was then cooled to RT and concentrated under reduced pressure. The residue was diluted with saturated aqueous NaHCO3 (50 mL) and extracted with EtOAc (3 x 50 mL). The combined EtOAc layers were washed with water (30 mL), brine (30 mL), dried over Na2SO4, and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography (100-200 mesh) using 3% MeOH / DCM as eluent to afford 250 mg (53.6%) of (S)-1-((2S,4R,5R)-5-(2-acetamido-7-(cyclopropylmethyl)-8-oxo-7,8-dihydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (16) as a pale yellow solid. 22 H 29 N5O7: ES + , m / z 476.1 [M+H] +

[0212] Process 2 2-Amino-7-(cyclopropylmethyl)-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7,9-dihydro-8H-purin-8-one, compound 5 To a stirred solution of (S)-1-((2S,4R,5R)-5-(2-acetamido-7-(cyclopropylmethyl)-8-oxo-7,8-dihydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (16) (250 mg, 0.526 mmol) in MeOH (15 mL) was added KCO (145 mg, 1.052 mmol) at 0 °C. The reaction mixture was stirred at RT for 16 h, and acetic acid (0.05 mL, 0.815 mmol) was added. The solution was stirred for an additional 20 min and concentrated under reduced pressure at 30 °C. The residue was purified by preparative HPLC (X-SELECT-C18 (250*19), 5μ mobile phase: 10 mM ammonium bicarbonate in HO:MeCN gradient: (T%B): -0 / 20, 8 / 35, 11 / 35, 11 / 98, 13 / 98, 13.1 / 20, 16 / 20 flow rate: 18 ml) to give 35 mg (46%) of 2-amino-7-(cyclopropylmethyl)-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7,9-dihydro-8H-purin-8-one, compound 5, as an off-white solid. 16 H 23 N5O4: ES + , m / z 350.1 [M+H] + . 1 H NMR (500MHz, DMSO-d6): δ 8.06 (s, 1H), 6.24 (brs, 2H), 5.64-5.60 (m, 1H), 5.44 (bs, 1H), 4.82-4.79 (m, 1H), 4.61 (bs, 1H), 4.03-4.02 (m, 1H), 4.03-4.01 (m, 1H), 3.62 (d, J = 7.0 Hz, 2H), 3.31 (s, 1H), 2.49 (m, 1H), 1.80-1.78 (m, 1H), 1.41-1.31 (m, 1H), 1.28-1.27 (m, 1H), 0.87 (t, J = 7.0 Hz, 3H)., 0.51-0.46 (m, 2H), 0.36-0.33 (m, 2H)

[0213] Example 52-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-8H-purin-8-one, compound 6 [ka]

[0214] Preparation of intermediate (21) Intermediate compound (21) was prepared by the following multi-step method. [ka]

[0215] Process 1 :N4-(4-Methoxybenzyl)pyrimidine-2,4,5-triamine (17) 10% Pd-C (10 g) was added to a solution of 6-chloro-N4-(4-methoxybenzyl)-pyrimidine-2,4,5-triamine (1) (2.5 g, 0.072 mol) in methanol (50 mL) and THF (150 mL). The reaction mixture was hydrogenated at room temperature for 24 hours at 80 psi H2 gas. The reaction mixture was filtered through a Celite pad, and the filtrate was evaporated. The crude compound was stirred in ethyl acetate (100 mL) for 15 minutes, and the solid compound was collected by filtration and dried under reduced pressure to give N4-(4-methoxybenzyl)pyrimidine-2,4,5-triamine (17) (21.0 g, 95.8%). ES + , m / z 246.2 [M+H] + ; C 12 H 15 N5O; 1 H NMR (400MHz, DMSO-d6): δ 11.71 (bs, 1H), 8.75-8.68 (m, 1H), 7.31 (d, J =8.4 Hz, 2H), 7.25 (bs, 2H),7.07 (s, 1H), 6.90 (d, J =8 Hz, 2H), 4.77 (bs, 2H), 4.56 (d, J = 5.6 Hz, 2H), 3.73 (s, 3H)

[0216] Process 2 :2-amino-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (18) A mixture of N4-(4-methoxybenzyl)pyrimidine-2,4,5-triamine (17) (23.0 g, 9.39 mol) and 1,1'-carbonyldiimidazole (18.3 g, 11.27 mol) in THF (250 mL) was stirred at room temperature for 18 hours. The reaction mixture was concentrated under reduced pressure. Ice-cold water was added to the resulting residue, and the mixture was stirred at room temperature for 30 minutes. The precipitated solid was collected by filtration, washed with water, and dried under reduced pressure to give 2-amino-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (18) (22.0 g, 86.6%) as a light brown solid. ES+, m / z 272.2 [M+H] + ; C 13 H 13 N5O2; 1 H NMR (400MHz, DMSO-d6): δ 10.81 (s, 1 H), 7.73 (s, 1H), 7.23 (d, J = 8.0 Hz, 2H), 6.88 (d, J = 8.4 Hz, 2H), 6.19 (s, 2H), 4.80 (s, 2H), 3.72 (s, 3H)

[0217] Process 3 2-Amino-9-(4-methoxybenzyl)-7-(prop-2-yn-1-yl)-7,9-dihydro-8H-purin-8-one (19) Propargyl bromide (87 mL, 77.49 mol) was added to a suspension of 2-amino-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (18) (17.5 g, 6.46 mol) and K2CO3 (13.4 g, 9.69 mol) in DMF (175 mL) at 0 °C and stirred at room temperature for 18 h. The reaction mixture was quenched with ice water (400 mL) and stirred for 30 min. The resulting precipitated solid was collected by filtration, washed with water, and dried under reduced pressure to give 2-amino-9-(4-methoxybenzyl)-7-(prop-2-yn-1-yl)-7,9-dihydro-8H-purin-8-one (19) (17.5 g, 86.68%) as a brown solid. ES+ , m / z 310.1 [M+H] + ; C 16 H 15 N5O2; 1 H NMR (400MHz, DMSO-d6): δ 7.97 (s, 1H), 7.24 (d, J =8.4 Hz, 2H), 6.89 (dd, J =8.8 Hz, J =4.8 Hz, 2H), 6.36 (s, 2H), 4.84 (s, 2H), 4.64 (d, J =2.4 Hz, 2H), 3.71 (s, 3H), 3.37 (s, 1H)

[0218] Process 4 2-Amino-7-(prop-2-yn-1-yl)-7,9-dihydro-8H-purin-8-one (20) Trifluoromethanesulfonic acid (21.84 g, 14.56 mol) was added to a suspension of 2-amino-9-(4-methoxybenzyl)-7-(prop-2-yn-1-yl)-7,9-dihydro-8H-purin-8-one (19) (15.0 g, 4.854 mol) in trifluoroacetic acid (16.65 g, 14.56 mol) at 0 °C under an argon atmosphere, and the resulting reaction mixture was stirred at room temperature for 18 h. The reaction mixture was quenched with ice-cold water, made basic with excess saturated NaHCO solution under vigorous stirring, and collected by filtration. The remaining solid was dissolved in ethyl acetate, stirred for 30 min, and collected by filtration. The solid residue was dried under reduced pressure to give 2-amino-7-(prop-2-yn-1-yl)-7,9-dihydro-8H-purin-8-one (20) (6.50 g, 70.85%) as a light yellow solid. ES+, m / z 190.1 [M+H] + ; C8H7N5O; 1 H NMR (400MHz, DMSO-d6): δ 11.32 (s, 1H), 7.57 (s, 1H), 5.60 (s, 2H), 4.46 (s, 2H), 3.22 (s, 1H)

[0219] Process 5 N-(8-oxo-7-(prop-2-yn-1-yl)-8,9-dihydro-7H-purin-2-yl)acetamide (21) Acetic anhydride (4.86 mL, 4.76 mol) was added to a solution of 2-amino-7-(prop-2-yn-1-yl)-7,9-dihydro-8H-purin-8-one (20) (6.0 g, 3.17 mol) in DMF (60 mL) at room temperature under an argon atmosphere, and the resulting reaction mixture was stirred at 140° C. for 10 h. The reaction mixture was cooled to 0° C., after which a solid formed with stirring for 1 h. The product was collected by filtration, washed with water, and dried under reduced pressure to give N-(8-oxo-7-(prop-2-yn-1-yl)-8,9-dihydro-7H-purin-2-yl)acetamide (21) (5.8 g, 79%) as a light yellow solid. ES+, m / z 232.1 [M+H] + ; C 10 H9N5O2; 1 H NMR (500MHz, DMSO-d6): δ 9.80 (s, 1H), 7.76 (s, 1H), 6.17 (s, 1H), 4.53 (s, 2H), 3.24 (s, 1H), 2.11 (s, 3H)

[0220] Process 6 (S)-1-((2S,4R,5R)-5-(2-acetamido-8-oxo-7-(prop-2-yn-1-yl)-7,8-dihydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (22) Compound 6 was prepared by the following two-step method. [ka]

[0221] A stirred mixture of N-(8-oxo-7-(prop-2-yn-1-yl)-8,9-dihydro-7H-purin-2-yl)acetamide (21) (300 mg, 1.298 mmol), (3R,5S)-5-((S)-1-acetoxypropyl)tetrahydrofuran-2,3-diyl diacetate (14S) (411 mg, 1.428 mmol), and bis(trimethylsilyl)acetamide (0.87 mL, 3.894 mmol) in 1,2-dichloroethane (30 mL) was heated at 80° C. for 30 min under argon. The reaction mixture was cooled to RT, and the 1,2-dichloroethane was removed under reduced pressure. The residue was dissolved in ACN (30 mL), and TMSOTf (0.36 mL, 1.945 mmol) was added. The stirred reaction mixture was heated at 80° C. for 16 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was diluted with saturated aqueous NaHCO3 (50 mL) and extracted with EtOAc (3 x 50 mL). The combined EtOAc layers were washed with water (30 mL), brine (30 mL), dried over Na2SO4, and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography (100-200 mesh) using 3% MeOH / DCM as eluent to afford 320 mg (53.6%) of (S)-1-((2S,4R,5R)-5-(2-acetamido-8-oxo-7-(prop-2-yn-1-yl)-7,8-dihydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (22) as a pale yellow solid. ES+, m / z 460.2 [M+H] + ; C 21 H 25 N5O7

[0222] Process 7 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-8H-purin-8-one, compound 6 To a stirred solution of (S)-1-((2S,4R,5R)-5-(2-acetamido-8-oxo-7-(prop-2-yn-1-yl)-7,8-dihydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (22) (150 mg, 0.326 mmol) in MeOH (15 mL) was added KCO (90 mg, 0.653 mmol) at 0 °C. Stirring was continued at RT for 16 h. Acetic acid (0.05 mL, 0.815 mmol) was added to the solution, which was stirred for 20 min and concentrated under reduced pressure at 30 °C. The residue was purified by normal phase GRACE flash chromatography (5% MeOH in DCM) to give 50 mg (46%) of 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-8H-purin-8-one, compound 6, as an off-white solid. ES+, m / z 334.2 [M+H] + ; C 15 H 19 N5O4: 1 H NMR (500MHz, DMSO-d6): δ 8.01 (s, 1H), 6.34 (brs, 2H), 5.60 (d, J = 3.0 Hz, 1H), 5.42 (d, J = 4.5 Hz, 1H), 4.80 (t, J = 3.5 Hz, 1H), 4.63 (d, J = 2.5 Hz, 2H), 4.54 (d, J = 6.5 Hz, 1H), 4.03-4.01 (m, 1H), 3.39 (t, J = 2.5 Hz, 1H), 3.29-3.28 (m, 1H), 2.45-2.42 (m, 1H), 1.81-1.78 (m, 1H), 1.45-1.41 (m, 1H), 1.28-1.26 (m, 1H), 0.87 (t, J = 7.5 Hz, 3H)

[0223] Example 62-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(propa-1,2-dien-1-yl)-7,9-dihydro-8H-purin-8-one, compound 7 [ka]

[0224] To a stirred solution of (S)-1-((2S,4R,5R)-5-(2-acetamido-8-oxo-7-(prop-2-yn-1-yl)-7,8-dihydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (22) (150 mg, 0.326 mmol) in MeOH (15 mL) was added KCO (135 mg, 0.980 mmol) at 0 °C. The reaction mixture was stirred at RT for 16 h and then concentrated under reduced pressure. The residue was purified by normal-phase GRACE flash chromatography (5% MeOH in DCM) and further purified by reverse-phase GRACE flash chromatography (0.01% HCOH in ACN). The compound thus obtained was washed with acetonitrile (10 mL) and dried to give 40 mg (36.6%) of 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(propa-1,2-dien-1-yl)-7,9-dihydro-8H-purin-8-one, compound 7. ES+, m / z 334.1 [M+H] + ; C 15 H 19 N5O4: 1H NMR (500MHz, DMSO-d6): δ 8.12 (s, 1H), 7.20 (t, J = 6.5 Hz, 1H), 6.47 (brs, 2H), 5.82 (d, J = 6.5 Hz, 2H), 5.63 (d, J = 3.0 Hz, 1H), 5.42 (d, J = 7.5 Hz, 1H), 4.81-4.78 (m, 1H), 4.50 (d, J = 6.5 Hz, 1H), 4.04-4.02 (m, 1H), 3.31-3.28 (m, 1H), 2.50-2.42 (m, 1H), 1.79-1.78 (m, 1H), 1.47-1.39 (m, 1H), 1.28-1.23 (m, 1H), 0.87 (t, J = 7.5 Hz, 3H)

[0225] Example 7 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(propa-1,2-dien-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 8 [ka]

[0226] To a stirred solution of (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (7A) (200 mg, 0.421 mmol) in MeOH (10 mL) was added KCO (290 mg, 2.10 mmol) at 0° C. The reaction mixture was stirred at rt for 16 h, and the methanol was concentrated under reduced pressure at rt. The resulting residue was directly purified by GRACE reverse-phase 0.1% FA:ACN (run twice), and the purified fractions were lyophilized to give 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(propa-1,2-dien-1-yl)-7,9-dihydro-1H-purine-6,8-dione as an off-white solid. 15 H 19 N5O 5: 1 H NMR (500MHz, DMSO-d6): δ 10.96 (brs, 1H), 7.18 (t, J = 6.5Hz, 1H), 6.60 (brs, 2H), 5.56 (d, J = 3.0 Hz, 1H), 5.51 (d, J = 3.0Hz, 2H), 5.45 (d, J = 8.0Hz, 1H), 4.74-4.72 (m, 1H), 4.48 (d, J = 6.5Hz, 1H), 3.98 (q, J = 3.0Hz, 1H), 3.29-3.26 (m, 1H), 2.36-2.33 (m, 1H), 1.76-1.73 (m, 1H), 1.39-1.38 (m, 1H), 1.27-1.24 (m, 1H), 0.87 (t, J = 7.5 Hz, 3H)

[0227] Example 8 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxyethyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 9 [ka]

[0228] Compound 9 was also prepared by the following multiple steps. [ka]

[0229] Process 1 (R)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (15S) To a stirred solution of (3aR,5S,6aR)-2,2-dimethyl-5-((R)-oxiran-2-yl)tetrahydrofuro[2,3-d][1,3]dioxole (9S) in dry THF (3 g, 15.9 mmol) was added LAH (1 M in THF, 53 mL, 159 mmol) at 0 °C under a N atmosphere. After stirring at 0 °C for 2 h, the reaction mixture was quenched with saturated NHCl solution (50 mL). The organic layer was separated, and the aqueous phase was extracted with EtOAc (2 × 200 mL). The combined organic layers were dried over NaSO and concentrated under reduced pressure. The crude material was purified by column silica gel chromatography (100-200 mesh, eluted with 40% EtOAc in petroleum ether) to give 2.7 g (89%) of (R)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol as a colorless liquid. 16 O4: 1 H NMR (400MHz, CDC13): δ 5.82 (d, J = 3.6Hz, 1H), 4.75 (t, J = 4.0 Hz, 1H), 4.19-4.08 (m, 2H), 2.05 (brs, 1H), 2.04-1.88 (m, 2H), 1.51 (s, 3H), 1.32 (s, 3H), 1.14 (d, J = 6.4 Hz, 3H)

[0230] Process 2(S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethyl 4-nitrobenzoate (16S) To a stirred solution of (R)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (15S) (2.7 g, 39 mmol) in THF (30 mL) were added triphenylphosphine (7.5 g, 28.7 mmol), 4-nitrobenzoic acid (4.7 g, 28.7 mmol), followed by the dropwise addition of diethyl azodicarboxylate (4.5 mL, 28.7 mmol) at 0 °C under a N atmosphere. After stirring at 25 °C for 10 h, the mixture was quenched with saturated NaHCO solution (50 mL) and extracted with EtOAc (2 × 100 mL). The combined organic layers were dried over NaSO and concentrated under reduced pressure. The residue was purified by column silica gel chromatography (100-200 mesh, eluted with 15% EtOAc in petroleum ether) to give 3 g (62%) of (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethyl 4-nitrobenzoate as a light yellow liquid. 16 H 19 NO7: ES+, m / z 338.2 [M+H] + . 1 H NMR (400MHz, CDC13): δ 8.30-8.20 (m, 4H), 5.86 (d, J = 3.6 Hz, 1H), 5.23 (quintet, J = 6.4 Hz, 1H), 4.76 (t, J = 4.4 Hz, 1H), 4.38 (ddd, J = 10.8, 6.0, 4.8 Hz, 1H), 2.10 (dd, J = 13.2, 4.4 Hz, 1H), 1.68 (m, 1H), 1.63 (s, 3H), 1.41 (d, J = 6.4 Hz, 3H), 1.33 (s, 3H)

[0231] Process 3(S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (17S) To a stirred solution of (R)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (16S) (5.5 g, 16.3 mmol) in methanol (50 mL) was added K2CO3 (4.5 g, 32.6 mmol). After stirring at room temperature for 30 min, the resulting reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column silica gel chromatography (100-200 mesh, eluted with 30% EtOAc in petroleum ether) to give 2.7 g (88%) of (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol as a light yellow solid. CH 16 O4: 1 H NMR (400MHz, CDC13): δ 5.81 (d, J = 3.6 Hz, 1H), 4.76 (t, J = 4.2 Hz, 1H), 4.12-4.05 (m, 1H), 3.68-3.65 (m, 1H), 2.19 (d, J = 4.0 Hz, 1H), 2.05-2.01 (m, 2H), 1.68-1.62 (m, 2H), 1.32 (s, 3H), 1.20 (s, 3H)

[0232] Process 4 (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethyl acetate (18S) To a stirred solution of (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (17S) (2.7 g, 14.0 mmol), TEA (3.9 mL, 28.0 mmol), and DMAP (0.122 g, 2.0 mmol) in anhydrous DCM (30 mL) was added acetic anhydride (2.1 mL, 21.0 mmol). After stirring at 25 °C for 10 h, the reaction mixture was quenched by the addition of saturated aqueous NaHCO (30 mL). The organic layer was separated, and the aqueous phase was extracted with CHCl (2 × 100 mL). The combined organic layers were dried over NaSO and concentrated under reduced pressure. The crude material was purified by column silica gel chromatography (100-200 mesh, eluted with 20% EtOAc in petroleum ether) to give 3.2 g (96%) of (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethyl acetate as a colorless oil. 11 H 18 O 5: 1 H NMR (400MHz, CDC13): δ 5.83 (d, J = 4.0 Hz, 1H), 4.97-4.92 (m, 1H), 4.73 (t, J = 3.2 Hz, 1H), 4.25-4.20 (m, 1H), 2.07-2.01(m, 4H), 1.67-1.60 (m, 1H), 1.52 (s, 3H), 1.32 (s, 3H), 1.25 (d, J = 4.0 Hz, 3H)

[0233] Process 5 (3R,5S)-5-((S)-1-acetoxyethyl)tetrahydrofuran-2,3-diyl diacetate (19S) To a solution of (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethyl acetate (18S) (3.2 g, 13.9 mmol), acetic acid (7.9 mL, 139.1 mmol), and acetic anhydride (6.9 mL, 69.5 mmol) in anhydrous DCM (30 mL) was added concentrated H2SO4 (0.3 mL) at 0 °C. After stirring at 25 °C for 3 h, the reaction mixture was quenched by the addition of saturated aqueous NaHCO3 (100 mL). The organic layer was separated, and the aqueous phase was extracted with EtOAc (2 × 100 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude mixture was purified by column silica gel chromatography (100-200 mesh, eluted with 30% EtOAc in petroleum ether) to afford 1.3 g (47%) of (3R,5S)-5-((S)-1-acetoxypropyl)tetrahydrofuran-2,3-diyl diacetate as a colorless oil. 12 H 18 O7: 1 H NMR (500MHz, CDC13): δ 6.39 (s, 1H), 5.18 (d, J = 3.6 Hz, 1H), 4.95-4.90 (m, 1H), 4.35-4.31 (m, 1H), 2.11-2.01 (m, 11H), 1.21(d, J = 5.2Hz, 3H)

[0234] Process 6 (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)ethyl acetate (23) N-(6,8-dioxo-7-(prop-2-yn-1-yl)-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (6) (200 mg, 0.8 mmol), (3R,5S)-5-((S)-1-acetoxypropyl)tetrahydrofuran-2,3-diyl diacetate (19S) (265 mg, 0.96 mmol), and BSA (0.61 mL, 2.4 mmol) were dissolved in 1,2-dichloroethane (20 mL), and the resulting reaction mixture was stirred at 80° C. for 30 min under argon. The reaction mixture was warmed to RT, and the 1,2-dichloroethane was removed under reduced pressure. The residue was redissolved in ACN (20 mL), followed by the addition of TMSOTf (0.22 mL, 1.2 mmol). The reaction mixture was heated at 80° C. for 16 h. The reaction mixture was then cooled to room temperature and concentrated under reduced pressure, and the residue was diluted with aqueous NaHCO (50 mL) and extracted with EtOAc (3 x 50 mL). The combined EtOAc layers were washed with water (30 mL), brine (30 mL), dried over anhydrous NaSO, and concentrated under reduced pressure. The crude compound was purified by GRACE (80% EtOAc in petroleum ether) to give 200 mg (53%) of (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)ethyl acetate as an off-white solid. 20 H 23 N5O 8: ES+, m / z 462.2 [M+H] +

[0235] Process 6 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxyethyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 9 To a solution of (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)ethyl acetate (23) (200 mg, 0.43 mmol) in MeOH (10 mL) was added KCO (59.6 mg, 0.43 mmol) at 0 °C. The resulting reaction mixture was stirred at RT for 16 h. Then, methanol was concentrated under reduced pressure at RT, and the resulting residue was purified by preparative HPLC. Lyophilization of the purified fractions afforded 35 mg (24%) of 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxyethyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 9, as an off-white solid. 14 H 17 N5O 5: ES+, m / z 336.1 [M+H] + ; 1 H NMR (400MHz, DMSO-d6): δ 10.93 (brs, 1H), 6.54 (brs, 2H), 5.22 (d, J = 4.0 Hz, 1H), 5.39 (d, J = 2.8 Hz, 1H), 5.39 (d, J = 4.4 Hz, 1H), 4.75-4.71 (m, 1H), 4.63-4.57 (m, 2H), 3.94-3.88 (m, 1H), 3.58-3.54 (m, 1H), 3.22(s, 1H), 2.38-2.33 (m, 1H), 1.75-1.70 (m, 1H), 1.06 (d, J = 6.4Hz, 3H)

[0236] Example 9 Compounds 10 and 11

[0237] 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((R)-2,2,2-trifluoro-1-hydroxyethyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 10 [ka]

[0238] 2-Amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-2,2,2-trifluoro-1-hydroxyethyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, Compound 11 [ka]

[0239] Compounds 10 and 11 were prepared in the following multiple steps. [ka]

[0240] Process 1 : 1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2,2,2-trifluoroethan-1-ol (20S) A stirred solution of (3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole-5-carbaldehyde (4S) (4.2 g, 24.41 mmol) in anhydrous dichloromethane (60 mL) was cooled to 0 °C, and trifluoromethyltrimethylsilane (3.81 g, 26.86 mmol) was added, followed by a catalytic amount of tetra-n-butylammonium difluorotriphenyltannate (1.53 g, 2.44 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 10 h. The reaction mixture was then treated with 1 equivalent of TBAF (1 M in THF) (24.4 mL), stirred for 1 h, and then quenched with aqueous NH4Cl and dichloromethane. The organic layer was separated, dried over Na2SO4, and concentrated under reduced pressure. The crude product was purified by column silica gel chromatography (100-200 mesh, eluted with 30% EtOAc in petroleum ether) to give 1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2,2,2-trifluoroethan-1-ol (20S) (2 g, 33%, as a diastereomeric mixture) as a colorless oil. 13 F3O4 (diastereomeric mixture): 1 H NMR (400MHz, CDCl3): δ 5.86-5.81 (m, 2H), 4.79-4.75 (m, 2H), 4.50-4.43 (m, 2H), 4.38-4.34 (m, 1H), 3.88-3.83 (m, 1H), 2.92 (d, J = 9.2 Hz, 1H), 2.79 (bs, 1H), 2.20-2.15 (m, 1H), 2.09-2.06 (m, 2H), 1.98-1.91(m, 1H), 1.52 (s, 6H), 1.34 (s, 6H)

