Modulators of 5'-nucleotidase, ecto and the use thereof

Abstract

To solve the problem in which: in view of the role played by CD73 in cancer, as well as a diverse array of other diseases, disorders and conditions, and the current lack of CD73 inhibitors available to medical practitioners, new CD73 inhibitors, and compositions and methods associated therewith, are needed.SOLUTION: Compounds that modulate the conversion of AMP to adenosine by 5'-nucleotidase, ecto, and compositions containing the compounds and methods for synthesizing the compounds, are described herein. The use of such compounds and compositions for the treatment and / or prevention of a diverse array of diseases, disorders and conditions, including cancer- and immune-related disorders, that are mediated by 5'-nucleotidase, ecto is also provided.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62 / 276,564, filed January 8, 2016, and U.S. Provisional Application No. 62 / 324,077, filed April 18, 2016, each of which is incorporated by reference in its entirety.

[0002] STATEMENT OF RIGHTS TO INVENTIONS MADE BY FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT Not applicable.

[0003] Reference to a "Sequence Listing," table, or computer program listing submitted on a compact disc Not applicable. FIELD OF THE INVENTION

[0004] Provided herein are compounds and compositions for the inhibition of adenosine by, for example, 5'-nucleotidase, ecto, also known as CD73, and pharmaceutical compositions comprising same. Also provided herein are methods for treating or preventing diseases, disorders, or conditions, symptoms thereof, mediated by, for example, the inhibition of adenosine by 5'-nucleotidase, ecto. [Background technology]

[0005] BACKGROUND OF THE INVENTION Purinergic signaling, a type of extracellular signaling mediated by purine nucleotides and nucleosides, such as ATP and adenosine, involves