[0241] Process 2 : 1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2,2,2-trifluoroethyl acetate (21S) To a stirred solution of 1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2,2,2-trifluoroethan-1-ol (20S) (2 g, 8.26 mmol), TEA (2.37 mL, 16.52 mmol), and DMAP (0.201 g, 1.65 mmol) in anhydrous DCM (30 mL) was added acetic anhydride (1.22 mL, 12.39 mmol). After stirring at 25 °C for 10 h, the reaction was quenched with saturated aqueous NaHCO3 (50 mL). The organic layer was separated and the aqueous phase was extracted with DCM (2 × 50 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude mixture was purified by column silica gel chromatography (100-200 mesh, eluted with 30% EtOAc in petroleum ether) to give 1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2,2,2-trifluoroethyl acetate (21S) (1.6 g, as a 69% diastereomeric mixture) as a colorless oil. 11 H 15 F3O5 (as a diastereomeric mixture): 1 H NMR (500MHz, CDCl3): δ 5.82 (d, J = 3.5 Hz, 1H), 4.77 (d, J = 4.0 Hz, 1H), 5.67-5.65 (m, 1H), 5.36-5.33 (m, 1H), 4.76-4.74 (m, 2H), 4.53-4.44 (m, 2H), 2.21-2.20 (m, 1H), 2.20 (s, 3H), 2.16 (s, 3H),2.15-2.12 (m, 1H),1.98-1.90 (m, 1H), 1.75-1.67(m, 1H), 1.52 (s, 6H), 1.34 (s, 6H)

[0242] Process 3 (2S,3R,5S)-5-(1-acetoxy-2,2,2-trifluoroethyl)tetrahydrofuran-2,3-diyl diacetate (22S) To a solution of 1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2,2,2-trifluoroethyl acetate (1.6 g, 4.87 mmol), acetic acid (2.92 mL, 48.7 mmol), and acetic anhydride (2.32 mL, 24.39 mmol) in anhydrous DCM (30 mL) was added concentrated H2SO4 (0.1 mL) at 0 °C. After stirring at 25 °C for 3 h, the reaction was quenched by the addition of saturated aqueous NaHCO3 (100 mL). The organic layer was separated, and the aqueous phase was extracted with DCM (2 × 70 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (100-200 mesh, eluted with 20% EtOAc in petroleum ether) to give (2S,3R,5S)-5-(1-acetoxy-2,2,2-trifluoroethyl)tetrahydrofuran-2,3-diyl diacetate (22S) (700 mg, 38%, as a diastereomeric mixture) as a colorless oil. 12 H 15 F3O7 (as a diastereomeric mixture): 1 H NMR (400MHz, CDCl3): δ 6.14 (d, J = 9.6 Hz, 2H), 5.61-5.58 (m, 1H), 5.33-5.27 (m, 1H), 5.22-5.19 (m, 2H), 4.71-4.66 (m, 1H), 4.61-4.55 (m, 1H), 2.44-2.37 (m, 1H), 2.36-2.20 (s, 2H), 2.19 (s, 3H), 2.17 (s, 3H), 2.12-2.09 (m, 4H), 2.06 (s, 9H)

[0243] Process 4(R)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (24A) and (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (24B). To N-(6,8-dioxo-7-(prop-2-yn-1-yl)-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (6) (400 mg, 1.61 mmol), (2S,3R,5S)-5-(1-acetoxy-2,2,2-trifluoroethyl)tetrahydrofuran-2,3-diyl diacetate (22S) (637 mg, 1.94 mmol) dissolved in 1,2-dichloroethane (20 mL) was added BSA (1.2 mL, 4.85 mmol). The reaction mixture was stirred at 80 °C for 30 min under argon, then cooled to room temperature, and the 1,2-dichloroethane was removed under reduced pressure. The residue was dissolved in acetonitrile (20 mL), followed by the addition of TMSOTf (0.44 mL, 2.42 mmol). The reaction mixture was heated at 80 °C for 16 h, cooled to room temperature, and concentrated under reduced pressure. The resulting residue was added with aqueous NaHCO (50 mL) and extracted with EtOAc (3 × 50 mL). The combined EtOAc layers were washed with water (30 mL), brine (30 mL), dried over NaSO, and concentrated under reduced pressure. The crude compound was purified by GRACE flash chromatography (using 80% EtOAc in petroleum ether as eluent) to afford 250 mg (LC / MS: 55%–25% diastereomeric mixture) of (R,S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (24A, 24B) as a brown solid. Further purification by preparative HPLC (X-SELECT-C18 (250*19), 5 μl mobile phase: 10 mM ammonium bicarbonate in HO:MeCN gradient: (T%B): -0 / 20, 8 / 58, 16 / 98, 16.1 / 20, 19 / 20; flow rate: 18 ml / min; diluent: MeCN + HO + THF) gave 130 mg of (R)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (24A) (C 20 H 20F3N5O8: ES+, m / z 516.2 [M+H] + ) and 80 mg (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (24B) (C 20 H 20 F3N5O8: ES+, m / z 516.1 [M+H] + ) was obtained.

[0244] Process 5 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((R)-2,2,2-trifluoro-1-hydroxyethyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 10 To a solution of (R)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (24A) (130 mg, 0.252 mmol) in MeOH (20 mL) was added KCO (34 mg, 0.252 mmol) at 0 °C, and the reaction mixture was stirred at RT for 16 h. Methanol was removed under reduced pressure at 30 °C. The residue was directly purified by reverse-phase GRACE flash chromatography using 10 mM ammonium bicarbonate in HO as eluent. Lyophilization of the purified fractions afforded 30 mg (50%) of 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((R)-2,2,2-trifluoro-1-hydroxyethyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 10, as a white solid. 14 H 14 F3N5O 5: ES-, m / z 388.0 [MH] - . 1H NMR (400MHz, DMSO-d6): δ 11.21 (brs, 1H), 6.62 (brs, 2H), 6.53 (d, J = 6.4 Hz, 1H), 5.58 (d, J = 6.4 Hz, 1H), 5.10 (d, J = 4.4 Hz, 1H), 4.833-4.79 (m, 1H), 4.59 (d, J = 2.0 Hz, 2H), 4.26 (q, J = 14.4 Hz, 14.8 Hz, 1H), 4.13 (q, J = 14.0 Hz, 14.0 Hz, 1H), 3.21 (t, J = 2.4 Hz, 1H), 2.72-2.65 (m, 1H), 1.96-1.90 (m, 1H)

[0245] Process 6 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-2,2,2-trifluoro-1-hydroxyethyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 11 A solution of (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (24B) (80 mg, 0.155 mmol) in MeOH (20 mL) was added to KCO (21 mg, 0.155 mmol) at 0 °C, and the reaction mixture was stirred at RT for 16 h. Methanol was removed under reduced pressure at 30 °C, and the resulting residue was directly purified by reverse-phase GRACE flash chromatography using 10 mM ammonium bicarbonate in HO as eluent. Lyophilization of the purified fractions afforded 30 mg (81%) of 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-2,2,2-trifluoro-1-hydroxyethyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 11, as a white solid. 14 H 14 F3N5O5: ES+, m / z 390.1 [M+H] + . 1 H NMR (400MHz, DMSO-d6): δ 11.02 (brs, 1H), 6.57 (brs, 2H), 6.30 (d, J = 8.4 Hz, 1H), 5.57-5.54 (m, 2H), 4.76-4.72 (m, 1H), 4.60 (d, J = 2.4 Hz, 2H), 4.34-4.29 (m, 1H), 4.05-4.00 (m, 1H), 3.24 (t, J = 2.4 Hz, 1H), 2.56-2.52 (m, 1H), 1.97-1.91 (m, 1H)

[0246] Example 10 Compounds 12 and 13

[0247] 2-Amino-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((R)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, Compound 12 [ka]

[0248] 2-Amino-9-((2R,3S,4R,5S)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 13 [ka]

[0249] Compounds 12 and 13 were prepared in the following multiple steps.

[0250] The synthesis of (27S) was carried out according to the method described in US9708607B2. [ka]

[0251] Process 1 (3aR,5R,6aS)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyldihydrofuro[2,3-d][1,3]dioxol-6(5H)-one (23S) To a stirring solution of (3aR,5S,6S,6aR)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-6-ol [582-52-5] (2 g, 7.68 mmol) and AcO (2 mL) in CHCl (20 mL) was added pyridinium dichromate (3.5 g, 9.30 mmol). The reaction mixture was stirred at room temperature for 16 h. At this point, the reaction mixture was concentrated under reduced pressure, and EtOAc (3 × 100 mL) was added with stirring. The remaining mixture was filtered through silica gel and concentrated under reduced pressure. The crude product was used in the next step without further purification to give (1.1 g, 55.44%) of (3aR,5R,6aS)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyldihydrofuro[2,3-d][1,3]dioxol-6(5H)-one (23S) [2847-00-9] as an off-white solid. 12 H 18 O 6: 1 H NMR (400MHz, CDCl3): δ 6.14 (d, J = 4.8 Hz, 1H), 4.39-4.35 (m, 3H), 4.02 (d, J = 0.8 Hz, 1H), 2.10 (s, 1H), 1.49 (s, 3H), 1.36 (s, 3H), 1.34 (s, 6H)

[0252] Process 2 (3aR,5S,6R,6aR)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-6-ol (24S) To a solution of (3aR,5R,6aS)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyldihydrofuro[2,3-d][1,3]dioxol-6(5H)-one (23S) (1.9 g, 7.36 mmol) in EtOH:water (10 mL:3 mL) was added NaBH4 (292 mg, 8.83 mmol). The reaction mixture was stirred at room temperature for 16 h and concentrated under reduced pressure. To the residue was added EtOAc (3 x 100 mL) with stirring. The mixture was then filtered through silica gel and concentrated under reduced pressure. The crude product was used in the next step without further purification to give (1 g, 52.2%) of (3aR,5S,6R,6aR)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-6-ol (24S) [2595-05-3] as an off-white solid. 12 H 20 O 6: 1 H NMR (500MHz, CDCl3): δ 5.65 (d, J =3.5 Hz, 1H), 5.09 (d, J = 7.0 Hz, 1H), 4.46 (t, J = 4.0 Hz, 1H), 4.24-4.21 (m, 1H), 3.93 (t, J = 8.0 Hz, 1H), 3.85-3.73 (m, 3H), 1.44 (s, 3H), 1.32 (s, 3H), 1.27 (s, 3H), 1.26 (s, 3H)

[0253] Process 3 (3aR,5R,6S,6aS)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole (25S) To a solution of (3aR,5S,6R,6aR)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-6-ol (24S) (22 g, 84.61 mmol) in pyridine (10 mL, 126.92 mmol) and anhydrous CHCl (200 mL) was slowly added DAST (16.8 mL, 126.92 mmol). The reaction mixture was stirred at room temperature for 48 hours, cooled to 0 °C, and poured into cold saturated aqueous NaHCO (100 mL). The aqueous layer was extracted with CHCl (3 × 300 mL). The combined organic layers were dried over NaSO, filtered, and evaporated under reduced pressure. The crude residue was purified by column chromatography (silica gel; hexane:ethyl acetate 7:3) to give 15 g (67.7%) of (3aR,5R,6S,6aS)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole (25S) [14049-05-9] as a dark yellow mass. 12 H 19 FO 5: 1 H NMR (400MHz, CDCl3): δ 5.95 (d, J = 3.6 Hz, 1H), 5.07 (dd, J = 2.0 Hz, 1H), 4.71-4.68 (dd, J = 4 Hz, 1H), 4.31-4.26 (m, 1H), 4.16-4.10 (m, 3H), 1.50 (s, 3H), 1.45 (s, 3H), 1.36 (s, 3H), 1.32 (s, 3H)

[0254] Process 4 (R)-1-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethane-1,2-diol (26S) A solution of (3aR,5R,6S,6aS)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole (25S) (2.0 g, 7.66 mmol) in 60% AcOH / water (12 mL) was stirred at room temperature for 24 hours. The reaction mixture was concentrated under reduced pressure to give 1.6 g (94%) of (R)-1-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethane-1,2-diol (26S) [18530-81-9] as a colorless oil. The crude product was used without further purification. CH 15 FO 5: 1 H NMR (400MHz, DMSO-d6): δ 5.94 (d, J = 3.6 Hz, 1H), 5.01 (dd, J = 30.0, 2.0 Hz, 1H), 4.72 (dd, J = 10.8, 4.0 Hz, 1H), 4.99 (ddd, 1H, J =30.0, 9.2, 2.0 Hz), 3.60-3.54 (m, 2H), 3.40-3.30 (m, 1H), 1.90 (s, 2H), 1.39 (s, 3H), 1.26 (s, 3H)

[0255] Process 5 (3aR,5R,6S,6aS)-6-Fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole-5-carbaldehyde (27S) To a solution of (R)-1-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethane-1,2-diol (26S) (2 g, 9.0 mmol) in methanol (20 mL) cooled in an ice bath, sodium periodate (2.3 g, 10.81 mmol) was added. After stirring at room temperature for 16 hours, the resulting suspension was filtered. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (EtOAc) to give 1.4 g (81.53%) of (3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole-5-carbaldehyde (27S) [70722-99-5] as a colorless oil, CH 11 Obtained as FO4 and used in the next step without further purification.

[0256] Process 6 : 1-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-1-ol (28S) To a solution of (3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole-5-carbaldehyde (27S) (1 g, 5.26 mmol) in THF (20 mL) was added ethylmagnesium bromide (1 M in THF, 5.26 mL, 5.26 mmol) at -20 °C under argon. After stirring at room temperature for 16 h, the reaction mixture was quenched with saturated NH4Cl solution and extracted with EtOAc (3 × 50 mL). The combined organic layers were filtered and concentrated under reduced pressure. The residue was purified by column silica gel chromatography (eluting with 1:4 EtOAc in petroleum ether) to afford 800 mg (69.14%) of a diastereomeric mixture of 1-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-1-ol (28S) as a colorless oil, which was used without further purification.

[0257] Process 7: 1-((3aR,5R,6S,6aS)-6-Fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propyl acetate (29S)

[0258] To a stirring solution of 1-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-1-ol (28S) (1.2 g, 5.42 mmol), TEA (1.52 mL, 10.85 mmol), and DMAP (133 mg, 1.08 mmol) in anhydrous DCM (20 mL) was added acetic anhydride (830 mg, 8.14 mmol). The resulting reaction mixture was stirred at 22 °C for 18 h and then quenched by the addition of saturated aqueous NaHCO (20 mL). The organic layer was separated and the aqueous phase was extracted with DCM (2 × 50 mL). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure. The crude product was purified by column silica gel chromatography (100-200 mesh, 20% EtOAc in petroleum ether) to afford 700 mg (49%) of 1-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propyl acetate (29S) as a colorless oil (diastereomeric mixture). 12 H 19 FO 5: 1 H NMR (400MHz, CDC13): δ 5.96 (d, J = 3.6 Hz, 1H), 5.13-5.07 (m, 1H), 4.90 (dd, J = 50.0, 2.4 Hz, 1H), 4.68 (dd, J = 10.6, 4.0 Hz, 1H), 4.24-4.01 (m, 2H), 2.04 (s, 3H), 1.92-1.85 (m, 1H), 1.71-1.64 (m, 1H), 1.51 (s, 3H), 1.32 (s, 3H), 0.95 (t, J = 7.6 Hz, 3H)

[0259] Process 8(3S,4S,5R)-5-(1-acetoxypropyl)-4-fluorotetrahydrofuran-2,3-diyl diacetate (30S) To a stirred solution of 1-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propyl acetate (29S) (600 mg, 2.28 mmol), acetic acid (1.3 mL, 22.81 mmol), and acetic anhydride (1.08 mL, 11.46 mmol) in anhydrous DCM (10 mL) was added concentrated H2SO4 (0.10 mL) at 0 °C. After stirring at RT for 3 h, the reaction mixture was quenched by the addition of saturated aqueous NaHCO3 (10 mL). The organic layer was separated, and the aqueous phase was extracted with DCM (2 × 50 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column silica gel chromatography (100-200 mesh, 30% EtOAc in petroleum ether) to give a diastereomeric mixture (300 mg, 42.85%) of (3S,4S,5R)-5-(1-acetoxypropyl)-4-fluorotetrahydrofuran-2,3-diyl diacetate (30S) as a colorless oil. 13 H 19 FO7: 1 H NMR (400MHz, CDCl3): δ 6.13 (s, 1H), 5.30-5.15 (m, 1H), 5.12-5.04 (m, 1H), 4.37-4.29 (m, 2H), 4.44-4.34 (m, 1H), 2.10-2.08 (m, 9H), 1.88-1.69 (m, 1H), 1.68-1.59 (m, 1H), 0.95-0.88 (m, 3H)

[0260] Process 9(R)-1-((2R,3S,4S,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (25A) and (S)-1-((2R,3S,4S,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (25B)

[0261] To N-(6,8-dioxo-7-(prop-2-yn-1-yl)-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (6) (340 mg, 1.28 mmol), (3S,4S,5R)-5-(1-acetoxypropyl)-4-fluorotetrahydrofuran-2,3-diyl diacetate (30S) (504 mg, 1.66 mmol) dissolved in 1,2-dichloroethane (10 mL) was added bis(trimethylsilyl)acetamide (77 mg, 3.84 mmol). The reaction mixture was stirred at 80° C. for 30 min under argon and cooled to RT, after which the 1,2-dichloroethane was removed under reduced pressure. The residue was then dissolved in MeCN (20 mL), followed by the addition of TMSOTf (0.43 mL, 1.92 mmol). The reaction mixture was heated at 80° C. for 16 hours, cooled to room temperature, and concentrated under reduced pressure. The residue was diluted with aqueous NaHCO (50 mL) and extracted with EtOAc (3×50 mL). The combined EtOAc layers were washed with water (30 mL), brine (30 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The crude compound was purified by preparative HPLC using a KROMOSIL C18 column (150*25MM), 10μ mobile phase: 0.1% HC0H:MeOH in HO gradient: (T%B): -0 / 50, 1 / 50, 8 / 70, 10 / 70, 10.1 / 98, 13 / 98, 13.1 / 50, 15 / 50 flow rate: 20 mL / min; diluent: MeCN+HO+THF to give 50 mg of (R)-1-((2R,3S,4S,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (25A) (C 21 H 24 FN5O8: ES+, m / z 494.2 [M+H] + ) and 50 mg of (S)-1-((2R,3S,4S,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (25B)(C 21 H 24FN5O8: ES+, m / z 494.2 [M+H] + ), both as yellow solids.

[0262] Step 10 2-amino-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((R)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 12 To a solution of (R)-1-((2R,3S,4S,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (25A) (50 mg, 0.10 mmol) in MeOH (4 mL) was added KCO (14 mg, 0.10 mmol) at 0 °C. The reaction mixture was stirred at RT for 16 h. Methanol was removed under reduced pressure at 30 °C. The residue was directly purified by reverse-phase GRACE flash chromatography using 0.01% aqueous HCOH and MeCN as eluent. The purified fractions were lyophilized to give 20 mg (53.7%) of 2-amino-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((R)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 12, as a white solid. 15 H 18 FN5O 5: ES+, m / z 368.2 [M+H] + . 1H NMR (400MHz, DMSO-d6): δ 11.04 (brs, 1H), 6.63 (brs, 2H), 5.91 (d, J = 5.6 Hz, 1H), 5.33-5.32 (m, 1H), 5.28-5.25 (m, 1H), 5.00 (d, J = 3.6 Hz, 1H), 4.85 (d, J = 6.0 Hz,, 1H), 4.59 (d, J = 2.4 Hz, 2H), 3.71-3.60 (m, 2H), 3.24-3.23 (t, J = 2.4 Hz, 1H), 1.59-1.53 ​​(m, 1H), 1.32-1.25 (m, 1H), 0.88 (t, J = 7.2 Hz, 3H)

[0263] Step 11 2-amino-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 13 To a solution of (S)-1-((2R,3S,4S,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (25B) (50 mg, 0.11 mmol) in MeOH (4 mL) was added KCO (16 mg, 0.11 mmol) at 0 °C. The reaction mixture was stirred at RT for 16 h, after which the methanol was removed under reduced pressure at 30 °C. The residue was purified by reverse-phase GRACE flash chromatography using 0.01% aqueous HCOH and MeCN as eluent. The purified fractions were lyophilized to give (15 mg, 40.3%) 2-amino-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 13, as a white solid. 15 H 18 FN5O 5:ES+, m / z 368.1 [M+H] + . 1 H NMR (400MHz, DMSO-d6): δ 11.0 (brs, 1H), 6.58 (brs, 2H), 5.94 (d, J = 5.6 Hz, 1H), 5.36 (m, 1H), 5.29 (m, 1H), 4.97 (ddd, 1H, J = 53.7, 4.6, 2.2 Hz, 1H), 4.82 (d, J = 6.8 Hz, 1H), 4.61(d, J = 2.4 Hz, 2H), 3.80 (ddd, J = 23.6, 7.0, 4.6 Hz, 1H), 3.54 (m, 1H), 3.25 (t, J = 2.4 Hz, 1H), 1.49 (m, 1H), 1.33 (m, 1H), 0.92 (t, J = 7.4 Hz, 3H)

[0264] Alternative Preparation of Compound 13 Example 11 2-amino-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 13 [ka]

[0265] Compound 13 was also prepared by the following stereoselective multi-step procedure. [ka]

[0266] Process 1 (R)-2-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2-hydroxyethyl 4-methylbenzenesulfonate (31S) To a solution of (R)-1-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethane-1,2-diol (26S) (4 g, 18.01 mmol) in dry pyridine (40 mL) was added p-toluenesulfonyl chloride (4.79 g, 25.22 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 12 h, and then the pyridine was removed under reduced pressure. The residue was diluted with water (100 mL) and extracted with EtOAc (2 × 200 mL). The combined EtOAc layers were dried over NaSO and concentrated. The residue was purified by column silica gel chromatography (100-200 mesh, eluted with 25% EtOAc in petroleum ether) to give (2 g, 29.5%) of (R)-2-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2-hydroxyethyl 4-methylbenzenesulfonate (31S) as a light yellow oil. 16 H 21 FO7S: ES+, m / z 394.2 [M+H2O] + . 1 H NMR (400MHz, CDC13): δ 7.81 (d, J = 6.4 Hz, 2H), 7.37(d, J = 8.00 Hz, 2H), 5.90 (d, J = 3.6 Hz, 1H), 5.13 (dd, J = 2.4 Hz, 1H), 4.69 (dd, J = 3.6 Hz, 1H), 4.32 (d, J = 7.2 Hz, 1H), 4.13-4.04 (m, 3H), 2.47 (d, J = 4.8 Hz, 1H), 2.45 (s, 3H), 1.46 (s, 3 H), 1.31 (s, 3 H)

[0267] Process 2 (3aR,5R,6S,6aS)-6-Fluoro-2,2-dimethyl-5-((R)-oxiran-2-yl)tetrahydrofuro[2,3-d][1,3]dioxole (32S) To a solution of (R)-2-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2-hydroxyethyl 4-methylbenzenesulfonate (31S) (2 g, 5.31 mmol) in anhydrous THF (10 mL) cooled to −78 °C under a N atmosphere was added potassium bis(trimethylsilyl)amide (6.43 mL, 6.43 mmol, 1 M in THF). After stirring at −78 °C for 1 h, the reaction mixture was poured into saturated NH4Cl solution. The organic layer was separated, and the aqueous phase was extracted with EtOAc (2 × 100 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (100-200 mesh; 25% EtOAc in petroleum ether) to give (900 mg, 82.9%) of (3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyl-5-((R)-oxiran-2-yl)tetrahydrofuro[2,3-d][1,3]dioxole (32) as a light yellow oil. 13 FO4: 1 H NMR: (400MHz, CDC13): δ 6.00 (d, J = 3.6 Hz, 1H), 5.06 (dd, J = 50.0, 2.0 Hz, 1H), 4.73 (dd, J = 10.8, 3.6 Hz, 1H), 3.80 (ddd, J = 28.6, 6.8, 2.2 Hz, 1H), 3.22 (m, 1H), 2.93 (dd, J = 5.0, 3.6 Hz, 1H), 2.80 (dd, J = 5.0, 2.6 Hz, 1H), 1.45 (s, 3H), 1.33 (s, 3H)

[0268] Process 3 (R)-1-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-1-ol (33S) To a stirred suspension of CuI (186 mg, 0.980 mmol) in dry THF (20 mL) was added methylmagnesium bromide (1.0 M in diethyl ether, 14.7 mL, 14.70 mmol) at −78 °C under a N atmosphere. After stirring at −78 °C for 1 h, a solution of (3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyl-5-((R)-oxiran-2-yl)tetrahydrofuro[2,3-d][1,3]dioxole (32S) (1.0 g, 4.90 mmol) in THF (10 mL) was added and stirred at −78 °C for an additional 2 h. The reaction mixture was poured into saturated NHCl solution (200 mL), the organic layer was separated, and the aqueous phase was extracted with EtOAc (2 × 200 mL). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure. The crude product was purified by column silica gel chromatography (100-200 mesh, 30% EtOAc in petroleum ether) to give 1.0 g (92.3%) of (R)-1-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-1-ol (33S) as a light yellow oil. 10 H 17 FO4: 1 H NMR (400MHz, CDC13): δ 5.98 (d, J = 3.6 Hz, 1H), 5.08 (dd, J = 50.0, 2.4 Hz, 1H), 4.69 (dd, J = 11.2, 3.6 Hz, 1H), 4.01 (dd, J = 29.8, 2.4 Hz, 1H), 3.85 (m, 1H), 1.86-1.80 (m, 1H), 1.75 (d, J = 6.0 Hz, 1H), 1.52 (s, 3H), 1.32 (s, 3H), 1.05 (t, J = 7.2 Hz, 3H)

[0269] Process 4 (S)-1-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propyl 4-nitrobenzoate (34S) To a stirred solution of (R)-1-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-1-ol (33S) (1.0 g, 4.52 mmol), triphenylphosphine (2.3 g, 9.04 mmol), and 4-nitrobenzoic acid (1.51 g, 9.04 mmol) in THF (240 mL) was added diethyl azodicarboxylate (1.57 mL, 9.04 mmol) dropwise at 0 °C under a N atmosphere. After stirring at RT for 10 h, the reaction mixture was quenched with saturated NaHCO solution (50 mL) and extracted with EtOAc (2 × 50 mL). The organic layers were combined, dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column silica gel chromatography (100-200 mesh, 15% EtOAc in petroleum ether) to give (650 mg, 38.9%) of (S)-1-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propyl 4-nitrobenzoate (34S) as a light yellow solid. 17 H 20 FNO7: ES+, m / z 370.2 [M+H] + . 1 H NMR (400MHz, CDC13): δ 8.30-8.21 (m, 4H), 6.00 (d, J = 3.6 Hz, 1H), 5.51 (m, 1H), 4.97 (dd, J = 50.0, 2.4 Hz, 1H), 4.73 (dd, J = 11.4, 3.6 Hz, 1H), 4.35 (ddd, J = 29.2, 8.2, 2.4 Hz, 1H), 1.89-1.80 (m, 2H), 1.51 (s, 3H), 1.33 (s, 3H), 1.03 (t, J = 7.2 Hz, 3H)

[0270] Process 5 (S)-1-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-1-ol (35S) To a stirred solution of (S)-1-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propyl 4-nitrobenzoate (34S) (650 mg, 1.76 mmol) in methanol (10 mL) was added K2CO3 (486 mg, 3.52 mmol). After stirring at room temperature for 3 hours, the resulting reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude material was purified by column silica gel chromatography (100-200 mesh, 30% EtOAc in petroleum ether) to give (S)-1-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-1-ol (35S) (389 mg, 100%) as a light yellow oil. 10 H 17 FO4: 1 H NMR (500MHz, CDC13): δ 6.00 (d, J = 4.0 Hz, 1H), 4.90 (dd, J = 50.0, 2.4 Hz, 1H), 4.71 (dd, J = 11.2, 3.6 Hz, 1H), 4.05 (ddd, J = 30.4, 8.0, 2.4 Hz, 1H), 3.85 (m, 1H), 2.17 (d, J = 1.6 Hz, 1H), 1.61-1.56 (m, 1H), 1.49 (s, 3H), 1.33 (s, 3H), 1.06 (t, J = 7.6 Hz, 3H)

[0271] Process 6 (S)-1-((3aR,5R,6S,6aS)-6-Fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propyl acetate (36S) To a stirred solution of (S)-1-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-1-ol (35S) (400 mg, 1.80 mmol), TEA (0.51 mL, 3.61 mmol), and DMAP (44 mg, 0.36 mmol) in anhydrous CHCl (10 mL) was added acetic anhydride (277 mg, 2.71 mmol). After stirring at 25 °C for 10 h, the reaction mixture was quenched with saturated aqueous NaHCO (20 mL). The organic layer was separated, and the aqueous phase was extracted with CHCl (2 × 50 mL). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure. The crude product was purified by column silica gel chromatography (100-200 mesh, 20% EtOAc in petroleum ether) to give (400 mg, 84%) of (S)-1-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propyl acetate (36S) as a colorless oil. 12 H 19 FO 5: 1 H NMR (500MHz, CDC13): δ 5.98 (d, J = 3.5 Hz, 1H), 5.23 (m, 1H), 4.89 (dd, J = 49.8, 2.5 Hz, 1H), 4.69 (dd, J = 11.5, 4.0 Hz, 1H), 4.19 (ddd, J = 29.8, 8.0, 2.5 Hz, 1H), 2.10 (s, 3H), 1.74 (m, 1H), 1.63 (m, 1H), 1.50 (s, 3H), 1.32 (s, 3H), 0.96 (t, J = 4.0 Hz, 3H)

[0272] Process 7 (3S,4S,5R)-5-((S)-1-acetoxypropyl)-4-fluorotetrahydrofuran-2,3-diyl diacetate (37S) To a solution of (S)-1-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propyl acetate (36S) (400 mg, 1.52 mmol), acetic acid (0.86 mL, 15.2 mmol), and acetic anhydride (0.72 mL, 7.60 mmol) in anhydrous CHCl (10 mL) was added concentrated HSO (0.010 mL) at 0 °C. The reaction mixture was stirred at RT for 3 h and then quenched by the addition of saturated aqueous NaHCO (10 mL). The organic layer was separated, and the aqueous phase was extracted with CHCl (2 × 50 mL). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure. The crude product was purified by column silica gel chromatography (100-200 mesh, 30% EtOAc in petroleum ether) to give (200 mg, 42.8%) of (3S,4S,5R)-5-((S)-1-acetoxypropyl)-4-fluorotetrahydrofuran-2,3-diyl diacetate (37S) as a colorless oil. 13 H 19 FO7: ( 1 2.4:1 mixture of α,β anomers by H NMR); 1 H NMR (400MHz, CDC13): δ 6.47 (d, J = 4.8 Hz, 0.294 H), 6.12 (s, 0.706 H), 5.37-4.94 (m, 3H), 4.35-4.25 (m, 1H), 2.12-2.08 (m, 9H), 1.74-1.57 (m, 2H), 0.96 (t, J = 7.2 Hz, 3H)

[0273] Process 8 (S)-1-((2R,3S,4S,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (25B) Using the method described in Example 11, Step 9, and substituting (3S,4S,5R)-5-(1-acetoxy-2,2,2-trifluoroethyl)tetrahydrofuran-2,3-diyl diacetate (37S) for (3R,5S)-5-((S)-1-acetoxypropyl)-4-fluorotetrahydrofuran-2,3-diyl diacetate (22S), (S)-1-((2R,3S,4S,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (25B) in 32% yield (0.1% HCOH in water and acetonitrile) after reverse-phase GRACE purification. 21 H 24 FN5O 8: ES+, m / z 494.2 [M+H] + . 1 H NMR (400MHz, CDC13): δ 11.95 (brs, 1H), 9.30 (s, 1H), 5.84 (m, 2H), 5.72 (m, 1H), 5.05 (ddd, J = 50.5, 3.4, 1.2 Hz, 1H), 4.84 (m, 2H), 4.24 (ddd, J = 28.7, 8.9, 3.4 Hz, 1H), 2.30 (s, 3H), 2.28 (m, 1H), 2.14 (s, 3H), 2.13 (s, 3H), 1.78 (m, 1H), 1.65 (m, 1H), 0.98 (t, J = 7.4, 3H)

[0274] Process 9 2-amino-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 13 Using the method described in Example 11, Step 11, 2-amino-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 13, was obtained in 31% yield as a white solid. 15 H 18 FN5O 5: ES+, m / z 368.3 [M+H] + . 1 H NMR (500MHz, DMSO-d6): δ 11.16 (brs, 1H), 6.65 (brs, 2H), 5.95 (bs, 1H), 5.36 (m, 1H), 5.33 (m, 1H), 4.97 (ddd, 1H, J = 53.3, 4.5, 2.5 Hz, 1H), 4.84 (d, J = 6.5 Hz, 1H), 4.61(d, J = 2.5 Hz, 2H), 3.80 (ddd, J = 23.5, 6.8, 4.8 Hz, 1H), 3.54 (m, 1H), 3.25 (t, J = 2.5 Hz, 1H), 1.49 (m, 1H), 1.34 (m, 1H), 0.91 (t, J = 7.3 Hz, 3H)

[0275] Example 12 2-amino-9-((2R,3R,5S)-5-((S)-1,2-dihydroxyethyl)-3-hydroxytetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 14 [ka]

[0276] Compound 14 was prepared in the following multiple steps. [ka]

[0277] Process 1(R)-2-(benzyloxy)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (38S) To a suspension of NaH (60% dispersion in mineral oil; 3.4 g, 86.2 mmol) in 1,2-dimethoxyethane (40 mL) was added benzyl alcohol (31.2 mL) at 0 °C. Subsequently, a solution of (3aR,5S,6aR)-2,2-dimethyl-5-((R)-oxiran-2-yl)tetrahydrofuro[2,3-d][1,3]dioxole (9S) (8 g, 43.0 mmol) in 1,2-dimethoxyethane (40 mL) was added under a N atmosphere at 0 °C. The reaction mixture was stirred at 60 °C for 4 h, then quenched with saturated NHCl solution (100 mL) and extracted with CHCl (2 × 300 mL). The combined organic layers were washed with water (100 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The crude mixture was purified by column silica gel chromatography (100-200 mesh, 60% EtOAc in petroleum ether) to give 10.0 g (79%) of (R)-2-(benzyloxy)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (38S) as a colorless liquid. 16 H 22 O 5: 1 H NMR (400MHz, CDC13): δ 7.32 (m, 5H), 5.80 (d, J = 3.6 Hz, 1H), 4.74 (t, J = 4.0 Hz, 1H), 4.56 (q, J a,b = 12.2 Hz, 2H), 4.23 (dt, J = 10.8, 5.0 Hz, 1H), 4.00 (m, 1H), 3.60 (dd, J = 9.6, 3.6 Hz, 1H), 3.48 (dd, J = 10.0, 6.8 Hz, 1H), 2.37 (d, J = 3.6 Hz, 1H), 2.07 (dd, J = 13.4, 4.8 Hz, 1H), 1.85 (m, 1H), 1.57 (s, 3H), 1.27 (s, 3H)

[0278] Process 2 (S)-2-(benzyloxy)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethyl 4-nitrobenzoate (39S) To a stirred solution of (R)-2-(benzyloxy)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (38S) (10.0 g, 32.4 mmol), triphenylphosphine (10.2 g, 38.9 mmol), and 4-nitrobenzoic acid (5.9 g, 35.7 mmol) in THF (100 mL) was added diethyl azodicarboxylate (6.1 mL, 38.9 mmol) dropwise at 0 °C under a N atmosphere. The reaction mixture was stirred at 18 °C for 10 h. The reaction mixture was then quenched with saturated NaHCO solution (100 mL) and extracted with EtOAc (2 × 300 mL). The organic layers were combined, dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column silica gel chromatography (100-200 mesh, 30% EtOAc in petroleum ether) to give 11.5 g (76%) of (S)-2-(benzyloxy)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethyl 4-nitrobenzoate (39S) as a light yellow liquid. 23 H 25 NO 8: 1 H NMR (400MHz, CDC13): δ 8.26 (m, 4H), 7.28 (m, 5H), 5.82 (d, J = 3.6 Hz, 1H), 5.40 (m, 1H), 4.72 (t, J = 4.2 Hz, 1H), 4.61-4.52 (m, 3H), 3.77 (d, J = 5.2 Hz, 2H), 2.12(dd, J = 13.2, 4.4 Hz, 1H), 1.69-1.66 (m, 1H), 1.53 (s, 3H), 1.32 (s, 3H)

[0279] Process 3(S)-2-(benzyloxy)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (40S) To a stirred solution of (S)-2-(benzyloxy)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethyl 4-nitrobenzoate (39S) (11.5 g, 25.9 mmol) in methanol (100 mL) was added K2CO3 (4.1 g, 51.9 mmol). After stirring at room temperature for 30 minutes, the resulting reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column silica gel chromatography (100-200 mesh, 30% EtOAc in petroleum ether) to give 7.0 g (92%) (S)-2-(benzyloxy)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (40S). 16 H 22 O 5: 1 H NMR (500MHz, CDC13): δ 7.33 (m, 5H), 5.81 (d, J = 3.5 Hz, 1H), 4.73 (t, J = 4.3 Hz, 1H), 4.57 (s, 2H), 4.29 (dt, J = 10.5, 4.3 Hz, 1H), 3.77 (m, 1H), 3.57 (m, 2H), 2.37 (d, J = 5.0 Hz, 1H), 2.02 (dd, J = 13.5, 4.5 Hz, 1H), 1.90 (dddd, J = 15.5, 9.8, 5.0, 2.5 Hz, 1H), 1.51 (s, 3H), 1.32 (s, 3H)

[0280] Process 4 (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethane-1,2-diol (41S) To a stirred solution of (S)-2-(benzyloxy)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (40S) (5.5 g, 18.7 mmol) in methanol (50 mL) was added 10% Pd / C (2.5 g, 50 mol%). The reaction mixture was stirred in a Parr shaker at rt under 70 psi of H2 for 16 h. The reaction mixture was then filtered through a pad of Celite. The Celite pad was washed with additional methanol, and the combined filtrates were concentrated under reduced pressure. The crude mixture was purified by column silica gel chromatography (100-200 mesh, 10% MeOH in CHCl) to give 3.0 g (78%) of (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethyl acetate (41S) as a colorless oil. 16 O 5: 1 H NMR (400MHz, CDC13): δ 5.82 (d, J = 3.6 Hz, 1H), 4.75 (t, J = 4.2 Hz, 1H), 4.29 (dt, J = 10.4, 4.2 Hz, 1H), 3.74 (d, J = 5.2 Hz, 2H), 3.64 (brs, 1H), 2.50 (s, 1H), 2.27 (s, 1H), 2.06 (dd, J = 13.4, 4.6 Hz, 1H), 1.93 (dddd, J = 15.4, 9.7, 4.6, 2.8 Hz, 1H), 1.52 (s, 3H), 1.32 (m, 3H)

[0281] Process 5 (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethane-1,2-diyl diacetate (42S) To a stirred solution of (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethyl acetate (41S) (2.0 g, 9.8 mmol), TEA (5.4 mL, 39.2 mmol), and DMAP (239.2 mg, 1.9 mmol) in anhydrous CHCl (20 mL) cooled to 0 °C was added acetic anhydride (2.9 mL, 29.4 mmol). The reaction mixture was stirred at 25 °C for 16 h and quenched with saturated aqueous NaHCO (30 mL). The organic layer was separated, and the aqueous phase was extracted with CHCl (2 × 100 mL). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure. The crude material was purified by column silica gel chromatography (100-200 mesh, 20% EtOAc in petroleum ether) to give 2.0 g (71%) of (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethane-1,2-diyl diacetate (42S) as a colorless oil. 13 H 20 O7: 1 H NMR (500MHz, CDC13): δ 5.81 (d, J = 3.5 Hz, 1H), 5.18 (dt, J = 8.0, 3.8 Hz, 1H), 4.73 (t, J = 4.3 Hz, 1H), 4.38 (m, 2H), 4.15 (dd, J = 12.0, 7.5 Hz, 1H), 2.12 (s, 3H), 2.08 (m, 1H), 2.05 (s, 3H), 1.65 (m, 1H), 1.52 (s, 3H), 1.34 (s, 3H)

[0282] Process 6 (2S,3R,5S)-5-((S)-1,2-diacetoxyethyl)tetrahydrofuran-2,3-diyl diacetate (43S) To a solution of (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethane-1,2-diyl diacetate (42S) (1.3 g, 4.5 mmol), acetic acid (2.58 mL, 45.1 mmol), and acetic anhydride (2.2 mL, 22.5 mmol) in anhydrous CHCl (20 mL) was added concentrated HSO (0.04 mL) at 0 °C. After stirring at 22 °C for 4 h, the reaction mixture was quenched by the addition of saturated aqueous NaHCO (100 mL). The organic layer was separated, and the aqueous phase was extracted with CHCl (2 × 100 mL). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure. The crude residue was purified by column silica gel chromatography (100-200 mesh, 30% EtOAc in petroleum ether) to afford 1.0 g (67%) of (2S,3R,5S)-5-((S)-1,2-diacetoxyethyl)tetrahydrofuran-2,3-diyl diacetate (43S) as a colorless oil. 14 H 20 O 9: 1 H NMR (500MHz, CDC13): δ 6.12 (s, 1H), 5.19 (d, J = 3.5 Hz, 1H), 5.13 (m, 1H), 4.50 (m, 1H), 4.32 (dd, J = 12.0, 4.0 Hz, 1H), 4.10 (dd, J = 12.0, 6.5 Hz, 1H), 2.10-2.06 (m, 14H)

[0283] Process 7 (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)ethane-1,2-diyl diacetate (26) N-(6,8-dioxo-7-(prop-2-yn-1-yl)-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (6) (300 mg, 1.2 mmol), (2S,3R,5S)-5-((S)-1,2-diacetoxyethyl)tetrahydrofuran-2,3-diyl diacetate (43S) (483.8 mg, 1.4 mmol), and BSA (0.92 mL, 3.6 mmol) were dissolved in 1,2-dichloroethane (20 mL), and the resulting reaction mixture was stirred at 80° C. for 30 minutes under argon. The reaction mixture was warmed to RT, and the 1,2-dichloroethane was removed under reduced pressure. The residue was dissolved in MeCN (20 mL), followed by the addition of TMSOTf (0.33 mL, 1.8 mmol). The reaction mixture was stirred and heated at 80 °C for 16 h, cooled to room temperature, and concentrated under reduced pressure. The resulting residue was diluted with aqueous NaHCO (50 mL) and extracted with EtOAc (3 × 50 mL). The combined EtOAc layers were washed with water (30 mL), brine (30 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The crude product was purified by column silica gel chromatography (100–200 mesh, 80% EtOAc in petroleum ether) to afford 300 mg (47%) of (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)ethane-1,2-diyl diacetate (26) as an off-white solid. 22 H 25 N5O 10: ES+, m / z 520.2 [M+H] +

[0284] Process 8 2-amino-9-((2R,3R,5S)-5-((S)-1,2-dihydroxyethyl)-3-hydroxytetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 14 To a solution of (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)ethane-1,2-diyl diacetate (26) (300 mg, 0.57 mmol) in MeOH (10 mL) was added KCO (119.6 mg, 0.86 mmol) at 0 °C. The reaction mixture was stirred at RT for 16 h, and then the methanol was removed under reduced pressure at RT. The resulting residue was purified using a preparative HPLC column: X-SELECT-C18 (150*19), 5μ mobile phase: 10 mM NH4HCO3:MeCN in HO gradient: (T%B): -0 / 2, 3 / 2, 8 / 20, 10 / 40, 10 / .1 / 98, 12 / 98, 13.1 / 2, 16 / 2; flow rate: 20 mL / min. The purified fraction was lyophilized to give 35 mg (17%) of 2-amino-9-((2R,3R,5S)-5-((S)-1,2-dihydroxyethyl)-3-hydroxytetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 14, as an off-white solid. 14 H 17 N5O 6: ES+, m / z 352.2 [M+H] + . 1 H NMR (400MHz, DMSO-d6): δ 11.76 (brs, 1H), 6.71(s, 2H), 5.50 (d, J = 3.6 Hz 1H), 5.38 (d, J = 4.4 Hz, 1H), 4.90 (bs, 1H), 4.75 (m, 1H), 4.60 (s, 2H), 4.47 (m, 1H), 4.17 (m, 1H), 3.37-3.32 (m, 3H), 3.20 (t, J = 2.2 Hz, 1H), 2.40 (m, 1H), 1.85 (m, 1H)

[0285] Example 132-amino-9-((2R,3R,5S)-5-((S)-2-amino-1-hydroxyethyl)-3-hydroxytetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 15 [ka]

[0286] Compound 15 was prepared in the following multiple steps. [ka]

[0287] Process 1 (R)-2-Azido-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (44S) To a solution of (3aR,5S,6aR)-2,2-dimethyl-5-((R)-oxiran-2-yl)tetrahydrofuro[2,3-d][1,3]dioxole (9S) (4.0 g, 21.71 mmol) in DMF (40 mL) were added TMSN3 (8 mL, 2 vol) and methanol (8 mL, 2 vol) at 60 °C. The reaction was monitored by TLC. After stirring at 60 °C for 48 h, the reaction mixture was poured into 400 mL of ice water and extracted with EtOAc (2 × 300 mL). The solution was further washed with saturated brine solution (2 x 100 mL), and the organic layer was separated, dried over anhydrous Na2SO4, filtered, and concentrated to give crude (R)-2-azido-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (44S) (ca. 4.0 g crude, 80%) as a liquid. 15 N3O4: The crude product was used directly in the next step. 1H NMR (400MHz, CDCl3): δ 5.80 (d, J = 3.2 Hz, 1H), 4.77 (t, J = 4.0 Hz, 1H), 4.19 (dt, J = 10.3, 4.6 Hz, 1H), 3.99 (m, 1H), 3.37 (m, 2H), 2.30 (m, 1H), 2.08 (dd, J = 13.2, 4.6 Hz, 1H), 1.86 (ddd, J = 15.4, 9.7, 4.8 Hz, 1H), 1.55 (s, 3H), 1.33 (s, 3H)

[0288] Process 2 (S)-2-Azido-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethyl 4-nitrobenzoate (45S) To a solution of (R)-2-azido-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (44S) (4 g, 17.44 mmol) in THF (60 mL) was added 4-nitrobenzoic acid (5.8 g, 34.8 mmol), triphenylphosphine (9.1 g, 34.89 mmol), and DEAD (5.4 mL, 34.8 mmol) at 0 °C. The reaction mixture was stirred at RT and monitored by TLC. After 16 h, the reaction mixture was poured into 200 mL of water and extracted with EtOAc (2 × 200 mL). The organic layer was further washed with saturated brine solution (2 × 60 mL), dried over anhydrous NaSO, filtered, and concentrated to give the crude product. The crude product was purified by column silica gel chromatography (100-200 mesh, 30% EtOAc in petroleum ether) to give (S)-2-azido-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethyl 4-nitrobenzoate (45S) (4.0 g, 60%) as a semi-solid. 16 H 18 N4O7: ES-, m / z 377.1 [MH] - . 1H NMR (400MHz, CDCl3): δ 8.33-8.24 (m, 4H), 5.84 (d, J = 3.6 Hz, 1H), 5.37 (m, 1H), 4.75 (t, J = 4.2 Hz, 1H), 4.51 (dt, J = 10.7, 4.6 Hz, 1H), 3.67 (d, J = 4.8 Hz, 2H), 2.16 (dd, J = 13.2, 4.8 Hz, 1H), 1.66(ddd, J = 15.6, 9.6, 3.6 Hz 1H), 1.55 (s, 3H), 1.33 (s, 3H)

[0289] Process 3 (S)-2-Azido-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (46S) To a solution of (S)-2-azido-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethyl 4-nitrobenzoate (45S) (4.0 g, 10.572 mmol) in methanol (40 mL, 10 volumes) was added K2CO3 (2.9 g, 21.1 mmol) at rt. The progress of the reaction was monitored by TLC. After stirring at rt for 1 h, the reaction mixture was filtered through a plug of silica gel, and the filtrate was concentrated under reduced pressure to give (S)-2-azido-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (46S) (2.2 g, 83%) as a liquid, which was used without further purification. CH 15 N3O4: 1 H NMR (400MHz, CDCl3): δ 5.81 (m, 1H), 4.75 (t, J = 3.8 Hz, 1H), 4.24-4.13 (m, 2H), 3.85 (m, 1H), 3.40 (m, 1H), 2.02 (m, 1H), 1.88 (m, 1H), 1.74 (m, 1H), 1.53 (s, 3H), 1.36 (s, 3H)

[0290] Process 4(S)-2-Azido-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethyl acetate (47S) To a stirred solution of (S)-2-azido-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (46S) (2.2 g, 9.59 mmol), TEA (2.67 mL, 19.19 mmol), and DMAP (0.23 g, 1.91 mmol) in anhydrous dichloromethane (22 mL) was added acetic anhydride (1.37 mL, 14.39 mmol). After stirring at 25 °C for 10 h, the reaction was quenched with saturated aqueous NaHCO (50 mL). The organic layer was separated and the aqueous phase was extracted with DCM (2 × 50 mL). The combined organic layers were dried over NaSO and concentrated under reduced pressure. The crude product was purified by column silica gel chromatography (100-200 mesh, eluted with 20% EtOAc in petroleum ether) to give (S)-2-azido-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethyl acetate (47S) (2 g, 86%) as a colorless oil. 11 H 17 N3O 5: 1 H NMR (500MHz, CDCl3): δ 5.81 (d, J = 4.0 Hz, 1H), 5.09 (q, J = 2.3 Hz, 1H), 4.74 (t, J = 4.5 Hz, 1H), 4.36 (dt, J= 6.0, 4.5 Hz, 1H), 4.27 (m, 1H), 3.50 (dd, J = 6.5, 4.0 Hz, 2H), 2.15 (s, 3H), 1.64-1.58 (m, 1H), 1.57 (s, 3H), 1.32 (s, 3H)

[0291] Process 5 (2S,3R,5S)-5-((S)-1-acetoxy-2-azidoethyl)tetrahydrofuran-2,3-diyl diacetate (48S) To a solution of (S)-2-azido-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethyl acetate (47S) (2.0 g, 7.3 mmol), acetic acid (4.21 mL, 73.72 mmol), and acetic anhydride (3.48 mL, 36.86 mmol) in anhydrous CHCl (40 mL) was added concentrated HSO (0.1 mL) at 0 °C. After stirring at 25 °C for 3 h, the reaction mixture was quenched by the addition of saturated aqueous NaHCO (100 mL). The organic layer was separated, and the aqueous phase was extracted with CHCl (2 × 100 mL). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure. The crude oil was purified by column silica gel chromatography (100-200 mesh, 30% EtOAc in petroleum ether) to give (2S,3R,5S)-5-((S)-1-acetoxy-2-azidoethyl)tetrahydrofuran-2,3-diyl diacetate (48S) (1.1 g, 45%) as a colorless oil. 12 H 17 N3O7: 1 H NMR (500MHz, CDCl3): δ 6.12 (s, 1H), 5.18 (d, J = 5.0 Hz, 1H), 5.04 (m, 1H), 4.52 (m, 1H), 3.46 (m, 2H), 2.15 (m, 2H), 2.09-2.07 (s, 9 H)

[0292] Process 6 (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2-azidoethyl acetate (27) To a suspension of N-(6,8-dioxo-7-(prop-2-yn-1-yl)-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (6) (700 mg, 2.83 mmol) and (2S,3R,5S)-5-((S)-1-acetoxy-2-azidoethyl)tetrahydrofuran-2,3-diyl diacetate (48S) (1.07 g, 3.40 mmol) in acetonitrile (30 mL) was added BSA (2.15 mL, 8.49 mmol). The reaction mixture was stirred at 70 °C for 1 h under argon to form a clear solution. TMSOTf (0.78 mL, 4.24 mmol) was added at 0 °C. After heating at 70 °C with stirring for 16 h, the reaction was quenched with water (60 mL) and extracted with EA (2 × 70 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified on a silica gel column (80% EtOAc in petroleum ether) to give (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2-azidoethyl acetate (27) (0.50 g, 40%) as an off-white solid. 20 H 22 N8O 8: ES+, m / z 503.3 [M+H] +

[0293] Process 7 2-Amino-9-((2R,3R,5S)-5-((S)-2-azido-1-hydroxyethyl)-3-hydroxytetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione (28) To a solution of (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2-azidoethyl acetate (27) (500 mg, 0.99 mmol) in methanol (30 mL) was added KCO (138 mg, 0.99 mmol) at rt. The reaction mixture was stirred at room temperature and monitored by LC / MS. After 16 h, the starting material was consumed. The reaction mixture was concentrated under reduced pressure to give crude 2-amino-9-((2R,3R,5S)-5-((S)-2-azido-1-hydroxyethyl)-3-hydroxytetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione (28) (370 mg, 55%) as a crude solid material, which was used in the next step without purification. 14 H 16 N8O5: ES+, m / z 377.3 [M+H] +

[0294] Process 8 2-2-amino-9-((2R,3R,5S)-5-((S)-2-amino-1-hydroxyethyl)-3-hydroxytetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 15 To a stirring solution of 2-amino-9-((2R,3R,5S)-5-((S)-2-azido-1-hydroxyethyl)-3-hydroxytetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione (28) (350 mg, 0.930 mmol) in THF (7 mL, 20 vol) at rt was added triphenylphosphine (487 mg, 1.86 mmol) and water (3.5 mL, 1 vol). The mixture was stirred at 60° C. The reaction was monitored by LCMS, and after 16 h the starting material was consumed. The reaction mixture was concentrated under reduced pressure to give a concentrated solid, which was subjected to GRACE reverse-phase chromatography (10 mmol NH4HCO3:MeCN) to give 2-amino-9-((2R,3R,5S)-5-((S)-2-amino-1-hydroxyethyl)-3-hydroxytetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione (compound 15) (30 mg, 20%) as an off-white solid. 14 H 18 N6O5: 1 H NMR (500MHz, DMSO-d6): δ 6.66 (bs, 2H), 5.50 (d, J = 3.0 Hz, 1H), 5.38 (d, J = 4.0 Hz, 1H), 4.95 (bs, 1H), 4.74 (d, J = 3.0 Hz, 1H), 4.64 (s, 2H), 4.09 (q, J = 6.7 Hz, 1H), 3.31 (s, 1H), 3.20 (t, J = 2.3 Hz, 1H), 2.59-2.54 (m, 1H), 2.46-2.37 (m, 2H), 1.91-1.71 (m, 1H). ES+, m / z 351.2 [M+H] +

[0295] Example 14 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxy-2-(methylamino)ethyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 16 [ka]

[0296] Compound 16 was prepared in the following multiple steps. [ka]

[0297] Process 1 (R)-2-((tert-butyldimethylsilyl)oxy)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (49S) To a solution of (R)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethane-1,2-diol (3S) (10.0 g, 49.01 mmol) in DMF (100 mL) was added TBDMS-Cl (7.35 g, 49.01 mmol) and imidizole (4.29 g, 63.21 mmol) at 0 °C. The reaction mixture was stirred at 25 °C and monitored by TLC. After 8 h, the reaction mixture was poured into 600 mL of ice-water and extracted with EtOAc (2 × 500 mL). After further washing with brine solution (2 × 200 mL), the combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was filtered through a silica pad to give (R)-2-((tert-butyldimethylsilyl)oxy)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (49S) (10.0 g, 65%) as a semi-solid, which was used as is. 15 H 30 O5Si: 1H NMR (400MHz, CDCl3): δ 5.80 (d, J = 3.6 Hz, 1H), 4.74 (t, J = 4.0 Hz, 1H), 4.20 (m, 1H), 3.75 (m, 2H), 3.71-3.65 (m, 1H), 2.15 (m, 1H), 1.83 (m, 1H), 1.56-1.53 ​​(m, 1H), 1.51 (s, 3H), 1.36 (s, 3H), 0.91 (s, 9H), 0.08 (s, 6H)

[0298] Process 2 (R)-2-((tert-butyldimethylsilyl)oxy)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethyl methanesulfonate (50S) To a solution of (R)-2-((tert-butyldimethylsilyl)oxy)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol) (49S) (10.0 g, 31.446 mmol) in CHCl (100 mL) cooled to 0 °C, TEA (14.24 mL, 110.06 mmol) and mesyl chloride (3.63 mL, 47.16 mmol) were added. The stirred reaction mixture was warmed to rt and monitored by TLC. After 16 h, the reaction mixture was poured into 500 mL of water and extracted with CHCl (2 × 500 mL). The mixture was washed with brine solution (2 x 200 mL), and the organic layer was collected, dried over anhydrous Na2SO4, filtered, and concentrated to give crude (R)-2-((tert-butyldimethylsilyl)oxy)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethyl methanesulfonate (50S) (11 g, 80%) as a liquid, which was used as is. 16 H 32 O7SSi: 1H NMR (500MHz, CDCl3): δ 5.78 (d, J = 3.5 Hz, 1H), 4.76 (d, J = 4.0 Hz, 1H), 4.42 (m, 1H), 3.83 (d, J = 5.0 Hz, 2H), 3.07 (s, 3H), 2.19 (m, 1H), 1.93 (m, 1H), 1.51 (s, 3H), 1.45-1.33 (m, 1H), 1.32 (s, 3H), 0.90 (s, 9H), 0.08 (s, 6H)

[0299] Process 3 (3aR,5S,6aR)-2,2-dimethyl-5-((S)-oxiran-2-yl)tetrahydrofuro[2,3-d][1,3]dioxole (51S) To a solution of (R)-2-((tert-butyldimethylsilyl)oxy)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethyl methanesulfonate (50S) (11 g, 27.7 mmol) in THF (55 mL) was added 1 M TBAF in THF (83.3 mL, 83.3 mmol) at 0 °C. The reaction mixture was concentrated to give the crude product, which was subjected to column chromatography on silica gel (100-200 mesh, eluted with 25% EtOAc in petroleum ether) to give (3aR,5S,6aR)-2,2-dimethyl-5-((S)-oxiran-2-yl)tetrahydrofuro[2,3-d][1,3]dioxole[20720-51-8] (51S) (3.0 g, 57%) as a liquid. C9H 14 O4: 1H NMR (400MHz, CDCl3): δ 5.81 (d, J = 3.6 Hz, 1H), 4.75 (t, J = 4.2 Hz, 1H), 4.18 (dt, J = 10.8, 4.6 Hz, 1H), 3.04 (q, J = 3.6 Hz, 1H), 2.81 (d, J = 5.6 Hz, 2H), 2.16 (dd, J = 13.4, 4.6 Hz, 1H), 1.85 (ddd, J = 15.4, 9.8, 3.6 Hz, 1H), 1.50 (s, 3H), 1.36 (s, 3H)

[0300] Process 4 (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2-(methylamino)ethan-1-ol (52S) To stirring (3aR,5S,6aR)-2,2-dimethyl-5-((S)-oxiran-2-yl)tetrahydrofuro[2,3-d][1,3]dioxole (51S) (3.0 g, 15.9 mmol) was added a solution of methylamine in THF (2 M, 45 mL, 15 vol) in anhydrous THF (30 mL). The reaction mixture was stirred at 75 °C in a sealed tube. After 24 h, TLC indicated the consumption of (51S). The mixture was concentrated under reduced pressure to give crude (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2-(methylamino)ethan-1-ol) (52S) (3.1 g, 86%) as a semisolid, which was used in the next step without purification. 10 H 19 NO4: 1 H NMR (500MHz, CDCl3): δ 5.81 (d, J = 3.5 Hz, 1H), 4.75 (m, 1H), 4.08-4.21 (m, 1H), 3.67-3.81 (m, 1H), 2.79-2.91 (m, 2H), 2.53 (s, 3H), 2.27-2.44 (b, 2H), 1.97-2.04 (m, 2H), 1.51 (s, 3H), 1.32 (s, 3H)

[0301] Process 5 (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2-(2,2,2-trifluoro-N-methylacetamido)ethyl 2,2,2-trifluoroacetate (53S) To a stirred solution of (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2-(methylamino)ethan-1-ol) (52S) (3.1 g, 14.28 mmol), TEA (7.4 mL, 57.142 mmol), and DMAP (0.234 g, 2.85 mmol) in anhydrous CHCl (31 mL) was added trifluoroacetic anhydride (3.99 mL, 28.57 mmol). The reaction mixture was heated at 25 °C for 16 h and then quenched with saturated aqueous NaHCO (60 mL). The organic layer was then separated, and the aqueous phase was extracted with CHCl (2 × 60 mL). The combined organic layers were dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude product was purified by column silica gel chromatography (100-200 mesh, 20% EtOAc in petroleum ether) to give (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2-(2,2,2-trifluoro-N-methylacetamido)ethyl 2,2,2-trifluoroacetate (53S) (2 g, 86%) as a viscous liquid. 14 H 17 F6NO6: ( 1 H NMR shows NEt3 salt impurity) 1H NMR (400MHz, CDCl3): δ 5.82 (d, J = 3.6 Hz, 1H), 4.76 (t, J = 4.2 Hz, 1H), 4.18 (m, 1H), 3.88 (m, 1H), 3.62 (dd, J = 13.8, 3.6 Hz, 1H), 3.49 (dd, J = 9.0, 2.4 Hz, 1H), 3.26 (m, 3H), 2.09 (dd, J = 13.4, 4.6 Hz, 1H), 1.88-1.95 (m, 1H), 1.51 (s, 3H), 1.30 (s, 3H)

[0302] Process 6 (2S,3R,5S)-5-((S)-1-acetoxy-2-(2,2,2-trifluoro-N-methylacetamido)ethyl)tetrahydrofuran-2,3-diyl diacetate (54S) To a solution of (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2-(2,2,2-trifluoro-N-methylacetamido)ethyl 2,2,2-trifluoroacetate (53S) (2.0 g, 4.8 mmol), acetic acid (1.78 mL, 29.33 mmol), and acetic anhydride (3.01 mL, 29.34 mmol) in anhydrous CHCl (40 mL) was added concentrated HSO (0.1 mL) at 0 °C. The resulting reaction mixture was stirred at 22 °C for 3 h and quenched by the addition of saturated aqueous NaHCO (200 mL). The organic layer was separated, and the aqueous phase was extracted with CHCl (2 × 200 mL). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure. The crude material was purified by column silica gel chromatography (100-200 mesh, 15% EtOAc in petroleum ether) to give (2S,3R,5S)-5-((S)-1-acetoxy-2-(2,2,2-trifluoro-N-methylacetamido)ethyl)tetrahydrofuran-2,3-diyl diacetate (1.1 g, 47%) as a viscous liquid, which was used without further purification. 15 H 20 F3NO8: ( 1 H NMR shows NEt3 salt impurity) 1H NMR (400MHz, CDCl3): δ 6.11 (s, 1H), 5.32 (m, 1H), 5.25 (m, 1H), 5.19 (d, J = 4.4 Hz, 1H), 4.41 (m, 1H), 3.68 (m, 1H), 3.60 (m, 1H), 3.18 (s, 3H), 3.04 (s,1H), 2.09 (m, 9H)

[0303] Process 7 (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2-(2,2,2-trifluoro-N-methylacetamido)ethyl acetate (29) To a mixture of N-(6,8-dioxo-7-(prop-2-yn-1-yl)-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (6) (400 mg, 1.619 mmol) and (2S,3R,5S)-5-((S)-1-acetoxy-2-(2,2,2-trifluoro-N-methylacetamido)ethyl)tetrahydrofuran-2,3-diyl diacetate (54S) (840 mg, 2.10 mmol) in 1,2-dichloroethane (40 mL) was added BSA (1.23 mL, 4.8 mmol). The reaction mixture was stirred at 80 °C for 30 min under argon, the resulting solution was cooled to RT, and the 1,2-dichloroethane was removed under reduced pressure. The residue was dissolved in MeCN (40 mL), followed by the addition of TMSOTf (0.449 mL, 2.4 mmol). The reaction mixture was heated at 80° C. for 16 hours, cooled to room temperature, and concentrated under reduced pressure. The residue was diluted with saturated aqueous NaHCO (60 mL) and extracted with EtOAc (3×80 mL). The combined EtOAc layers were washed with water (50 mL), brine (40 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude compound was purified by flash column silica gel chromatography (100-200 mesh, 80% EtOAc in petroleum ether) to give (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2-(2,2,2-trifluoro-N-methylacetamido)ethyl acetate (29) (260 mg, 28%) as an off-white solid. 23 H 25 F3N6O 9: 1H NMR (400MHz, DMSO-d6): δ 11.20-11.80 (b, 1H), 5.71 (s, 1H), 5.66 (d, J = 6.0 Hz, 1H), 5.26-5.37 (m, 1H), 4.68 (s, 2H), 4.32 (m, 1H), 3.78 (m, 1H), 3.55 (m, 1H), 3.08 (s, 3H), 2.89 (m, 2H), 2.81 (m, 1H), 2.17 (s, 3H), 2.07 (s, 3H), 2.02 (m, 1H), 1.93 (s, 3H). ES+, m / z 586.9 [M+H] +

[0304] Process 8 2-Amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxy-2-(methylamino)ethyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione Compound 16 To a solution of (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2-(2,2,2-trifluoro-N-methylacetamido)ethyl acetate (29) (260 mg, 0.443 mmol) in methanol (10 mL) was added KCO (91.8 mg, 0.665 mmol) at rt. The resulting reaction mixture was stirred at room temperature and monitored by LC / MS. After 16 h, the starting material was consumed. The reaction mixture was concentrated under reduced pressure, and the resulting thick slurry was subjected to GRACE reverse-phase chromatography (10 mM aqueous (NH4)HCO3:MeCN) to afford 2-amino-9-(2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxy-2-(methylamino)ethyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione (compound 16) (71 mg, 44%) as an off-white solid. 15 H 20 NO 5:1 H NMR (400MHz, DMSO-d6; D2O): δ 5.53 (d, J = 3.6 Hz, 1H), 4.74 (m, 1H), 4.62 (s, 2H), 4.12 (m, 1H), 3.58 (m, 1H), 3.16 (t, J = 2.4 Hz, 1H), 2.36-2.48 (m, 3H), 2.96 (s, 3H), 1.86 (m, 1H). ES+, m / z 365.3 [M+H] +

[0305] Example 15 2-Amino-7-(cyclopropylmethyl)-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 17 [ka]

[0306] Following the procedures described in Examples 3 and 11, compound 17 was prepared from (12) and (37S) by the following method. [ka]

[0307] Process 1 (S)-1-((2R,3S,4S,5R)-5-(2-acetamido-7-(cyclopropylmethyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (30) N-(7-(cyclopropylmethyl)-6,8-dioxo-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (12) (300 mg, 1.1 mmol), (3S,4S,5R)-5-((S)-1-acetoxypropyl)-4-fluorotetrahydrofuran-2,3-diyl diacetate (37S) (523.5 mg, 1.7 mmol), and BSA (0.86 mL, 3.42 mmol) were dissolved in 1,2-dichloroethane (20 mL), and the resulting reaction mixture was stirred at 80° C. for 30 min under argon. The reaction mixture was cooled to RT, and the 1,2-dichloroethane was removed under reduced pressure. The residue was dissolved in MeCN (20 mL), followed by the addition of TMSOTf (0.31 mL, 1.71 mmol). The reaction mixture was heated at 80° C. for 16 h, cooled to RT, and concentrated under reduced pressure. The residue was diluted with saturated aqueous NaHCO3 (60 mL) and extracted with EtOAc (3 x 60 mL). The combined EtOAc layers were washed with water (30 mL), brine (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by flash column chromatography (silica gel, 100-200 mesh, 80% EtOAc in petroleum ether) to afford 230 mg (39%) of (S)-1-((2R,3S,4S,5R)-5-(2-acetamido-7-(cyclopropylmethyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (30) as a pale yellow solid. 22 H 28 FN5O 5: ES+, m / z 510.8 [M+H] +

[0308] Process 2 2-Amino-7-(cyclopropylmethyl)-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 17 To a solution of (S)-1-((2R,3S,4S,5R)-5-(2-acetamido-7-(cyclopropylmethyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (30) (230 mg, 0.45 mmol) in methanol (10 mL) was added KCO (62.3 mg, 0.45 mmol) at rt. The reaction mixture was stirred at room temperature for 16 h and concentrated under reduced pressure to give a solid mass. The crude product was purified by preparative HPLC using a Luna@omega (250*21.2) column with a 5μ mobile phase of 0.1% HCOH:MeCN in HO gradient (T%B): -0 / 5, 8 / 50, 10.5 / 50, 10.6 / 98, 12 / 98, 12.1 / 5, 15 / 5 at a flow rate of 17 mL / min to give 16 mg (approximately 9%) of 2-amino-7-(cyclopropylmethyl)-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7,9-dihydro-1H-purine-6,8-dione (compound 17) as a white solid. 16 H 22 FN5O5: 1 H NMR spectrum showed that the product was the formate salt; 1 H NMR (400MHz, DMSO-d6, D2O): δ 8.48 (s, 1H), 5.39 (d, J = 6.4 Hz, 1H), 5.38-5.29 (m, 1H), 5.00 (ddd, J = 52.8, 4.8, 2.6 Hz, 1H), 3.85 (dt, J = 22.0, 5.7 Hz, 1H), 3.67 (d, J = 5.6 Hz, 2H), 3.52 (m, 1H), 1.51 (m, 1H), 1.36 (m, 1H), 1.19 (m, 1H), 0.91 (t, J = 7.2 Hz, 3H), 0.43 (m, 2H), 0.38 (m, 2H). ES+, m / z 384.2 [M+H] +

[0309] Example 162-amino-9-((2R,3R,5S)-3-hydroxy-5-(2-hydroxypropan-2-yl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 18 [ka]

[0310] Compound 18 was prepared in the following multiple steps. [ka]

[0311] Process 1 : 1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-one (55S) To a stirred solution of (R)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (15S) (7 g, 37.2 mmol) in CHCl (70 mL) at 0 °C under a N atmosphere, pyridinium dichromate (16.7 g, 44.6 mmol) was added, followed by the dropwise addition of acetic anhydride (7 mL, 1 vol). The reaction mixture was stirred at rt for 16 h, concentrated under reduced pressure, and the residue was purified by column silica gel chromatography (100-200 mesh, 50% EtOAc in petroleum ether) to give 5 g (66%) of 1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-one (55S) as a light yellow liquid. 14 O4: 1H NMR (400MHz, CDC13): δ 5.93 (d, J = 3.2 Hz, 1H), 4.76 (t, J = 4.0 Hz 1H), 4.61 (dd, J = 11.2, 5.2 Hz, 1H), 2.37 (dd, J = 13.4, 5.0 Hz, 1H), 2.24 (s, 3H), 1.77 (m, 1H), 1.52 (s, 3H), 1.34 (s, 3H)

[0312] Process 2 : 2-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-2-ol (56S) To a stirred solution of 1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-one (55S) (5.0 g, 26.8 mmol) in diethyl ether (50 mL) was added methylmagnesium bromide (3.0 M) (22.4 mL 67.20 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 3 h and then quenched with saturated aqueous ammonium chloride solution. The organic layer was separated and the aqueous phase was extracted with EtOAc (2 × 200 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude oil was purified by column silica gel chromatography (100-200 mesh, 50% EtOAc in petroleum ether) to give 2 g (37%) of 2-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-2-ol (56S). 10 H 18 O4: 1 H NMR (400MHz, CDC13): δ 5.81 (d, J = 2.4 Hz, 1H), 4.72 (s, 1H), 4.07 (dd, J = 10.4, 4.4 Hz, 1H), 1.99 (dd, J = 13.2, 4.4 Hz, 1H), 1.95 (m, 1H), 1.85 (m, 1H), 1.53 (s, 3H), 1.33 (s, 3H), 1.31 (s, 3H), 1.13 (s, 3H)

[0313] Process 3 2-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-2-yl 2,2,2-trifluoroacetate (57S) To a stirring, ice-cold solution of 2-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-2-ol (56S) (2.0 g, 9.9 mmol) in anhydrous CHCl (30 mL) was added TEA (4.1 mL, 29.70 mmol), DMAP (0.24 g, 1.9 mmol), and trifluoroacetic anhydride (3.4 mL, 24.7 mmol). The reaction mixture was stirred at rt for 16 h and then quenched with saturated aqueous NaHCO (30 mL). The organic layer was separated, and the aqueous phase was extracted with CHCl (2 × 100 mL). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure. The crude product was purified by column silica gel chromatography (100-200 mesh, 20% EtOAc in petroleum ether) to give 1.5 g (51%) of 2-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-2-yl 2,2,2-trifluoroacetate (57S) as a colorless oil. 12 H 17 F3O 5: 1 H NMR (400MHz, CDC13): δ 5.82 (d, J = 3.6 Hz, 1H), 4.76 (t, J = 4.2 Hz, 1H), 4.26 (dd, J = 10.8, 4.8 Hz, 1H), 2.10 (dd, J = 13.6, 4.8 Hz, 1H), 1.84 (ddd, J = 15.4, 9.5, 4.0 Hz, 1H), 1.65 (s, 3H), 1.57 (s, 3H), 1.52 (s, 3H), 1.33 (s, 3H)

[0314] Process 4(2S,3R,5S)-5-(2-(2,2,2-trifluoroacetoxy)propan-2-yl)tetrahydrofuran-2,3-diyl diacetate (58S) To a solution of 2-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-2-yl 2,2,2-trifluoroacetate (57S) (1.5 g, 5.03 mmol), acetic acid (1.4 mL, 25.1 mmol), and acetic anhydride (2.5 mL, 25.1 mmol) in anhydrous CHCl (15 mL) was added concentrated HSO (0.1 mL) at 0 °C. The reaction mixture was stirred at 25 °C for 3 h and then quenched by the addition of saturated aqueous NaHCO (100 mL). The organic layer was separated, and the aqueous phase was extracted with CHCl (2 × 100 mL). The combined organic layers were dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude oil was purified by column silica gel chromatography (100-200 mesh, 30% EtOAc in petroleum ether) to give 0.7 g (41%) of (2S,3R,5S)-5-(2-(2,2,2-trifluoroacetoxy)propan-2-yl)tetrahydrofuran-2,3-diyl diacetate (58S). 13 H 17 F3O7: 1 H NMR (400MHz, CDC13): δ 6.16 (s, 1H), 5.20 (d, J = 4.8 Hz, 1H), 4.38 (dd, J = 10.4, 6.0 Hz, 1H), 2.25-2.21 (m, 2H), 2.10 (s, 3H), 2.07 (s, 3H), 1.65 (s, 3H), 1.57 (s, 3H)

[0315] Process 5 2-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propan-2-yl 2,2,2-trifluoroacetate (31) N-(6,8-dioxo-7-(prop-2-yn-1-yl)-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (6) (350 mg, 1.4 mmol), (2S,3R,5S)-5-(2-(2,2,2-trifluoroacetoxy)propan-2-yl)tetrahydrofuran-2,3-diyl diacetate (58S) (726.9 mg, 2.12 mmol), and BSA (1.07 mL, 4.25 mmol) were dissolved in 1,2-dichloroethane (15 mL). The resulting reaction mixture was stirred at 80 °C for 30 min under argon, cooled to room temperature, and the solvent was removed under reduced pressure. The residue was dissolved in MeCN (20 mL), followed by the addition of TMSOTf (0.39 mL, 2.1 mmol). The reaction mixture was heated at 70° C. for 16 hours, cooled to room temperature, and concentrated under reduced pressure. The residue was diluted with saturated aqueous NaHCO (50 mL) and extracted with EtOAc (3×50 mL). The combined EtOAc layers were washed with water (30 mL), brine (20 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The crude compound was purified by GRACE chromatography (80% EtOAc in petroleum ether) to afford 270 mg (49%) of 2-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propan-2-yl 2,2,2-trifluoroacetate (31) as an off-white solid. 21 H 22 F3N5O 8: ES+, m / z 529.9 [M+H] +

[0316] Process 6 2-amino-9-((2R,3R,5S)-3-hydroxy-5-(2-hydroxypropan-2-yl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 18 To a solution of 2-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propan-2-yl 2,2,2-trifluoroacetate (31) (270 mg, 0.51 mmol) in MeOH (10 mL) was added KCO (105.6 mg, 0.76 mmol) at 0 °C. The reaction mixture was stirred at RT for 16 h and neutralized with acetic acid at 0 °C. Methanol was removed under reduced pressure at RT, and the resulting residue was purified by preparative HPLC (column: X-SELECT-C18 (250*19), 5μ mobile phase: 0.1% HCOH in HO:MeOH gradient: (T%B): -0 / 20, 8 / , 50, 10.5 / 50, 10.6 / 98, 13 / 98, 13.1 / 20, 16 / 20, flow rate: 18 mL / min). The purified fraction was lyophilized to give 45 mg (25%) of 2-amino-9-((2R,3R,5S)-3-hydroxy-5-(2-hydroxypropan-2-yl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione (compound 18) as an off-white solid. 15 H 19 N5O 5: 1 H NMR (500MHz, DMSO-d6): δ 10.80 (brs, 1H), 6.53 (brs, 2H), 6.51 (brs, 1H), 5.49 (d, J = 3.5 Hz, 1H), 5.37 (d, J = 4.5 Hz, 1H), 4.76 (m, 1H), 4.59 (d, J = 1.5 Hz, 2H), 4.38 (s, 1H), 3.91 (t, J = 7.3 Hz, 1H), 3.23 (s, 1H), 2.46-2.41 (m, 1H), 1.75 (m, 1H), 1.03 (d, J = 6.0 Hz, 6H). ES+, m / z 350.0 [M+H] +

[0317] Example 172-amino-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-(2-hydroxypropan-2-yl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 19 [ka]

[0318] Compound 19 was prepared in the following multiple steps. [ka]

[0319] Process 1 : 1-((3aR,5R,6S,6aS)-6-Fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (59S) To a solution of (3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxole-5-carbaldehyde (27S) (1 g, 5.26 mmol) in THF (20 mL) was added methylmagnesium bromide (1 M in THF, 15.8 mL, 15.78 mmol) at −20 °C under argon. The reaction mixture was stirred at room temperature for 6 h, then quenched with saturated aqueous NH4Cl and extracted with EtOAc (3 × 100 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude product, which was purified by column silica gel chromatography (1:4 EtOAc:petroleum ether). 1-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (59S) (600 mg, 55.35%) was obtained as a concentrated mass: CH 15 FO4: 1 H NMR shows preferential formation of one enantiomer; C-1 stereochemistry not determined: 1H NMR (500MHz, CDCl3): δ 5.98 (d, J = 3.5 Hz, 1H), 5.08 (dd, J = 50.3, 2.3 Hz, 1H), 4.70 (dd, J = 13.5, 4.0 Hz, 1H), 4.10 (m, 1H), 3.97 (ddd, J = 29.8, 8.0, 2.3 Hz, 1H), 1.77 (d, J = 5.0 Hz, 1H), 1.50 (s, 3H), 1.38 (d, J = 6.5 Hz, 3H), 1.33 (s, 3H)

[0320] Process 2 : 1-((3aR,5R,6R,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-one (60S) To a solution of 1-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-ol (59S) (100 mg, 0.48 mmol) and AcO (0.1 mL) in CHCl (10 mL) was added pyridinium dichromate (219 mg, 0.58 mmol). The reaction mixture was stirred at room temperature for 16 h, concentrated under reduced pressure, and EtOAc was added in portions (3 × 100 mL) with vigorous stirring. The organic layer was passed through a silica gel plug and concentrated under reduced pressure to give (80 mg, 80.88%) 1-((3aR,5R,6R,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-one (60S) as a colorless oil, which was used without further purification. 13 FO4: 1 H NMR (400MHz, CDCl3): δ 6.13 (d, J = 4.0 Hz, 1H), 5.18 (dd, J = 49.8, 2.6 Hz, 1H), 4.72 (dd, J = 9.8, 3.8 Hz, 1H), 4.64 (dd, J = 33.0, 2.6 Hz, 1H), 2.28 (d, J = 0.4 Hz, 3H), 1.49 (s, 3H), 1.35 (s, 3H)

[0321] Process 3 2-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-2-ol (61S) To a solution of 1-((3aR,5R,6R,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)ethan-1-one (60S) (100 mg, 0.49 mmol) in THF (10 mL) was added methylmagnesium bromide (1 M in THF, 0.75 mL, 0.73 mmol) at −20° C. under argon. The reaction mixture was then stirred at room temperature for 16 h. The reaction mixture was quenched with saturated aqueous NH4Cl and extracted with EtOAc (3 × 50 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude product, which was purified by column silica gel chromatography (1:4 EtOAc / petroleum ether) to give (50 mg, 46.4%) of 2-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-2-ol (61S) as a colorless oil. 10 H 17 FO4: 1 H NMR (500MHz, CDCl3): δ 6.04 (d, J = 4.0 Hz, 1H), 5.03 (dd, J = 50.3, 2.3 Hz, 1H), 4.68 (dd, J = 12.3, 3.8 Hz, 1H), 3.99 (dd, J = 34.0, 2.5 Hz, 1H), 2.18 (d, J = 5.0 Hz, 1H), 1.50 (s, 3H), 1.37 (s, 3H) 1.35 (s, 3H), 1.34 (s, 3H)

[0322] Process 4 2-((3aR,5R,6S,6aS)-6-Fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-2-yl 2,2,2-trifluoroacetate (62S) To a solution of 2-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-2-ol (61S) (1.0 g, 4.54 mmol) in CHCl (20 mL) was added EtN (1.28 mL, 9.09 mmol) at 0 °C. After stirring for 10 min, trifluoroacetic anhydride (1.43 mL, 6.81 mmol) was added at 0 °C and stirred at room temperature for an additional 16 h. The reaction mixture was then quenched with saturated aqueous NaHCO and extracted with CHCl (3 × 100 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue that was purified by column silica gel chromatography (1:4 EtOAc petroleum ether) to give (500 mg, 34.8%) of 2-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-2-yl 2,2,2-trifluoroacetate (62S) as a colorless oil. 12 H 16 F4O 5: 1 H NMR (400MHz, CDCl3): δ 6.03 (d, J = 4.0 Hz, 1H), 5.01 (dd, J = 50.4, 2.4 Hz, 1H), 4.67 (dd, J = 11.8, 3.8 Hz, 1H), 4.43 (dd, J = 32.4, 2.4 Hz, 1H), 1.72 (s, 3H), 1.69 (s, 3H), 1.50 (s, 3H), 1.34 (s, 3H)

[0323] Process 5 (2S,3S,4S,5R)-4-fluoro-5-(2-(2,2,2-trifluoroacetoxy)propan-2-yl)tetrahydrofuran-2,3-diyl diacetate (63S) To 2-((3aR,5R,6S,6aS)-6-fluoro-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)propan-2-yl 2,2,2-trifluoroacetate (62S) (800 mg, 2.53 mmol) dissolved in CHCl (20 mL) was added AcOH (1.46 mL, 25.32 mmol), AcO (1.20 mL, 12.65 mmol), and concentrated HSO (0.1 mL) at 0 °C. The resulting reaction mixture was stirred at room temperature for 3 h, quenched with saturated aqueous NaHCO, and extracted with CHCl (3 × 100 mL). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography (SiO, 0–15% EtOAc-petroleum ether) to afford 150 mg of (2S,3S,4S,5R)-4-fluoro-5-(2-(2,2,2-trifluoroacetoxy)propan-2-yl)tetrahydrofuran-2,3-diyl diacetate (63S) (22.6% yield) as a colorless oil. 13 H 16 F4O7: 1 H NMR (400MHz, CDCl3): δ 6.20 (s, 1H), 5.31 (d, J =11.6 Hz, 1H), 5.06 (d, J = 50.4 Hz, 1H), 4.54 (d, J = 31.6 Hz, 1H), 2.14 (s, 3H), 2.10 (s, 3H), 1.74 (s, 3H), 1.69 (s, 3H)

[0324] Process 6 2-((2R,3S,4S,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propan-2-yl 2,2,2-trifluoroacetate (32) N-(6,8-dioxo-7-(prop-2-yn-1-yl)-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (6) (300 mg, 1.21 mmol), (2S,3S,4S,5R)-4-fluoro-5-(2-(2,2,2-trifluoroacetoxy)propan-2-yl)tetrahydrofuran-2,3-diyl diacetate (63S) (654 mg, 1.82 mmol), and BSA (0.61 mL, 3.03 mmol) were dissolved in 1,2-dichloroethane (10 mL), and the reaction mixture was stirred at 80° C. for 30 minutes under argon. The reaction mixture was cooled to room temperature, followed by the addition of TMSOTf (0.40 mL, 1.82 mmol). The reaction mixture was stirred at 80° C. for 30 minutes under argon, cooled to room temperature, and the solvent was removed under reduced pressure. The residue was dissolved in MeCN (15 mL), followed by the addition of TMSOTf (0.40 mL, 1.82 mmol). The reaction mixture was heated at 80° C. for 16 h, cooled to room temperature, diluted with water, and extracted with EtOAc (3×100 mL). The combined organic layers were washed with water (100 mL), brine (100 mL), dried over Na2SO4, filtered, and concentrated. The crude product was purified by GRACE reverse-phase flash chromatography (column: FLASH PURE-C18 (40 μm irregular), mobile phase: 0.1% HCOH:MeCN in HO T%B: -30 min; flow rate: 12 mL / min) to give (160 mg, 24.1%) 2-((2R,3R,4S,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propan-2-yl 2,2,2-trifluoroacetate (32) as a yellow solid. 21 H 21 F4N5O 8: 1H NMR (400MHz, CDCl3): δ 11.97 (bs, 1H), 8.18 (s, 1H), 6.19 (dd, J = 22.8, 5.2 Hz, 1H), 5.77 (d, J = 5.6 Hz, 1H), 5.17 (d, J = 51.4, 3.8 Hz, 1H), 4.86 (d, J = 2.4 Hz, 2H), 4.27 (dd, J = 29.6, 4.0 Hz, 1H), 2.32 (t, J = 2.4 Hz, 1H), 2.21 (s, 3H), 2.13 (s, 3H), 1.76 (s, 3H), 1.72 (s, 3H). ES+, m / z 548.40 [M+H] +

[0325] Process 7 2-amino-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-(2-hydroxypropan-2-yl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 19 To a solution of 2-((2R,3R,4S,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propan-2-yl 2,2,2-trifluoroacetate 32 (160 mg, 0.29 mmol) in methanol (10 mL) was added KCO (48 mg, 0.35 mmol) at 0° C. The reaction mixture was stirred at room temperature for 16 h, concentrated, and subjected to GRACE flash chromatography (reverse phase using 0.01% formic acid in acetonitrile). The purified fractions were concentrated to give 2-amino-9-((2R,3S,4S,5R)-4-fluoro-3-hydroxy-5-(2-hydroxypropan-2-yl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 19 (53 mg, 49.4%) as a white solid. 15 H 18 FN5O 5: 1H NMR (400MHz, DMSO-d6): δ 10.96 (brs, 1H), 6.56 (s, 2H), 5.93 (d, J = 5.6 Hz, 1H), 5.35-5.32 (m, 1H), 5.26 (m, 1H), 5.01 (ddd, J = 53.8, 4.8, 2.6 Hz, 1H), 4.64 (s, 1H), 4.60 (d, J = 2.0 Hz, 2H), 3.75 (dd, J = 26.2, 4.6 Hz, 1H), 3.24 (t, J = 2.2 Hz, 1H), 1.17 (s, 6H). ES+, m / z 368.0 [M+H] +

[0326] Example 18 2-amino-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-8H-purin-8-one, compound 20 and 2-amino-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(propa-1,2-dien-1-yl)-7,9-dihydro-8H-purin-8-one, compound 21 [ka]

[0327] Using the method described in Example 5, compounds 20 and 21 were synthesized from (21) and (37S) as follows. [ka]

[0328] Process 1 (S)-1-((2R,3S,4S,5R)-5-(2-acetamido-8-oxo-7-(prop-2-yn-1-yl)-7,8-dihydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (33) N-(8-oxo-7-(prop-2-yn-1-yl)-8,9-dihydro-7H-purin-2-yl)acetamide (21) (400 mg, 1.731 mmol), (3S,4S,5R)-5-((S)-1-acetoxypropyl)-4-fluorotetrahydrofuran-2,3-diyl diacetate (37S) (627 mg, 2.079 mmol), and BSA (1.049 g, 5.194 mmol) were dissolved in dichloroethane (20 mL). The reaction mixture was stirred at 80 °C for 30 min under argon. The reaction mixture was then concentrated under reduced pressure, and the residue was dissolved in MeCN (50 mL). TMSOTf (577 mg, 2.594 mmol) was added to the reaction flask and placed in an oil bath preheated to 80 °C. After 18 h, the reaction was cooled to room temperature, and the solvent was removed by rotary evaporation. The resulting solid was dissolved in ethyl acetate (50 mL) and washed with saturated aqueous NaHCO3 (2 x 30 mL). The organic phase was dried over Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (SiO2, 0-80% ethyl acetate-petroleum ether) to give (S)-1-((2R,3S,4S,5R)-5-(2-acetamido-8-oxo-7-(prop-2-yn-1-yl)-7,8-dihydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (33) as a light yellow solid (400 mg, 48.43% yield). 21 H 24 FN5O7: ES+, m / z 477.8 [M+H]+. LC / MS also showed loss of the 1Ac group (ES+, m / z 435.8 [M+H]+). The product was used without further purification.

[0329] Process 22-amino-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-8H-purin-8-one, compound 20 and 2-amino-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(propa-1,2-dien-1-yl)-7,9-dihydro-8H-purin-8-one, compound 21

[0330] (S)-1-((2R,3S,4S,5R)-5-(2-acetamido-8-oxo-7-(prop-2-yn-1-yl)-7,8-dihydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (33) (400 mg, 0.8385 mmol) was dissolved in methanol (10 mL), followed by the addition of KCO (173 mg, 1.257 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h and concentrated. The residue was purified by GRACE flash chromatography (reverse phase; 0.01% formic acid in acetonitrile) to give (53 mg, 38.93%) of 2-amino-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-8H-purin-8-one (compound 20) as an off-white solid and (20 mg, 14.69%) of 2-amino-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(prop-1,2-dien-1-yl)-7,9-dihydro-8H-purin-8-one (compound 21) as an off-white solid.

[0331] Compound 20:C 15 H 18 FN5O4: ES+, m / z 352.2 [M+H] + ; 1H NMR (500MHz, DMSO-d6): δ 8.06 (s, 1H), 6.39 (s, 2H), 5.97 (d, J = 5.5 Hz, 1H), 5.46 (d, J = 6.5 Hz, 1H), 5.40 (m, 1H), 5.00 (ddd, J = 53.3, 4.3, 2.0 Hz, 1H), 4.80 (d, J = 6.5 Hz, 1H), 4.66 (d, J = 5.5 Hz, 2H), 3.85 (ddd, J = 24.3, 7.0, 4.8 Hz, 1H), 3.56 (m, 1H), 3.41 (t, J = 2.5 Hz, 1H), 1.49 (m, 1H), 1.35 (m, 1H), 0.91 (t, J = 7.3 Hz, 3H)

[0332] Compound 21:C 15 H 18 FN5O4: ES+, m / z 352.1 [M+H] + ; 1 H NMR (500MHz, DMSO-d6): δ 8.18 (s, 1H), 7.23 (t, J = 6.8 Hz, 1H), 6.52 (s, 2H), 5.95 (brs, 1H), 5.83 (d, J = 6.5 Hz, 2H), 5.48 (d, J = 6.0 Hz, 1H), 5.33 (ddd, J = 25.0, 6.0, 2.0 Hz, 1H), 5.01 (ddd, J = 53.0, 4.5, 2.0 Hz, 1H), 4.77 (s, 1H), 3.85 (ddd, J = 24.3, 7.3, 4.5 Hz, 1H), 3.57 (m, 1H), 1.51 (m, 1H), 1.34 (m, 1H), 0.92 (t, J = 7.3 Hz, 3H)

[0333] Example 19 :2-アミノ-9-((2R,3R,5S)-3-ヒドロキシ-5-((S)-1-ヒドロキシプロピル)テトラヒドロフラン-2-イル)-7-(2-(メチルチオ)エチル)-7,9-ジヒドロ-1H-プリン-6,8-ジオン, compound 22 [ka]

[0334] Compound 22 was prepared in the following multiple steps. [ka]

[0335] Process 1 2-Amino-6-(benzyloxy)-9-(4-methoxybenzyl)-7-(2-(methylthio)ethyl)-7,9-dihydro-8H-purin-8-one (34) To a suspension of 2-amino-6-(benzyloxy)-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (4.0 g, 10.61 mmol) (3) in DMF (60 mL) was added (2-chloroethyl)(methyl)sulfane (2.56 g, 23.34 mmol) and KCO (4.39 g, 31.83 mmol) at 0 °C. The reaction mixture was stirred at 70 °C for 16 h. The reaction mixture was quenched with ice water (200 mL), and diethyl ether (100 mL) was added and stirred for 15 min. The resulting precipitated solid was collected by filtration, washed with water, and dried to give 2-amino-6-(benzyloxy)-9-(4-methoxybenzyl)-7-(2-(methylthio)ethyl)-7,9-dihydro-8H-purin-8-one (34) (4.0 g, 85%) as an off-white solid. 23 H 25 N5O3S: 1H NMR (400MHz, DMSO-d6): δ 7.47 (d, J = 7.2 Hz, 1H), 7.40 (d, J = 6.8 Hz, 2H), 7.39-7.22 (m, 2H), 7.19 (d, J = 6.8 Hz, 2H), 6.87 (d, J = 8.8 Hz, 2H), 6.42 (s, 2H), 5.42 (s, 2H), 4.83 (s, 2H), 3.93 (t, J = 7.0 Hz, 2H), 3.71 (s, 3H), 2.68 (t, J = 7.0 Hz, 2H), 1.90 (s, 3H). ES+, m / z 452.3 [M+H] +

[0336] Process 2 2-Amino-6-(benzyloxy)-7-(2-(methylthio)ethyl)-7,9-dihydro-8H-purin-8-one (35) Trifluoromethanesulfonic acid (3.56 mL, 35.47 mmol) was added to a suspension of 2-amino-6-(benzyloxy)-9-(4-methoxybenzyl)-7-(2-(methylthio)ethyl)-7,9-dihydro-8H-purin-8-one (34) (4.0 g, 8.86 mmol) in TFA (2.71 mL, 35.47 mmol) at room temperature under an argon atmosphere, and the resulting reaction mixture was stirred at room temperature for 16 h under an argon atmosphere. The reaction mixture was quenched with ice-cold water (200 mL), basified with saturated aqueous NaHCO3 (300 mL) under vigorous stirring, and collected by filtration. The filtered solid was dissolved in ethyl acetate, stirred for 30 minutes, filtered, and dried to give 2-amino-6-(benzyloxy)-7-(2-(methylthio)ethyl)-7,9-dihydro-8H-purin-8-one (35) (1.8 g, 62%) as a brown solid. 15 H 17 N5O2S; 1H NMR (400MHz, DMSO-d6): δ 10.87 (s, 1H), 7.47 (s, 5H), 6.48 (s, 2H), 4.84 (d, J = 12.8 Hz, 1H), 4.64 (d, J = 12.8 Hz, 1H), 4.23 (m, 2H), 3.67-3.58 (m, 2H), 2.81 (s, 3H). ES+, m / z 332.2 [M+H] +

[0337] Process 3 2-Amino-7-(2-(methylthio)ethyl)-7,9-dihydro-1H-purine-6,8-dione (36) LiBr (0.701 g, 8.15 mmol) and TMSCl (2.7 mL, 21.7 mmol) were added to a suspension of 2-amino-6-(benzyloxy)-7-(2-(methylthio)ethyl)-7,9-dihydro-8H-purin-8-one (35) (1.8 g, 5.4 mmol) in acetonitrile (36 mL) at 0 °C under an argon atmosphere. The reaction mixture was stirred at 50 °C for 16 h under an argon atmosphere, quenched with ice-cold water (200 mL), and basified with saturated aqueous NaHCO (300 mL) under vigorous stirring. The solid formed was collected by filtration, EtOAc was added, stirred for 30 min, filtered, and dried to give 2-amino-7-(2-(methylthio)ethyl)-7,9-dihydro-1H-purine-6,8-dione (36) (1.0 g, 76%) as a brown solid. 11 N5O2S: 1 H NMR (400MHz, DMSO-d6): δ 11.09 (s, 1H), 10.66 (s, 1H), 6.37 (s, 2H), 3.90 (t, J = 6.8 Hz, 2H), 2.76 (t, J = 6.8 Hz, 2H), 2.07 (s, 3H). ES+, m / z 242.0 [M+H] +

[0338] Process 4 N-(7-(2-(methylthio)ethyl)-6,8-dioxo-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (37) Acetic anhydride (2.53 mL, 24.89 mmol) was added to a solution of 2-amino-7-(2-(methylthio)ethyl)-7,9-dihydro-1H-purine-6,8-dione (36) (1 g, 4.14 mmol) in AcOH (10 mL) at ambient temperature under an argon atmosphere, and the resulting reaction mixture was heated at 120° C. for 16 hours under an argon atmosphere. The reaction mixture was stirred and cooled to 0° C. for 30 minutes, after which a dark solid precipitated. The product was collected by filtration, washed with diethyl ether, and dried under reduced pressure to give N-(7-(2-(methylthio)ethyl)-6,8-dioxo-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (37) (1.0 g, 85%) as a brown solid. The dried product was used directly in the next step. 10 H 13 N5O3S: 1 H NMR (500MHz, DMSO-d6): δ 12.01 (s, 1H), 11.69 (s, 1H), 11.64 (s, 1H), 3.99 (t, J = 7.0 Hz, 2H), 2.79 (t, J = 7.0 Hz, 2H), 2.18 (s, 3H), 2.08 (s, 3H). ES+, m / z 283.8 [M+H] +

[0339] Process 5 (S)-1-((2S,4R,5R)-5-(2-acetamido-7-(2-(methylthio)ethyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (38) N-(7-(2-(methylthio)ethyl)-6,8-dioxo-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (37) (760 mg, 2.663 mmol), (3R,5S)-5-((S)-1-acetoxypropyl)tetrahydrofuran-2,3-diyl diacetate (14S) (923 mg, 3.20 mmol), and BSA (1.33 mL, 5.28 mmol) were dissolved in 1,2-dichloroethane (35 mL), and the resulting reaction mixture was stirred at 80° C. for 30 minutes under argon. The reaction mixture was cooled to rt, and the 1,2-dichloroethane was removed under reduced pressure. The residue was dissolved in MeCN (35 mL), followed by the addition of TMSOTf (0.488 mL, 2.64 mmol). The reaction mixture was heated at 80° C. for 16 hours, cooled to room temperature, and concentrated under reduced pressure. The residue was diluted with saturated aqueous NaHCO (60 mL) and extracted with EtOAc (3 × 80 mL). The combined EtOAc layers were washed with water (50 mL), brine (40 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The crude compound was purified by flash column silica gel chromatography (80% EtOAc in petroleum ether) to give (S)-1-((2S,4R,5R)-5-(2-acetamido-7-(2-(methylthio)ethyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (38) (250 mg, 28%) as an off-white solid, which was used directly in the final step.

[0340] Process 6 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(2-(methylthio)ethyl)-7,9-dihydro-1H-purine-6,8-dione, compound 22 To a solution of (S)-1-((2S,4R,5R)-5-(2-acetamido-7-(2-(methylthio)ethyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate 38 (250 mg, 0.4892 mmol) in methanol (10 mL) was added KCO (135 mg, 0.978 mmol) at room temperature. The reaction was monitored by LC / MS until 38 was consumed. After stirring at room temperature for 16 hours, the reaction mixture was concentrated under reduced pressure to give a crude solid, which was purified by GRACE reverse-phase chromatography (0.1% aqueous HCOH:MeCN) to give 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(2-(methylthio)ethyl)-7,9-dihydro-1H-purine-6,8-dione (compound 22) (25 mg, 14%) as an off-white solid. 15 H 23 N5O5S: 1 H NMR (500MHz, DMSO-d6): δ 11.6 (s, 1H), 6.62 (s, 2H), 5.52 (d, J = 3.5 Hz, 1H), 5.35 (d, J = 4.5 Hz, 1H), 4.75 (m, 2H), 4.00 (m, 1H), 3.95 (t, J = 7.0 Hz, 2H), 3.25 (m, 1H), 2.78 (t, J = 7.0 Hz, 2H), 2.38 (m, 1H), 2.08 (s, 3H), 1.78 (m, 1H), 1.39 (m, 1H), 1.28 (m, 1H), 0.87 (t, J = 7.5 Hz, 3H). ES+, m / z 386.2 [M+H] +

[0341] Example 20 2-amino-9-((2R,3R,5S)-5-((S)-2-fluoro-1-hydroxyethyl)-3-hydroxytetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 23 [ka]

[0342] Compound 23 was prepared in the following multiple steps. [ka]

[0343] Process 1 (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2-fluoroethyl acetate (64S) A 40% HF solution (0.69 mL, 16.1 mmol) was added to TBAF (1 M in THF) (16 mL, 16.1 mmol), and after 5 min, the solution was concentrated under reduced pressure (1 mbar, 100 °C). To this concentrated mixture was added a solution of (3aR,5S,6aR)-2,2-dimethyl-5-((R)-oxiran-2-yl)tetrahydrofuro[2,3-d][1,3]dioxole (9S) (1 g, 5.3 mmol) and KHF (125.8 mg, 1.61 mmol) in dry toluene (20 mL), and the mixture was maintained at 120 °C for 18 h. The reaction mixture was cooled to rt, and AcO (1.6 mL, 16.1 mmol) and dry pyridine (3.5 mL, 16.1 mmol) were added, and the mixture was maintained under stirring at 40 °C for 2 h. The reaction mixture was quenched with water and extracted with EtOAc (2 x 75 mL). The organic phase was washed with 2M HCl and saturated aqueous NaHCO3 (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by column silica gel chromatography (100-200 mesh, 20% EtOAc in petroleum ether) to give 1 g (76%) of (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2-fluoroethyl acetate (64S). 11 H 17 FO 5: 1H NMR (400MHz, CDC13): δ 5.80 (d, J = 3.6 Hz, 1H), 5.11 (m, 1H), 4.74 (t, J = 4.2 Hz, 1H), 4.61 (ddd, J = 47.6, 10.6, 3.0 Hz, 1H), overlapping with 4.56 (ddd, J = 46.8, 10.6, 4.8 Hz, 1H), 4.36 (ddd, J = 10.6, 6.2, 4.8 Hz, 1H), 2.17 (dd, J = 13.6, 4.4 Hz, 1H), 2.11 (s, 3H), 1.80 (dd, J = 13.4, 10.6, 4.8Hz, 1H), 1.51 (s, 3H), 1.32 (s, 3H)

[0344] Process 2 (3R,5S)-5-((S)-1-acetoxy-2-fluoroethyl)tetrahydrofuran-2,3-diyl diacetate (65S) To a solution of (S)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2-fluoroethyl acetate (64S) (1 g, 4.03 mmol), acetic acid (2.3 mL, 40.3 mmol), and acetic anhydride (2 mL, 20.1 mmol) in anhydrous CHCl (30 mL) was added concentrated HSO (0.1 mL) at 0 °C. After stirring at 25 °C for 4 h, the reaction was quenched by adding ice-cold water. The organic layer was separated, and the aqueous phase was extracted with CHCl (2 × 75 mL). The combined organic layers were washed with saturated aqueous NaHCO (50 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The crude product was purified by column silica gel chromatography (100-200 mesh, 30% EtOAc in petroleum ether) to give 0.4 g (36%) of (3R,5S)-5-((S)-1-acetoxypropyl)tetrahydrofuran-2,3-diyl diacetate (64S) as a colorless oil. 12 H 17 FO7: 1 H NMR shows a mixture of α (minor) and β (major) anomers; 1H NMR (400MHz, CDC13): δ 6.16 (s, 1H), 5.18 (m, 1H), 5.08-5.02 (m, 1H), 4.66-4.64 (m, 1H), 4.55-4.51 (m, 2H), 2.23-2.12 (m, 2H), 2.10-2.08 (m, 9H)

[0345] Process 3 (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2-fluoroethyl acetate (39) N-(6,8-dioxo-7-(prop-2-yn-1-yl)-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (6) (250 mg, 1.01 mmol), (3R,5S)-5-((S)-1-acetoxypropyl)tetrahydrofuran-2,3-diyl diacetate (64S) (354.6 mg, 1.2 mmol), and BSA (0.76 mL, 3.03 mmol) were dissolved in 1,2-dichloroethane (15 mL), and the solution was stirred at 80° C. under argon for 30 minutes. The reaction mixture was then cooled to rt, and the 1,2-dichloroethane was removed under reduced pressure. The residue was dissolved in MeCN (20 mL), followed by the addition of TMSOTf (0.28 mL, 1.5 mmol). The reaction mixture was heated at 80° C. for 16 hours, cooled to room temperature, and concentrated under reduced pressure. The residue was diluted with saturated aqueous NaHCO3 (50 mL) and extracted with EtOAc (3 x 50 mL). The combined EtOAc layers were washed with water (30 mL), brine (30 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by GRACE flash chromatography (80% EtOAc in petroleum ether) to afford 190 mg (39%) of (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2-fluoroethyl acetate (39) as an off-white solid. 20 H22 FN5O 8: ES+, m / z 480.2 [M+H] +

[0346] Process 4 2-amino-9-((2R,3R,5S)-5-((S)-2-fluoro-1-hydroxyethyl)-3-hydroxytetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 23 A solution of (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2-fluoroethyl acetate (39) (190 mg, 0.39 mmol) in MeOH (10 mL) was added to KCO (54.7 mg, 0.43 mmol) at 0 °C, and the reaction mixture was stirred at RT for 16 h. The solvent was removed under reduced pressure at RT, and the residue was purified by preparative HPLC (column: X-SELECT-C18 (250*19), 5μ mobile phase: 10 mM ammonium bicarbonate in HO:MeCN gradient: (T%B): -0 / 5, 1 / 5, 8 / 50, 8.1 / 98, 11 / 98, 11.1 / 5, 14 / 5; flow rate: 18 mL / min; diluent: MeCN + HO + THF). The purified fraction was lyophilized to give 55 mg (39%) of 2-amino-9-((2R,3R,5S)-5-((S)-2-fluoro-1-hydroxyethyl)-3-hydroxytetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione (compound 23) as an off-white solid. 14 H 16 FN5O 5: 1H NMR (400MHz, DMSO-d6): δ 11.09 (brs, 1H), 6.59 (bs, 2H) 5.52 (d, J = 3.6 Hz, 1H), 5.43 (d, J = 4.8 Hz, 1H), 5.28 (d, J = 5.6 Hz, 1H), 4.80 (m, 1H), 4.58 (d, J = 2.4 Hz, 2H), 4.49-4.36 (m, 1H), 4.38-4.22 (m, 1H), 4.05 (q, J = 7.2 Hz, 1H), 3.77-3.70 (m, 1H), 3.22 (t, J = 2.2 Hz, 1H), 2.55 (m, 1H), 1.93 (m, 1H). ES+, m / z 354.1 [M+H] +

[0347] Example 21 2-amino-9-((2R,3R,5S)-5-((R)-2-fluoro-1-hydroxyethyl)-3-hydroxytetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 24 [ka]

[0348] Similar to the method used to synthesize compound 23, compound 24 was prepared in the following multiple steps. [ka]

[0349] Process 1 (R)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2-fluoroethyl acetate (66S) A 40% HF solution (1.38 mL, 32.2 mmol) was added to TBAF (32 mL, 32.2 mmol, 1 M in THF), and after 5 min, the solution was concentrated under reduced pressure (1 mbar, 100 °C). To this concentrated mixture was added a solution of (3aR,5S,6aR)-2,2-dimethyl-5-((S)-oxiran-2-yl)tetrahydrofuro[2,3-d][1,3]dioxole (51S) (2 g, 10.7 mmol) and KHF (251.6 mg, 3.2 mmol) in dry toluene (20 mL), and the mixture was maintained at 120 °C for 18 h. The reaction mixture was then cooled to room temperature, and AcO (3.2 mL, 32.2 mmol) and dry pyridine (6.1 mL, 86.0 mmol) were added. The reaction mixture was stirred at 40 °C for 2 h, then quenched with water and EtOAc (2 × 75 mL). The combined organic phase was washed with 2M HCl, saturated aqueous NaHCO3 (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude mixture was purified by silica gel column chromatography (100-200 mesh, 20% EtOAc in petroleum ether) to afford 1.6 g (61%) of (R)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2-fluoroethyl acetate (66S). 11 H 17 FO 5: 1 H NMR (400MHz, CDC13): δ 5.82 (d, J = 3.6 Hz, 1H), 5.19-5.14 (m, 1H), 4.74 (t, J = 4.2 Hz, 1H), 4.63 (m, 1H), 4.51 (m, 1H), 4.41 (dt, J = 10.8, 4.6 Hz, 1H), 2.14 (s, 3H), 2.11 (m, 1H) 1.66 (ddd, J = 13.2, 10.8, 4.8 Hz, 1H), 1.51 (s, 3H), 1.32 (s, 3H)

[0350] Process 2 (3R,5S)-5-((R)-1-acetoxy-2-fluoroethyl)tetrahydrofuran-2,3-diyl diacetate (67S) To a solution of (R)-1-((3aR,5S,6aR)-2,2-dimethyltetrahydrofuro[2,3-d][1,3]dioxol-5-yl)-2-fluoroethyl acetate (66S) (1.6 g, 4.03 mmol), acetic acid (3.6 mL, 64.5 mmol), and acetic anhydride (3.2 mL, 32.2 mmol) in anhydrous CHCl (20 mL) was added concentrated HSO (0.1 mL) at 0 °C. The resulting reaction mixture was stirred at 22 °C for 4 h and then quenched by the addition of ice-cold water. The organic layer was separated, and the aqueous phase was extracted with CHCl (2 × 75 mL). The combined organic layers were washed with saturated aqueous NaHCO (50 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (100-200 mesh, 30% EtOAc in petroleum ether) to give 0.8 g (44%) of (3R,5S)-5-((R)-1-acetoxy-2-fluoroethyl)tetrahydrofuran-2,3-diyl diacetate (67S) as a colorless oil. 12 H 17 FO7: 1 H NMR showed a mixture of α (minor) and β (major) anomers; 1 H NMR (400MHz, CDC13): δ 6.13 (s, 1H), 5.20 (d, J = 4.4 Hz, 2H), 5.12 (ddd, J = 20.4, 4.4, 1.3 Hz, 1H), 4.61-4.47 (m, 4H), 2.14 (s, 3H), 2.10 (s, 3H), 2.08 (s, 3H)

[0351] Process 3 (R)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2-fluoroethyl acetate (40) N-(6,8-dioxo-7-(prop-2-yn-1-yl)-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (6) (300 mg, 1.2 mmol), (3R,5S)-5-((R)-1-acetoxy-2-fluoroethyl)tetrahydrofuran-2,3-diyl diacetate (67S) (425.5 mg, 1.4 mmol), and BSA (0.9 mL, 3.6 mmol) were dissolved in 1,2-dichloroethane (15 mL) and stirred at 80° C. for 30 minutes under argon. The reaction mixture was cooled to RT, and the 1,2-dichloroethane was removed under reduced pressure. The residue was dissolved in MeCN (20 mL), and to the solution was added TMSOTf (0.33 mL, 1.5 mmol). The reaction mixture was stirred at 80° C. for 16 hours, cooled to room temperature, and concentrated under reduced pressure. The residue was added with saturated aqueous NaHCO3 (50 mL) and extracted with EtOAc (3 x 50 mL). The combined EtOAc layers were washed with water (30 mL), brine (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by GRACE flash chromatography (80% EtOAc in petroleum ether) to give 120 mg (20%) of (R)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2-fluoroethyl acetate (40) as an off-white solid. 20 H 22 FN5O 8: ES+, m / z 480.2 [M+H] +

[0352] Process 4 2-Amino-9-((2R,3R,5S)-5-((R)-2-fluoro-1-hydroxyethyl)-3-hydroxytetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione (compound 24) To a solution of (R)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(prop-2-yn-1-yl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2-fluoroethyl acetate (40) (120 mg, 0.25 mmol) in MeOH (10 mL) was added KCO (34.5 mg, 0.43 mmol) at 0 °C. The reaction mixture was stirred at RT for 16 h, and then the solvent was removed under reduced pressure at RT. The residue was purified by preparative HPLC (column: X-SELECT-C18 (150*19), 5μ mobile phase: 0.1% HCOH:MeCN in HO gradient: (T%B): -0 / 5, 8 / 40, 8.1 / 98, 10 / 98, 10.1 / 5, 12 / 5; flow rate: 17 mL / min; diluent: MeOH + HO + THF), and the purified fraction was lyophilized to give 35 mg (39%) of 2-amino-9-((2R,3R,5S)-5-((R)-2-fluoro-1-hydroxyethyl)-3-hydroxytetrahydrofuran-2-yl)-7-(prop-2-yn-1-yl)-7,9-dihydro-1H-purine-6,8-dione (compound 24) as an off-white solid. 14 H 16 FN5O 5: 1 H NMR (400MHz, DMSO-d6): δ 11.36 (brs, 1H), 6.69 (s, 2H), 5.53 (d, J = 2.8 Hz, 1H), 5.46 (brs, 1H), 5.24 (m, 1H), 4.73 (m, 1H), 4.59 (brs, 2H), 4.48-4.39 (m, 1H), 4.36-4.27 (m, 1H), 4.16 (m, 1H), 3.71-3.67 (m, 1H), 3.22 (s, 1H), 2.45 (m, 1H), 1.83 (m, 1H). 19 F NMR (376MHz, DMSO-d6): δ-228.9 (ddd, J = 47.4, 46.2, 19.4 Hz). ES+, m / z 354.1 [M+H] +

[0353] Example 222-amino-9-((2R,3R,5S)-3-hydroxy-5-((R)-2,2,2-trifluoro-1-hydroxyethyl)tetrahydrofuran-2-yl)-7-propyl-7,9-dihydro-1H-purine-6,8-dione, compound 25 [ka]

[0354] Compound 25 was prepared in the following multiple steps. [ka]

[0355] Process 1 2-Amino-6-(benzyloxy)-9-(4-methoxybenzyl)-7-propyl-7,9-dihydro-8H-purin-8-one (41) 1-Iodopropane (1.59 mL, 15.91 mmol) was added to a suspension of 2-amino-6-(benzyloxy)-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (3) (4 g, 10.61 mmol) and K2CO3 (3.66 g, 26.52 mmol) in DMF (50 mL) at 0 °C and stirred at room temperature for 16 h. The reaction mixture was quenched with ice water (100 mL), diluted with diethyl ether (80 mL), and stirred for 15 min. The resulting precipitate was collected by filtration, washed with water, and dried to give 2-amino-6-(benzyloxy)-9-(4-methoxybenzyl)-7-propyl-7,9-dihydro-8H-purin-8-one (41) (3.5 g, 75%) as a brown solid; C 23 H 25 N5O3: 1H NMR (400MHz, DMSO-d6): δ 7.48-7.34 (m, 5H), 7.20 (d, J = 8.8 Hz, 2H), 6.87 (d, J = 8.8 Hz, 2H), 6.39 (s, 2H), 5.41 (s, 2H), 4.82 (s, 2H), 3.73-3.69 (m, 2H), 3.71 (s, 3H), 1.58 (m, 2H), 0.73 (t, J = 7.4 Hz, 3H). ES+, m / z 420.2 [M+H] +

[0356] Process 2 :2-Amino-7-propyl-7,9-dihydro-1H-purine-6,8-dione (42) Trifluoromethanesulfonic acid (4 mL, 50.11 mmol) was added to a suspension of 2-amino-6-(benzyloxy)-9-(4-methoxybenzyl)-7-propyl-7,9-dihydro-8H-purin-8-one (41) (3.5 g, 8.35 mmol) in trifluoroacetic acid (4.4 mL, 50.11 mmol) at 0 °C under an argon atmosphere, and the resulting reaction mixture was slowly warmed to room temperature and stirred for 3 h. The reaction mixture was quenched with ice-cold water, and the pH was made basic with excess saturated aqueous NaHCO3 under vigorous stirring and collected by filtration. The residual solid was dissolved in diethyl ether (150 mL), stirred for 30 min, filtered, and dried to give 2-amino-7-propyl-7,9-dihydro-1H-purine-6,8-dione (42) (1.6 g; 91%) as a pale yellow solid: CH 11 N5O2: 1 H NMR (400MHz, DMSO-d6): δ 11.04 (s, 1H), 10.64 (s, 1H), 6.33 (s, 2H), 3.67 (t, J = 7.2 Hz, 2H), 1.62 (sextet, J = 7.2 Hz, 2H), 0.81 (t, J = 7.6 Hz, 3H). ES+, m / z 210.1 [M+H] +

[0357] Process 3N-(6,8-dioxo-7-propyl-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (43) Acetic anhydride (2.27 mL, 22.96 mmol) was added to a solution of 2-amino-7-propyl-7,9-dihydro-1H-purine-6,8-dione (42) (1.6 g, 7.65 mmol) in AcOH (20 mL) at ambient temperature under an argon atmosphere, and the resulting reaction mixture was heated at 120° C. for 10 h. With vigorous stirring, the reaction mixture was cooled to 0° C., after which a solid formed. After stirring at 0° C. for an additional 30 min, the solid was collected by filtration and dried under reduced pressure to give N-(6,8-dioxo-7-propyl-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (43) (1.3 g, 68%) as a pale yellow solid: C 10 H 13 N5O3: 1 H NMR (400MHz, DMSO-d6): δ 11.98 (s, 1H), 11.65 (s, 1H), 11.64 (s, 1H), 3.76 (t, J = 7.0 Hz, 2H), 2.15 (s, 3H), 1.67-1.62 (m, 2H), 0.83 (t, J = 7.4 Hz, 3H). ES+, m / z 252.1 [M+H] +

[0358] Process 4 (R)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-propyl-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (44A) N-(6,8-Dioxo-7-propyl-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (43) (500 mg, 1.99 mmol), (2S,3R,5S)-5-(1-acetoxy-2,2,2-trifluoroethyl)tetrahydrofuran-2,3-diyl diacetate (22S) (784 mg, 2.39 mmol), and BSA (1.5 mL, 5.97 mmol) were dissolved in 1,2-dichloroethane (20 mL), and the resulting reaction mixture was stirred at 80° C. for 30 minutes under argon. The reaction mixture was cooled to RT, and the 1,2-dichloroethane was removed under reduced pressure. The residue was dissolved in MeCN (20 mL), followed by the addition of TMSOTf (0.54 mL, 2.98 mmol). The reaction mixture was heated at 80° C. for 16 hours with stirring, cooled to room temperature, and concentrated under reduced pressure. The residue was added with saturated aqueous NaHCO3 (50 mL) and extracted with EtOAc (3 x 50 mL). The combined EtOAc layers were washed with water (30 mL), brine (30 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by GRACE flash chromatography (80% EtOAc in petroleum ether) to give 400 mg of a diastereomeric mixture (estimated ca. 3:1 44A:44B) by LC / MS, which was purified by preparative HPLC (KROMOSIL-C18 (150*25MM), 7µ mobile phase: 10 mM Methyl 44A in HO). Further purification with ammonium bicarbonate:MeCN gradient: (T % B): -0 / 20, 8 / 50, 13 / 50, 13.1 / 98, 15 / 98, 15.1 / 20, 17 / 20; flow rate: 22 mL / min; diluent: (MeCN + HO + THF) gave approximately 100 mg of (R)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-propyl-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy C)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-propyl-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (44A) and approximately 50 mg of (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-propyl-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (44B), both as pale yellow solids, were obtained after lyophilization. 20 H24 F3N5O 8: 1 H NMR (500MHz, DMSO-d6): δ 12.15 (brs, 1H), 11.75 (brs, 1H), 5.75 (d, J = 1.5 Hz, 1H), 5.66 (m, 2H), 4.51 (m, 1H), 3.82 (t, J = 7.0 Hz, ES+, m / z 520.1 [M+H] + . (44B): 1 H NMR (500MHz, CDCl3): δ 11.96 (brs, 1H), 9.50 (brs, 1H), 6.16 (s, 1H), 6.02 (t, J = 7.0 Hz, 1H), 5.41 (d, J = 4.5 Hz, 1H), 4.65 (m, 1H), 3.97 (t, J = 7.3 Hz, 2H), 2.94 (m, 1H), 2.29 (s, 3H), 2.25 (s, 3H), 2.16 (m, 1H), 2.14 (s, 6H) 1.77 (m, 2H), 0.94 (t, J = 7.0 Hz, 3H)

[0359] Process 5 2-Amino-9-((2R,3R,5S)-3-hydroxy-5-((R)-2,2,2-trifluoro-1-hydroxyethyl)tetrahydrofuran-2-yl)-7-propyl-7,9-dihydro-1H-purine-6,8-dione (compound 25) To a solution of (R)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-propyl-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (44A) (100 mg, 0.192 mmol) in MeOH (20 mL) was added KCO (40 mg, 0.289 mmol) at 0 °C, and the reaction mixture was stirred at rt for 16 h. Methanol was removed under reduced pressure at 30 °C. The residue was purified by reverse-phase GRACE flash chromatography using 10 mM ammonium bicarbonate in HO as eluent. The purified fractions were lyophilized to give 60 mg (80%) of 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((R)-2,2,2-trifluoro-1-hydroxyethyl)tetrahydrofuran-2-yl)-7-propyl-7,9-dihydro-1H-purine-6,8-dione (compound 25) as a white solid. 14 H 18 F3N5O 5: 1 H NMR (500MHz, DMSO-d6): δ 11.06 (brs, 1H), 6.53 (brs, 2H), 6.39 (d, J = 8.0 Hz, 1H), 5.58 (d, J = 3.50 Hz, 1H), 5.52 (d, J = 4.5 Hz, 1H), 4.76 (m, 1H), 4.34 (m, 1H), 4.02 (m, 1H), 3.74 (t, J = 7.3 Hz, 2H), 2.54 (m, 1H), 1.97 (m, 1H), 1.63 (sextet, J = 7.3 Hz, 2H), 0.82 (t, J = 7.3 Hz, 3H). ES+, m / z 394.0 [M+H] +

[0360] Example 23 2-amino-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-propyl-7,9-dihydro-1H-purine-6,8-dione, compound 26 [ka]

[0361] Compound 26 was prepared in two steps. [ka]

[0362] Process 1 (S)-1-((2R,3S,4S,5R)-5-(2-acetamido-6,8-dioxo-7-propyl-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (45) N-(6,8-Dioxo-7-propyl-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (43) (300 mg, 1.1 mmol), (3S,4S,5R)-5-((S)-1-acetoxypropyl)-4-fluorotetrahydrofuran-2,3-diyl diacetate (37S) (548.6 mg, 1.7 mmol), and BSA (0.9 mL, 3.58 mmol) were dissolved in 1,2-dichloroethane (20 mL), and the resulting reaction mixture was stirred at 80° C. for 30 minutes under argon. The reaction mixture was cooled to RT, and the 1,2-dichloroethane was removed under reduced pressure. The residue was dissolved in MeCN (20 mL), followed by the addition of TMSOTf (0.33 mL, 1.79 mmol). The reaction mixture was heated at 80° C. for 16 hours, cooled to room temperature, and concentrated under reduced pressure. The residue was added with saturated aqueous NaHCO3 (60 mL) and extracted with EtOAc (3 x 60 mL). The combined EtOAc layers were washed with water (30 mL), brine (30 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (80% EtOAc in petroleum ether) to give 200 mg (33%) (S)-1-((2R,3S,4S,5R)-5-(2-acetamido-6,8-dioxo-7-propyl-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (45) as an off-white solid. 21H 28 FN5O 8: ES+, m / z 498.2 [M+H] +

[0363] Process 2 2-Amino-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-propyl-7,9-dihydro-1H-purine-6,8-dione (compound 26) To a solution of (S)-1-((2R,3S,4S,5R)-5-(2-acetamido-6,8-dioxo-7-propyl-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (45) (200 mg, 0.4 mmol) in methanol (10 mL) was added KCO (111 mg, 0.80 mmol) at rt. The stirred reaction mixture was monitored by LC / MS. After 16 hours, the mixture was concentrated under reduced pressure to give a concentrated mass, which was subjected to preparative HPLC (Column: LUNA@OMEGA (250*21.2), 5μ mobile phase: 0.1% HCOH in HO:MeCN gradient: (T%B): -0 / 10, 8 / 50, 9 / 50, 9.1 / 98, 12 / 98, 12.1 / 10, 15 / 10; flow rate: 17 mL / min) to give 30 mg (21%) of 2-amino-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-propyl-7,9-dihydro-1H-purine-6,8-dione (Compound 26) as an off-white solid; C 15 H 22 FN5O5: 1 1 H NMR showed the product to be the formate salt. 1H NMR (500MHz, DMSO-d6): δ 11.59 (brs, 1H), 8.48 (s, 1H), 6.79 (s, 2H), 5.95 (m, 1H), 5.37 (d, J = 6.5 Hz, 1H) 5.32 (ddd, J = 24.8, 6.5, 2.8 Hz, 1H), 5.00 (dd, J = 50.5, 4.5 Hz, 1H), 4.91 (m, 1H), 3.82 (dt, J = 21.5, 5.8 Hz, 1H), 3.75 (t, J = 7.0 Hz, 2H), 3.51 (brs, 1H), 1.64 (m, 2H), 1.50-1.43 (m, 1H), 1.36-1.31 (m, 1H), 0.91 (t, J = 7.3 Hz, 3H), 0.84 (t, J = 7.3 Hz, 3H). ES+, m / z 372.2 [M+H] +

[0364] Example 24 2-amino-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(3,3,3-trifluoropropyl)-7,9-dihydro-1H-purine-6,8-dione, compound 27 [ka]

[0365] Compound 27 was prepared by the following multiple steps. [ka]

[0366] Process 1 : 3,3,3-trifluoropropyl methanesulfonate (46) To a solution of 3,3,3-trifluoropropan-l-ol [2240-88-2] (5.0 g, 43.8 mmol) in CHCl (10 mL) was added TEA (12.3 mL, 87.7 mmol) followed by methanesulfonyl chloride (5 mL, 65.8 mmol) dropwise at 0 °C. The resulting mixture was stirred at room temperature for 3 h. The reaction mixture was diluted with CHCl, and the organic phase was washed with brine, dried over NaSO, filtered, and concentrated to give the crude product, which was purified by silica gel chromatography eluting with petroleum ether / ethyl acetate (30%) to give 3,3,3-trifluoropropyl methanesulfonate [911116-16-0] (46) (7.0 g, 83% yield) as a light yellow oil. CHFOS: 1 H NMR (400MHz, CDCl3): 4.43 (t, J = 5.6 Hz, 2H), 3.05(s, 3H), 2.66-2.55 (m, 2H)

[0367] Process 2 2-Amino-6-(benzyloxy)-9-(4-methoxybenzyl)-7-(3,3,3-trifluoropropyl)-7,9-dihydro-8H-purin-8-one (47) To a stirred suspension of 2-amino-6-(benzyloxy)-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one 3 (8.0 g, 21.22 mmol) and KCO (5.85 g, 42.44 mmol) in DMF (100 mL) was added 3,3,3-trifluoropropyl methanesulfonate 46 (6.11 g, 31.83 mmol) at 0° C. The reaction mixture was warmed to room temperature and stirred for 16 h. The reaction was quenched with ice water (200 mL), and diethyl ether (80 mL) was added and stirred for 15 min. The resulting precipitate was collected by filtration, washed with water, and dried to give 2-amino-6-(benzyloxy)-9-(4-methoxybenzyl)-7-(3,3,3-trifluoropropyl)-7,9-dihydro-8H-purin-8-one (47) (6.5 g, 75%) as a brown solid; C 23 H 22 F3N5O3: 1H NMR (400MHz, DMSO-d6): 7.48 (d, J = 6.8 Hz, 2H), 7.40 (d, J = 6.8 Hz, 2H), 7.37 (m, 1H), 7.20 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.4 Hz, ES+, m / z 474.1 [M+H] +

[0368] Process 3 2-Amino-7-(3,3,3-trifluoropropyl)-7,9-dihydro-1H-purine-6,8-dione (48) Trifluoromethanesulfonic acid (2.06 mL, 25.36 mmol) was added to a suspension of 2-amino-6-(benzyloxy)-9-(4-methoxybenzyl)-7-(3,3,3-trifluoropropyl)-7,9-dihydro-8H-purin-8-one (47) (3.0 g, 6.3424 mmol) in trifluoroacetic acid (2.12 mL, 25.36 mmol) at room temperature under an argon atmosphere, and the resulting reaction mixture was stirred at room temperature for 8 hours. The reaction mixture was quenched with ice-cold water. The pH of the reaction mixture was made basic with saturated aqueous NaHCO3 under vigorous stirring, and the insoluble material was collected by filtration. The collected solid was dissolved in ethyl acetate, stirred for 30 minutes, filtered, and dried to give 2-amino-7-(3,3,3-trifluoropropyl)-7,9-dihydro-1H-purine-6,8-dione (48) (1.2 g, 75%) as a brown solid. C8H8F3N5O2: 1 H NMR (400MHz, DMSO-d6): δ 11.17 (s, 1H), 10.74 (s, 1H), 6.41 (s, 2H), 3.97 (t, J = 6.8 Hz, 2H), 2.73-2.70 (m, 2H). ES+, m / z 264.1 [M+H] +

[0369] Process 4N-(6,8-dioxo-7-(3,3,3-trifluoropropyl)-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (49) Acetic anhydride (0.69 mL, 6.844 mmol) was added to a solution of 2-amino-7-(3,3,3-trifluoropropyl)-7,9-dihydro-1H-purine-6,8-dione (48) (1.2 g, 4.562 mmol) in AcOH (15 mL) at room temperature under an argon atmosphere, and the resulting reaction mixture was stirred at 130° C. for 8 hours. The reaction mixture was cooled to 0° C., and the solid formed was stirred for 30 minutes. The product was collected by filtration, washed with EtOH, and dried under reduced pressure to give N-(6,8-dioxo-7-(3,3,3-trifluoropropyl)-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (49) (800 mg, 61%) as a brown solid. 10 H 10 F3N5O3: 1 H NMR (400MHz, DMSO-d6): δ 12.05 (s, 1H), 11.77 (s, 1H), 11.68 (s, 1H), 4.06 (t, J = 6.8 Hz, 2H), 2.75 (m, 2H), 2.15 (s, 3H). ES+, m / z 306.1 [M+H] +

[0370] Process 5 (S)-1-((2R,3S,4S,5R)-5-(2-acetamido-6,8-dioxo-7-(3,3,3-trifluoropropyl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (50) N-(6,8-dioxo-7-(3,3,3-trifluoropropyl)-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (49) (350 mg, 1.147 mmol), (3S,4S,5R)-5-((S)-1-acetoxypropyl)-4-fluorotetrahydrofuran-2,3-diyl diacetate (37S) (450 mg, 1.491 mmol), and BSA (0.873 mL, 3.441 mmol) were dissolved in 1,2-dichloroethane (20 mL), and the reaction mixture was stirred at 80 °C for 30 min under argon. The reaction mixture was cooled to rt, and the 1,2-dichloroethane was removed under reduced pressure. The residue was dissolved in MeCN (20 mL), followed by the addition of TMSOTf (0.318 mL, 1.720 mmol). The stirred reaction mixture was heated at 80 °C for 16 h, cooled to room temperature, and concentrated under reduced pressure. Saturated aqueous NaHCO (60 mL) was added to the residue, which was then extracted with EtOAc (3 × 60 mL). The combined EtOAc layers were washed with water (30 mL), brine (30 mL), dried over NaSO, filtered, and concentrated under reduced pressure. The crude compound was purified by flash column (80% EtOAc in petroleum ether) to give (S)-1-((2R,3S,4S,5R)-5-(2-acetamido-6,8-dioxo-7-(3,3,3-trifluoropropyl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (50) (210 mg, 33%) as an off-white solid. 21 H 25 F4N5O 8: ES+, m / z 552.3 [M+H] +

[0371] Process 6 2-Amino-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(3,3,3-trifluoropropyl)-7,9-dihydro-1H-purine-6,8-dione (compound 27) To a solution of (S)-1-((2R,3S,4S,5R)-5-(2-acetamido-6,8-dioxo-7-(3,3,3-trifluoropropyl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (50) (210 mg, 0.381 mmol) in methanol (6 mL) was added KCO (79 mg, 0.571 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 hours and then concentrated under reduced pressure to give a concentrated mass. The crude product was purified by GRACE reverse-phase chromatography (0.1% HCOH:MeCN) to give 2-amino-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(3,3,3-trifluoropropyl)-7,9-dihydro-1H-purine-6,8-dione (compound 27) (30 mg, 19%) as an off-white solid. 15 H 19 F4N5O5: 1 1 H NMR showed that the compound existed as the formate salt. 1 H NMR (500MHz, DMSO-d6): δ 11.25 (brs, 1H), 8.42 (s, 1H), 6.69 (s, 2H), 5.91 (s, 1H), 5.39 (d, J = 6.5 Hz, 1H), 5.32-5.25 (m, 1H), 4.98 (dt, J = 48.5, 3.5 Hz, 1H), 4.84 (d, J = 6.0 Hz, 1H), 4.04 (t, J = 7.0 Hz, 2H), 3.81 (dt, J = 23.0, 5.8 Hz, 1H), 3.52 (m, 1H), 2.75 (m, 2H), 1.49 (m, 1H), 1.34 (m, 1H), 0.91 (t, J = 7.5 Hz, 3H). ES+, m / z 426.2 [M+H] +

[0372] Example 252-amino-9-((2R,3R,5S)-3-hydroxy-5-((R)-2,2,2-trifluoro-1-hydroxyethyl)tetrahydrofuran-2-yl)-7-(3,3,3-trifluoropropyl)-7,9-dihydro-1H-purine-6,8-dione, compound 28 [ka]

[0373] Compound 28 was prepared in two steps. [ka]

[0374] Process 1 (R)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(3,3,3-trifluoropropyl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (51A) and (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(3,3,3-trifluoropropyl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (51B) To a suspension of N-(6,8-dioxo-7-(3,3,3-trifluoropropyl)-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (49) (500 mg, 1.63 mmol) and (2S,3R,5S)-5-(1-acetoxy-2,2,2-trifluoroethyl)tetrahydrofuran-2,3-diyl diacetate (22S) (645 mg, 1.96 mmol) in 1,2-dichloroethane (20 mL) was added BSA (1.24 mL, 4.91 mmol). The reaction mixture was stirred at 80 °C for 30 min under argon and then cooled to RT. The 1,2-dichloroethane was removed under reduced pressure, and the residue was dissolved in MeCN (20 mL), followed by the addition of TMSOTf (0.44 mL, 2.45 mmol). The reaction mixture was heated at 80 °C for 16 h, cooled to room temperature, and concentrated under reduced pressure. Saturated aqueous NaHCO (50 mL) was added to the concentrate and extracted with EtOAc (3 × 50 mL). The combined EtOAc layers were washed with water (30 mL), brine (30 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude product was purified by GRACE flash chromatography (80% EtOAc in petroleum ether) to afford 350 mg (approximately 62:38 diastereomeric mixture by LC / MS) of (R,S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(3,3,3-trifluoropropyl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (51A:51B) as an off-white solid.Further purification by preparative HPLC (LUNA OMEGA C18 (250*21.2), 5μ mobile phase: 10 mM ammonium bicarbonate in HO:MeCN gradient: (T%B): -0 / 40, 8 / 65, 10 / 65, 10.1 / 98, 14 / 98, 14.1 / 40, 17 / 40; flow rate: 17 mL / min; diluent: MeCN+HO+THF) gave 60 mg of (R)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(3,3,3-trifluoropropyl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(3,3,3-trifluoropropyl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (51A) and 40 mg of (S)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(3,3,3-trifluoropropyl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (51A) were obtained after lyophilization, both as off-white solids. (51A):C 20 H 21 F6N5O8: ES+, m / z 574.4 [M+H] + . (51B): C 20 H 21 F6N5O8: ES+, m / z 574.4 [M+H] +

[0375] Process 2 2-Amino-9-((2R,3R,5S)-3-hydroxy-5-((R)-2,2,2-trifluoro-1-hydroxyethyl)tetrahydrofuran-2-yl)-7-(3,3,3-trifluoropropyl)-7,9-dihydro-1H-purine-6,8-dione (compound 28) To a solution of (R)-1-((2S,4R,5R)-5-(2-acetamido-6,8-dioxo-7-(3,3,3-trifluoropropyl)-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (51A) (60 mg, 0.1 mmol) in MeOH (20 mL) was added KCO (21.6 mg, 1.5 mmol) at 0 °C, and the reaction mixture was stirred at RT for 16 h. Methanol was removed under reduced pressure at 30 °C. The residue was directly purified by reverse-phase GRACE flash chromatography using 10 mM ammonium bicarbonate in HO. The purified fractions were lyophilized to give 25 mg (54%) of 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((R)-2,2,2-trifluoro-1-hydroxyethyl)tetrahydrofuran-2-yl)-7-(3,3,3-trifluoropropyl)-7,9-dihydro-1H-purine-6,8-dione (compound 28) as a white solid. 14 H 15 F6N5O 5: 1 H NMR (400MHz, DMSO-d6): δ 11.07 (brs, 1H), 6.56 (brs, 2H), 6.31 (d, J = 8.0 Hz, 1H), 5.58 (d, J = 3.2 Hz, 1H), 5.53 (d, J = 4.8 Hz, 1H), 4.73 (m, 1H), 4.33 (m, 1H), 4.05 (t, J = 6.8 Hz, 2H), 4.02(m, 1H), 2.74 (m, 2H), 2.55 (m, 1H), 1.96 (m, 1H). ES+, m / z 448.3 [M+H] +

[0376] Example 26 2-Amino-7-(cyclopropylmethyl)-9-((2R,3R,5S)-3-hydroxy-5-((R)-2,2,2-trifluoro-1-hydroxyethyl)tetrahydrofuran-2-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 29 [ka]

[0377] Compound 29 was prepared in two steps. [ka]

[0378] Process 1 (R)-1-((2S,4R,5R)-5-(2-acetamido-7-(cyclopropylmethyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (52A) and (S)-1-((2S,4R,5R)-5-(2-acetamido-7-(cyclopropylmethyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (52B). 2-Amino-7-(cyclopropylmethyl)-7,9-dihydro-1H-purine-6,8-dione (12) (350 mg, 1.33 mmol), (2S,3R,5S)-5-(1-acetoxy-2,2,2-trifluoroethyl)tetrahydrofuran-2,3-diyl diacetate (22S) (567 mg, 1.73 mmol), and BSA (1.0 mL, 3.9 mmol) were dissolved in 1,2-dichloroethane (20 mL), and the mixture was stirred at 80 °C for 30 minutes under argon. The reaction mixture was cooled to rt, and the 1,2-dichloroethane was removed under reduced pressure. The residue was dissolved in MeCN (20 mL), followed by the addition of TMSOTf (0.36 mL, 1.99 mmol). The reaction mixture was heated at 80 °C for 16 hours, cooled to room temperature, and concentrated under reduced pressure. The resulting residue was added with saturated aqueous NaHCO3 (50 mL) and then extracted with EtOAc (3 x 50 mL). The combined EtOAc layers were washed with water (30 mL), brine (30 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by GRACE flash chromatography (using 80% EtOAc in petroleum ether as eluent) to afford 260 mg (approximately 5:3 diastereomeric mixture) of (R,S)-1-((2S,4R,5R)-5-(2-acetamido-7-(cyclopropylmethyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (52A:52B) as a brown solid.Further purification by preparative HPLC (LUNA OMEGA C18 (250*21.2), 5μ mobile phase: 10 mM ammonium bicarbonate in HO:MeCN gradient: (T%B): -0 / 30, 8 / 60, 12 / 60, 12.1 / 98, 14 / 98, 14.1 / 30, 17 / 30; flow rate: 17 mL / min; diluent: MeCN+HO+THF) gave 130 mg of (R)-1-((2S,4R,5R)-5-(2-acetamido-7-(cyclopropylmethyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy (tetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (52A) and 60 mg (S)-1-((2S,4R,5R)-5-(2-acetamido-7-(cyclopropylmethyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (52B) were obtained after lyophilization, both as off-white solids: C. 21 H 24 F3N5O 8: ES+, m / z 532.4 [M+H] + . (52B): C 21 H 24 F3N5O 8: ES+, m / z 532.4 [M+H] +

[0379] Process 2 2-Amino-7-(cyclopropylmethyl)-9-((2R,3R,5S)-3-hydroxy-5-((R)-2,2,2-trifluoro-1-hydroxyethyl)tetrahydrofuran-2-yl)-7,9-dihydro-1H-purine-6,8-dione (compound 29) To a solution of (R)-1-((2S,4R,5R)-5-(2-acetamido-7-(cyclopropylmethyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (52A) (130 mg, 0.24 mmol) in MeOH (20 mL) was added KCO (50.6 mg, 0.36 mmol) at 0 °C. The reaction mixture was stirred at rt for 16 h, after which the methanol was removed under reduced pressure at 30 °C. The residue was purified by reverse-phase GRACE flash chromatography using 0.01% aqueous NHHCO and MeCN. The purified fractions were lyophilized to give 75 mg (75%) of 2-amino-7-(cyclopropylmethyl)-9-((2R,3R,5S)-3-hydroxy-5-((R)-2,2,2-trifluoro-1-hydroxyethyl)tetrahydrofuran-2-yl)-7,9-dihydro-1H-purine-6,8-dione (compound 29) as a white solid. 15 H 18 F3N5O 5: 1 H NMR (400MHz, DMSO-d6): δ 10.95 (brs, 1H), 6.50 (brs, 2H), 6.33 (d, J = 8.4 Hz, 1H), 5.59 (d, J = 3.6 Hz, 1H), 5.53 (d, J = 4.8 Hz, 1H), 4.77 (m, 1H), 4.34 (m, 1H), 4.03 (m, 1H), 3.65 (d, J = 7.2 Hz, 2H), 2.54 (m, 1H), 1.97 (m, 1H), 1.18 (m, 1H), 0.41 (m, 2H), 0.35 (m, 2H). ES+, m / z 406.0 [M+H] +

[0380] Example 27 2-amino-7-butyl-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 30 [ka]

[0381] Compound 30 was prepared in the following multiple steps. [ka]

[0382] Process 1 2-Amino-6-(benzyloxy)-7-butyl-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (53) Butyl bromide (2.15 mL, 15.9 mmol) was added to a suspension of 2-amino-6-(benzyloxy)-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (3) (5 g, 13.25 mmol) and K2CO3 (2.7 g, 19.85 mmol) in DMF (80 mL) at 0 °C and stirred at room temperature for 18 h. The reaction mixture was quenched with ice water (120 mL), diluted with diethyl ether (80 mL), and stirred for 15 min. The resulting precipitated solid was collected by filtration, washed with water, and dried to give 2-amino-6-(benzyloxy)-7-butyl-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (53) (4.9 g, 85%) as a brown solid. 24 H 27 N5O3: 1 H NMR (400MHz, DMSO-d6): δ 7.47 (d, J = 6.8 Hz, 2H), 7.40 (d, J = 6.8 Hz, 2H), 7.37 (m, 1H), 7.20 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.8 Hz, 2H), 6.39 (s, 2H), 5.41 (s, 2H), 4.82 (s, 2H), 3.74 (m, 2H), 3.71 (s, 3H), 1.53 (m, 2H), 1.15 (m, 2H), 0.77 (t, J = 7.4 Hz, 3H). ES+, m / z 434.1 [M+H] +

[0383] Process 2 2-Amino-7-butyl-7,9-dihydro-1H-purine-6,8-dione (54) Trifluoromethanesulfonic acid (2.99 mL, 33.9 mmol) was added to a suspension of 2-amino-6-(benzyloxy)-7-butyl-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (53) (4.9 g, 11.31 mmol) in trifluoroacetic acid (2.59 mL, 33.9 mmol) at 0 °C under an argon atmosphere. The reaction mixture was slowly warmed to room temperature and stirred for 3 h. Ice-cold water was added to the reaction mixture, and the pH was made basic with saturated aqueous NaHCO3 solution under vigorous stirring. The resulting solid was collected by filtration, dissolved in ethyl acetate (50 mL), and stirred for 30 min. The insoluble material was collected by filtration and dried to give 2-amino-7-butyl-7,9-dihydro-1H-purine-6,8-dione (54) (2.0 g, 79%) as a brown solid. CH 13 N5O2: 1 H NMR (400MHz, DMSO-d6): δ 11.05 (s, 1H), 10.62 (s, 1H), 6.33 (s, 2H), 3.71 (t, J = 7.0 Hz, 2H), 1.57 (m, 2H), 1.23 (m, 2H), 0.8 (t, J = 7.4 Hz, 3H). ES+, m / z 224.1 [M+H] +

[0384] Process 3 N-(7-butyl-6,8-dioxo-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (55) Acetic anhydride (2.54 ml, 26.90 mmol) was added to a solution of 2-amino-7-butyl-7,9-dihydro-1H-purine-6,8-dione (54) (2 g, 8.96 mmol) in AcOH (20 mL) at ambient temperature under an argon atmosphere. The reaction mixture was stirred at 130° C. for 3 hours under argon and cooled to 0° C. A solid formed, and stirring was continued for 30 minutes. The product was collected by filtration, washed with ethanol, and dried under reduced pressure to give N-(7-butyl-6,8-dioxo-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (55) (1.5 g, 66%) as a brown solid. 11 H 13 N5O3: ES+, m / z 266.3 [M+H] +

[0385] Process 4 (S)-1-((2R,3S,4S,5R)-5-(2-acetamido-7-butyl-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (56) To a solution of N-(7-butyl-6,8-dioxo-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (55) (340 mg, 1.28 mmol) and (3S,4S,5R)-5-((S)-1-acetoxypropyl)-4-fluorotetrahydrofuran-2,3-diyl diacetate (37S) (504 mg, 1.66 mmol) in 1,2-dichloroethane (10 mL) was added BSA (777 mg, 3.84 mmol). The reaction mixture was stirred at 80° C. for 30 min under argon, cooled to RT, and the 1,2-dichloroethane was removed under reduced pressure. The residue was dissolved in MeCN (20 mL) and TMSOTf (0.43 mL, 1.92 mmol) was added. The reaction mixture was heated at 80° C. for 16 h, cooled to RT, and concentrated under reduced pressure. The residue was added with saturated aqueous NaHCO3 (50 mL) and then extracted with EtOAc (3 x 50 mL). The combined EtOAc layers were washed with water (30 mL), brine (30 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by GRACE flash chromatography (80% EtOAc in petroleum ether) to give (150 mg, 22.9%) (S)-1-((2R,3S,4S,5R)-5-(2-acetamido-7-butyl-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (56) as a yellow gummy solid. 22 H 30 FN5O 8: ES+, m / z 512.4 [M+H] +

[0386] Process 5 2-Amino-7-butyl-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7,9-dihydro-1H-purine-6,8-dione (compound 30) To a solution of (S)-1-((2R,3S,4S,5R)-5-(2-acetamido-7-butyl-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxy-3-fluorotetrahydrofuran-2-yl)propyl acetate (56) (150 mg, 0.29 mmol) in MeOH (10 mL) was added KCO (60 mg, 0.44 mmol) at 0 °C, and the reaction mixture was stirred at RT for 16 h. Methanol was removed under reduced pressure at 30 °C. The residue was directly subjected to normal-phase GRACE flash chromatography (using 7% MeOH in CHCl) followed by reverse-phase GRACE flash chromatography (using 0.01% aqueous HCOH in MeCN). The purified fractions were collected and lyophilized to give (20 mg, 17.69%) 2-amino-7-butyl-9-((2R,3S,4R,5R)-4-fluoro-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7,9-dihydro-1H-purine-6,8-dione (compound 30) as a white solid. 16 H 24 FN5O 5: 1 H NMR (500MHz, DMSO-d6): δ 10.90 (s, 1H), 6.51 (brs, 2H), 5.90 (d, J = 5.5 Hz, 1H), 5.37-5.32 (m, 2H), 5.00 (dt, J = 55.5, 2.5 Hz, 1H), 4.84 (d, J = 7.0 Hz, 1H), 3.83 (m, 1H), 3.78 (t, J =3.5 Hz, 2H), 3.51 (m, 1H), 1.60 (m, 2H), 1.51 (m, 1H), 1.33 (m, 1H), 1.26 (m, 2H), 0.91 (t, J = 7.5 Hz, 3H), 0.88 (t, J = 7.5 Hz, 3H). ES+, m / z 386.2 [M+H] +

[0387] Example 282-Amino-7-butyl-9-((2R,3R,5S)-3-hydroxy-5-((R)-2,2,2-trifluoro-1-hydroxyethyl)tetrahydrofuran-2-yl)-7,9-dihydro-1H-purine-6,8-dione, compound 31 [ka]

[0388] Compound 31 was prepared in two steps. [ka]

[0389] Process 1 (R)-1-((2S,4R,5R)-5-(2-acetamido-7-butyl-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (57A) and (S)-1-((2S,4R,5R)-5-(2-acetamido-7-butyl-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (57B). N-(7-butyl-6,8-dioxo-6,7,8,9-tetrahydro-1H-purin-2-yl)acetamide (55) (350 mg, 1.32 mmol), (2S,3R,5S)-5-(1-acetoxy-2,2,2-trifluoroethyl)tetrahydrofuran-2,3-diyl diacetate (22S) (563 mg, 1.71 mmol), and BSA (1 mL, 3.9 mmol) were dissolved in 1,2-dichloroethane (20 mL), and the resulting reaction mixture was stirred at 80 °C for 30 min under argon. The reaction mixture was cooled to rt, and the 1,2-dichloroethane was removed under reduced pressure. The residue was dissolved in MeCN (20 mL), followed by the addition of TMSOTf (0.36 mL, 1.98 mmol), and the reaction mixture was heated at 80 °C for 16 h. The reaction mixture was then cooled to rt and concentrated under reduced pressure. A saturated aqueous solution of NaHCO3 (50 mL) was added to the residue and extracted with EtOAc (3 × 50 mL). The combined EtOAc layers were washed with water (30 mL), brine (30 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude compound was purified by GRACE flash chromatography (80% EtOAc in petroleum ether) to give 250 mg (46% and 29% diastereomeric mixture by LC / MS) of (R,S)-1-((2S,4R,5R)-5-(2-acetamido-7-butyl-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate as a brown solid.Further purification by preparative HPLC (X-SELECT-C18 (250*19MM), 5μ mobile phase: 10 mM ammonium bicarbonate in HO:MeCN gradient: (T%B): -0 / 30, 8 / 5, 13 / 55, 13.1 / 98, 15 / 98, 15.1 / 30, 18 / 30; flow rate: 17 mL / min; diluent: MeCN+HO+THF) gave 90 mg of (R)-1-((2S,4R,5R)-5-(2-acetamido-7-butyl-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (57A) (peak 1) and 60 mg of 57B. (S)-1-((2S,4R,5R)-5-(2-acetamido-7-butyl-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (57B) (peak 2) was obtained after lyophilization of the collected purified fractions, both as an off-white solid: C. 21 H 26 F3N5O8: 1 H NMR (500MHz, DMSO-d6): δ 12.15 (bs, 1H), 11.61 (brs, 1H), 5.75 (s, 1H), 5.65 (m, 2H), 4.53 (m, 1H), 3.86 (t, J = 6.8 Hz, 2H), 2.83 (bs, ES+, m / z 534.0 [M+H] + . (57B): C 21 H 26 F3N5O8: 1H NMR (500MHz, CDCl3): δ 12.10 (bs, 1H), 9.47 (brs, 1H), 6.19 (s, 1H), 6.04 (m, 1H), 5.38 (d, J = 6.0 Hz, 1H), 4.66 (m, 1H), 4.00 (t, J = 7.3 Hz, 2H), 2.93 (m, 1H), 2.30 (s, 3H), 2.26 (s, 3H), 2.17 (m, 1H), 2.12 (s, 3H), 1.74 (m, 2H), 1.37 (m, 2H), 0.94 (t, J = 7.3 Hz, 3H). ES+, m / z 534.4 [M+H] +

[0390] Process 2 2-Amino-7-butyl-9-((2R,3R,5S)-3-hydroxy-5-((R)-2,2,2-trifluoro-1-hydroxyethyl)tetrahydrofuran-2-yl)-7,9-dihydro-1H-purine-6,8-dione (compound 31) To a solution of (R)-1-((2S,4R,5R)-5-(2-acetamido-7-butyl-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)-2,2,2-trifluoroethyl acetate (57A) (90 mg, 0.16 mmol) in MeOH (20 mL) was added KCO (34.9 mg, 0.25 mmol) at 0 °C. The reaction mixture was stirred at rt for 16 h, and the methanol was removed under reduced pressure at 30 °C. The residue was directly subjected to reverse-phase GRACE flash chromatography using 10 mM ammonium bicarbonate in HO. Lyophilization of the purified fractions afforded 50 mg (73%) of 2-amino-7-butyl-9-((2R,3R,5S)-3-hydroxy-5-((R)-2,2,2-trifluoro-1-hydroxyethyl)tetrahydrofuran-2-yl)-7,9-dihydro-1H-purine-6,8-dione (compound 31) as an off-white solid. 15 H 20 F3N5O5: 1H NMR (500MHz, DMSO-d6): δ 11.00 (brs, 1H), 6.51 (brs, 2H), 6.35 (d, J = 8.5 Hz, 1H), 5.58 (d, J = 3.5 Hz, 1H), 5.52 (d, J = 5.0 Hz, 1H), 4.74 (m, 1H), 4.34 (dt, J = 11.3, 3.6 Hz, 1H), 4.02 (m, 1H), 3.78 (t, J = 7.0 Hz, 2H), 2.51 (m, 1H), 1.97 (m, 1H), 1.60 (m, 2H), 1.24 (m, 2H), 0.88 (t, J = 7.5 Hz, 3H). ES+, m / z 408.3 [M+H] +

[0391] Example 29 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(2-hydroxyethyl)-7,9-dihydro-1H-purine-6,8-dione, compound 32 [ka]

[0392] Compound 32 was prepared in the following multiple steps. [ka]

[0393] Process 1 2-Amino-6-(benzyloxy)-7-(2-hydroxyethyl)-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (58) 2-Bromoethan-1-ol (2.4 mL, 33.1 mmol) was added to a suspension of 2-amino-6-(benzyloxy)-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (3) (5.0 g, 13.2 mmol) and CsCO (5.4 g, 39.8 mmol) in DMF (50 mL) at 0 °C. The reaction mixture was stirred at 80 °C for 72 h, after which ice water (100 mL) was added and stirring was continued for 1 h. The resulting precipitated solid was collected by filtration, washed with water, EtOAc, and dried to give 4 g of 2-amino-6-(benzyloxy)-7-(2-hydroxyethyl)-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (58) by LC / MS and NMR (MS / MS). 1 It was obtained as an off-white solid, contaminated with approximately 10% starting purine (3) by H NMR. 22 H 23 N5O4: ES+, m / z 422.0 [M+H] + and C 20 H 19 NO: 378.0 [M+H] + . 1 H NMR (400MHz, DMSO-d6): δ 10.80 (s, N 7 H(4)), 7.49-7.18 (m, 9H), 6.87 (dd, J = 8.4, 1.2 Hz, 2H), 6.36 (s, 1H), 6.39 (s, 1H), 5.42 (d, J = 7.2 Hz, 2H), 4.81 (d, J = 15.6 Hz, 2H), 4.76 (m, 1H), 3.84 (t, J = 6.2 Hz, 1H), 3.71 (s, 3H), 3.57 (q, J = 6.0 Hz, 1H)

[0394] Process 2 :2-Amino-7-(2-hydroxyethyl)-7,9-dihydro-1H-purine-6,8-dione (59) Trifluoromethanesulfonic acid (5.0 mL, 57.0 mmol) was added to a suspension of 2-amino-6-(benzyloxy)-7-(2-hydroxyethyl)-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (58) (4.0 g, 9.5 mmol) in trifluoroacetic acid (4.6 mL, 57.0 mmol) at 0 °C under an argon atmosphere. The reaction mixture was slowly warmed to room temperature and stirred for 16 h, after which ice-cold water was added. With vigorous stirring, the pH of the mixture was made basic with concentrated aqueous NH3. The solid that formed was filtered, dissolved in ethyl acetate, stirred for 30 min, filtered, and dried to give 1.8 g of 2-amino-6-hydroxy-7-(2-hydroxyethyl)-7,9-dihydro-8H-purin-8-one (59) as a brown solid. C7H9N5O3: ES+, m / z 212.0 [M+H] + . 1 H NMR (500MHz, DMSO-d6): δ 10.82 (s, 1H), 10.53 (s, 1H), 6.29 (s, 2H), 4.75 (t, J = 5.8 Hz, 1H), 3.78 (t, J = 6.0 Hz, 2H), 3.57 (m, 2H)

[0395] Process 3 2-(2-acetamido-6,8-dioxo-1,6,8,9-tetrahydro-7H-purin-7-yl)ethyl acetate (60) Acetic anhydride (3.3 mL, 34.1 mmol) was added to a solution of 2-amino-6-hydroxy-7-(2-hydroxyethyl)-7,9-dihydro-8H-purin-8-one (59) (1.8 g, 8.53 mmol) in AcOH (20 mL) at room temperature, and the reaction mixture was heated at 120° C. for 16 hours under an argon atmosphere. The reaction mixture was cooled to 0° C. and stirred for 30 minutes. The precipitated solid was collected by filtration, washed with EtOAc, and dried under reduced pressure to give 1.3 g of 2-(2-acetamido-6-hydroxy-8-oxo-8,9-dihydro-7H-purin-7-yl)ethyl acetate (60) as a brown solid. 11 H 13 N5O5: ES+, m / z 296.0 [M+H] +The crude product was used directly in the next step without further purification.

[0396] Process 4 (S)-1-((2S,4R,5R)-5-(2-acetamido-7-(2-acetoxyethyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (61) 2-(2-Acetamido-6-hydroxy-8-oxo-8,9-dihydro-7H-purin-7-yl)ethyl acetate (60) (1.0 g, 3.3 mmol), (3R,5S)-5-((S)-1-acetoxypropyl)tetrahydrofuran-2,3-diyl diacetate (14S) (1.17 g, 4.06 mmol), and BSA (2.5 mL, 10.1 mmol) were dissolved in 1,2-dichloroethane (20 mL). The reaction mixture was stirred at 80 °C for 30 min under argon and concentrated under reduced pressure. MeCN (20 mL) was added to the remaining residue, and to this solution was added TMSOTf (0.94 mL, 5.08 mmol). The reaction mixture was placed in an oil bath preheated to 80 °C and stirred for 16 h, then cooled to room temperature. The solvent was removed under reduced pressure, and the solid was dissolved in ethyl acetate (100 mL) and washed with saturated aqueous NaHCO3 (1 x 30 mL). The organic phase was dried over Na2SO4, filtered, and concentrated. The crude compound was purified by column chromatography (SiO2, 0-60% EtOAc-petroleum ether) to afford 250 mg (13%) of (S)-1-((2S,4R,5R)-5-(2-acetamido-7-(2-acetoxyethyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (61) as a pale yellow solid. 22 H 29 N5O 10 : ES+, m / z 523.9 [M+H] +

[0397] Process 52-Amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(2-hydroxyethyl)-7,9-dihydro-1H-purine-6,8-dione (compound 32) To a solution of (S)-1-((2S,4R,5R)-5-(2-acetamido-7-(2-acetoxyethyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (61) (250 mg, 0.47 mmol) in methanol (15 mL) was added KCO (98.9 mg, 0.71 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 hours. After LC / MS showed complete consumption of the starting material, the reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC using a LUNA OMEGA C18 (250*21.2) column with a 5μm column, mobile phase: 10 mM NH4HCO3:MeOH in HO gradient (T%B): -0 / 10, 8 / 50, 12 / 60, 12.1 / 98, 14 / 98, 14 / 10, 18 / 10; flow rate: 16 mL / min; diluent: MeCN + HO + THF. Lyophilization of pure fractions afforded 20 mg of 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-(2-hydroxyethyl)-7,9-dihydro-1H-purine-6,8-dione (compound 32) as an off-white solid. 14 H 21 N5O6: 1H NMR (400MHz, DMSO-d6): δ 11.10 (brs, 1H), 6.51 (s, 2H), 5.52 (d, J = 3.2 Hz, 1H), 5.34 (d, J = 4.8 Hz, 1H), 4.87 (brs, 1H), 4.75 (m, 1H), 4.63 (d, J = 6.8 Hz, 1H), 4.01 (m, 1H), 3.83 (t, J = 6.0 Hz, 2H), 3.59 (m, 2H), 3.27 (m, 1H), 2.38 (m, 1H), 1.77 (m, 1H), 1.39 (m, 1H), 1.28 (m, 1H), 0.87 (t, J = 7.2 Hz, 3H). ES+, m / z 356.0 [M+H] +

[0398] Example 30 Compounds 33 and 34

[0399] 2-Amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-((R)-2-hydroxypropyl)-7,9-dihydro-1H-purine-6,8-dione, compound 33 [ka]

[0400] 2-Amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-((S)-2-hydroxypropyl)-7,9-dihydro-1H-purine-6,8-dione, compound 34 [ka]

[0401] Compounds 33 and 34 were prepared in the following multi-step process. [ka]

[0402] Process 1 2-Amino-6-(benzyloxy)-7-(2-hydroxypropyl)-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (62) To a stirring suspension of 2-amino-6-(benzyloxy)-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (3) (5 g, 13.26 mmol) and KCO (2.74 g, 19.89 mmol) in DMF (50 mL) was added 1-bromopropan-2-ol (2.765 g, 19.89 mmol) at 0 °C. Stirring was continued at 90 °C for 48 h. To the stirring reaction mixture was added ice water (500 mL) followed by diethyl ether (80 mL). After approximately 15 min, the resulting precipitated solid was collected by filtration, washed with water, and dried to give 2-amino-6-(benzyloxy)-7-(2-hydroxypropyl)-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (62) (2 g, 34.66%) as an off-white solid. C 23 H 25 N5O4: 1 H NMR (400MHz, DMSO-d6): δ 7.48 (d, J = 7.2 Hz, 2H), 7.41-7.32 (m, 3H), 7.21 (d, J = 8.8 Hz, 2H), 6.87 (d, J = 8.4 Hz, 2H), 6.36 (s, 2H), 5.41 (q, J = 12.4 Hz, 2H), 4.82 (s, 2H), 4.74 (s, 1H), 3.90 (m, 1H), 3.77 (m, 1H), 3.71 (s, 3H), 3.57 (dd, J = 13.6, 5.2 Hz, 1H), 0.941(d, J = 6.4 Hz, 3H). ES+, m / z 436.4 [M+H] +

[0403] Process 2 :2-Amino-7-(2-hydroxypropyl)-7,9-dihydro-1H-purine-6,8-dione (63) Trifluoromethanesulfonic acid (1.55 mL, 10.34 mmol) was added to a stirred suspension of 2-amino-6-(benzyloxy)-7-(2-hydroxypropyl)-9-(4-methoxybenzyl)-7,9-dihydro-8H-purin-8-one (62) (1.5 g, 3.44 mmol) in trifluoroacetic acid (1.18 mL, 10.34 mmol) at 0 °C under an argon atmosphere. The reaction mixture was slowly warmed to room temperature and stirred for 16 h. The crude product was purified by reverse-phase GRACE flash chromatography using water and 0.1% HCOH in MeCN to give 2-amino-7-(2-hydroxypropyl)-7,9-dihydro-1H-purine-6,8-dione (63) (0.4 g, 47.4%) as an off-white solid. 11 N5O3: 1 H NMR (400MHz, DMSO-d6): δ 11.08 (s, 1H), 10.69 (s, 1H), 6.35 (s, 2H), 4.8 (bs, 1H), 3.94 (m, 1H), 3.69 (dd, J = 13.4, 6.6 Hz, 1H), 3.59 (dd, J = 13.6, 6.0 Hz, 1H), 0.99 (d, J = 5.6 Hz, 3H). ES+, m / z 226.0 [M+H] +

[0404] Process 3 1-(2-acetamido-6,8-dioxo-1,6,8,9-tetrahydro-7H-purin-7-yl)propan-2-yl acetate (64) Acetic anhydride (0.79 mL, 7.73 mmol) was added to a solution of 2-amino-7-(2-hydroxypropyl)-7,9-dihydro-1H-purine-6,8-dione (63) (0.58 g, 2.57 mmol) in AcOH (10 mL) at ambient temperature under argon, and the resulting reaction mixture was stirred at 120° C. for 8 hours under argon. The reaction mixture was concentrated, EtOAc (20 mL) was added, and stirring was continued for 30 minutes. The product was filtered, washed with EtOAc, and dried under reduced pressure to give 1-(2-acetamido-6,8-dioxo-1,6,8,9-tetrahydro-7H-purin-7-yl)propan-2-yl acetate (64) (500 mg, 62.81%) as a light brown solid. 12 H 15 N5O5: 1 H NMR (400MHz, DMSO-d6): δ 12.00 (s, 1H), 11.70 (s, 1H), 11.66 (s, 1H), 5.15 (m, 1H), 3.97 (dd, J = 14.2, 7.4 Hz, 1H), 3.90 (dd, J = 14.0, 4.4 Hz, 1H), 2.15 (s, 3H), 1.88 (s, 3H), 1.17 (d, J = 6.4 Hz, 3H). ES+, m / z 310.0 [M+H] +

[0405] Process 4 (S)-1-((2S,4R,5R)-5-(2-acetamido-7-((R)-2-acetoxypropyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (65A) and (S)-1-((2S,4R,5R)-5-(2-acetamido-7-((S)-2-acetoxypropyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (65B) 1-(2-Acetamido-6,8-dioxo-1,6,8,9-tetrahydro-7H-purin-7-yl)propan-2-yl acetate (64) (400 mg, 1.29 mmol), (3R,5S)-5-((S)-1-acetoxypropyl)tetrahydrofuran-2,3-diyl diacetate (14S) (559 mg, 1.94 mmol), and BSA (1.30 mL, 6.47 mmol) were dissolved in 1,2-dichloromethane (15 mL). The reaction mixture was stirred at 80 °C under argon for 30 minutes. The reaction mixture was then concentrated under reduced pressure, and the residue was dissolved in MeCN (30 mL), followed by the addition of TMSOTf (0.71 mL, 3.2 mmol). The stirred reaction mixture was placed in an oil bath preheated to 80 °C. After 3 hours, the reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure. The resulting solid was dissolved in ethyl acetate (80 mL), and to the stirred solution was added saturated aqueous NaHCO3 (2 × 30 mL). The organic phase was separated, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (SiO2, 0–80% ethyl acetate-petroleum ether) to give a 1:1 mixture of (S)-1-((2S,4R,5R)-5-(2-acetamido-7-((R,S)-2-acetoxypropyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (220 mg).The racemic mixture was further purified by chiral FC-150-080 HPLC (Lux; Cellulose-4 OX-H; 250 × 30 × 5 μm; 75% CO , 25% MeOH; total flow: 60.0 g / min; back pressure: 120.0 bar; 30 °C; UV: 214.0 nm; stack time: 7.0 min; load / inject: 14.57 mg) to give 50 mg of (S)-1-((2S,4R,5R)-5-(2-acetamido-7-((R)-2-acetoxypropyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran. After evaporation of methanol, 50 mg of (S)-1-((2S,4R,5R)-5-(2-acetamido-7-((S)-2-acetoxypropyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (65A; 7(R) stereochemistry arbitrarily assigned) and 50 mg of (S)-1-((2S,4R,5R)-5-(2-acetamido-7-((S)-2-acetoxypropyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (65B; 7(S) stereochemistry arbitrarily assigned) were obtained, both as off-white solids. Analytical SFC was used to confirm the diastereomeric purity; (Chiralcel OX-H; 250 × 4.6 × 5 μm; 75% CO , 25% MeOH; total flow rate: 3.0 g / min; back pressure: 100.0 bar; 30 °C; UV: 214.0 nm) 65A:C. 23 H 31 N5O 10 : ES+, m / z 538.0 [M+H] + . 65B:C 23 H 31 N5O 10 : ES+, m / z 537.9 [M+H] + .

[0406] Process 5 2-Amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-((R)-2-hydroxypropyl)-7,9-dihydro-1H-purine-6,8-dione (compound 33) To a solution of (S)-1-((2S,4R,5R)-5-(2-acetamido-7-((R)-2-acetoxypropyl)-6,8-dioxo-1,6,7,8-tetrahydro-9H-purin-9-yl)-4-acetoxytetrahydrofuran-2-yl)propyl acetate (65A) (50 mg, 0.09 mmol) in methanol (5 mL) was added KCO (19 mg, 0.13 mmol) at room temperature, and the reaction mixture was stirred at room temperature for 16 hours. The solvent was then removed under reduced pressure, and the resulting crude compound was purified by GRACE flash chromatography (0.1% HCOH in water, acetonitrile) to give 2-amino-9-((2R,3R,5S)-3-hydroxy-5-((S)-1-hydroxypropyl)tetrahydrofuran-2-yl)-7-((R)-2-hydroxypropyl)-7,9-dihydro-1H-purine-6,8-dione (compound 33) (24 mg, 69.66%) as a yellow solid. 15 H 23 N5O6: 1 1 H NMR indicates the formation of the formate salt. 1 H NMR (500MHz, DMSO-d6): δ 8.52 (s, 1H), 6.70-6.50 (b, 2H), 5.52 (d, J = 3.5 Hz, 1H), 5.35 (s, 1H), 4.75 (s, ...

Claims

1. Formula I: 【Chemistry 1】 [During the ceremony, R １ are independently —H, —OH, —O—C(O)—R ８ or -F; R ２ are independently —H, —OH, —O—C(O)—R ８ or -F; R ３ is -OH or -O-C(O)-R ８ and R ４ -H, -OH, -O-C(O)-R ８ Or -(C １ -C ８ ) alkyl, Here, R ３ and R ４ may be in the form of a carbonyl oxygen (=O); R ５ -H, -OH, -O-C(O)-R ８ , -(C １ -C ８ ) alkyl, —O—(C １ -C ８ ) alkyl, -NH ２ or -NHR ８ and Here, R ４ and R ５ can form a 3- to 6-membered cycloalkyl ring; R ６ -H, -(C １ -C ８ ) alkyl, —C(H)═CH ２ , -C(H)=C(H)(C １ -C ８ ) alkyl), -C(H)=C((C １ -C ８ ) alkyl)(C １ -C ８ ) alkyl), -C(H)=C=CH ２ , -C(H)=C=C((C １ -C ８ ) alkyl) H, —CH ２ C≡CH, —OH or —O(C １ -C ８ ) alkyl; R ７ is -H, -OH, -OCH ３ , —SH or —Cl; R ８ is independently -(C １ -C ８ ) alkyl, aryl, -(CH ２ ) ｎ (aryl), heteroaryl or -(CH ２ ) ｎ (heteroaryl); n is an integer 1, 2, 3, 4, or 5; Here, R ４ or R ５ is not —H; and wherein each alkyl, cycloalkyl, aryl, and heteroaryl independently represents CN, NO ２ , halogen, (C １ -C ３ ) alkyl, (C １ -C ３ ) haloalkyl, (C ３ -C １５ ) cycloalkyl, aryl, heteroaryl, OH, alkenyl, alkynyl, O—(C １ -C ３ ) alkyl, O—C(O)—R ９ , O-(alkylene)aryl, O-(alkylene)heteroaryl, C(O)R ９ , S(C １ -C ８ ) alkyl, S(O)(C １ -C ８ ) alkyl, SO ２ (C １ -C ８ ) alkyl, C(O)OR ９ , C(O)NR ９ R ９ , C(O)NR ９ SO ２ (C １ -C ８ ) alkyl, NR ９ R ９ , N.R. ９ (CO)OR ９ , NH(CO)R ９ , NH(SO ２ )(C １ -C ８ ) alkyl or NH(SO ２ )NR ９ R ９ and R ９ are independently -H, -OH, -(C １ -C ８ ) alkyl, cycloalkyl, heterocyclyl or C(O)NR ９ R ９ or NR ９ R ９ The two R's ９ together with the nitrogen atom form a heterocyclyl. or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof.

2. R １ and R ２ 2. The compound of claim 1, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein is -H, -OH, or F.

3. R ３ is —OH or —O—C(O)—CH ３ 2. The compound of claim 1, wherein:

4. R ４ is -H or -(C １ -C ８ 2. The compound of claim 1, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein:

5. R ４ is -H or -CH ２ CH ３ 2. The compound of claim 1, wherein:

6. R ５ is -H or -(C １ -C ８ 2. The compound of claim 1, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein:

7. R ６ -C(H)=CH ２ or -CH ２ 2. The compound of claim 1, wherein C≡CH, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof.

8. R ７ 2. The compound of claim 1, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein is -H or -OH.

9. R ８ But-(C １ -C ８ 2. The compound of claim 1, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein:

10. R ８ Ga-CH ３ 2. The compound of claim 1, wherein: 【Request Item 11】 【Chemistry 2】 1. A compound selected from the group consisting of: or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof. 【Request Item 12】 【Chemistry 3】 1. A compound selected from the group consisting of: or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof.

13. structure 【Chemistry 4】 or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof.

14. structure 【Transformation 5】 or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof.

15. A pharmaceutical composition comprising one or more compounds of any one of claims 1 to 14 or stereoisomers, tautomers or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable excipient.

16. 15. A medicament for treating or preventing an infectious disease or cancer, comprising an effective amount of one or more compounds of any of claims 1 to 14, or stereoisomers, tautomers, or pharmaceutically acceptable salts thereof.

17. The pharmaceutical of claim 16, wherein the infectious disease is a viral infection.

18. 18. The pharmaceutical composition of claim 17, wherein the viral infection is an infection with a virus selected from the group consisting of HIV, hepatitis viruses (A, B, C, and D), VZV, HSV-I, HAV-6, HSV-II, CMV, Epstein-Barr virus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, arboviral encephalitis virus, SARS-CoV, MERS, and SARS-CoV-2.

19. The pharmaceutical of claim 16, which is for the treatment or prevention of cancer.

20. 20. The pharmaceutical agent of claim 19, wherein the cancer is selected from the group consisting of osteosarcoma, rhabdomyosarcoma, neuroblastoma, kidney cancer, leukemia, renal transitional cell carcinoma, bladder cancer, Wilms' carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, bone cancer, lung cancer, non-small cell lung cancer, colorectal cancer, cervical cancer, synovial sarcoma, head and neck cancer, squamous cell carcinoma, multiple myeloma, renal cell carcinoma, retinoblastoma, hepatoblastoma, hepatocellular carcinoma, melanoma, renal rhabdoid tumor, Ewing's sarcoma, chondrosarcoma, brain cancer, glioblastoma, meningioma, pituitary adenoma, vestibular schwannoma, primitive neuroectodermal tumor, medulloblastoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, ependymoma, choroid plexus papilloma, polycythemia vera, thrombocythemia, idiopathic myelofibrosis, soft tissue sarcoma, thyroid cancer, endometrial cancer, carcinoid cancer, liver cancer, breast cancer, and gastric cancer.

21. The pharmaceutical agent of claim 16, further comprising one or more additional agents, wherein the one or more additional agents are each independently an immune checkpoint inhibitor, an OX40 agonist, a 4-1BB agonist, an ICOS agonist, a GITR agonist, an IL-2-receptor agonist, or an antibody mediated by ADCC.

22. The pharmaceutical agent of claim 21, wherein the one or more additional agents are each independently an inhibitor of PD-1, PD-L1, CTLA4, TIM3, LAG3, SIRPα, TIGIT, or CD47.

23. 17. The pharmaceutical of claim 16, further comprising one or more additional agents, wherein the one or more agents are each independently a small molecule therapeutic, a protein or peptide therapeutic, an antibody, serum from a person who has recovered from a viral infection, or a therapeutic or prophylactic vaccine.

24. 15. A medicament for treating or preventing cancer in a subject having a red blood cell and / or white blood cell deficiency, comprising an effective amount of a compound of any one of claims 1 to 14, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof.

25. 25. The medicament of claim 24, wherein the compound is in combination with an additional agent selected from filgrastim, PEG-filgrastim, erythropoietin, epoetin alfa, or darbepoetin alfa.

26. 26. The medicament of claim 25, wherein the compound and the additional medicament are formulated in a single composition.

27. 26. The medicament of claim 25, wherein the compound and the additional medicament are formulated separately in two or more compositions.

28. 28. The pharmaceutical of claim 27, wherein the two or more compositions are formulated for delivery other than simultaneously.

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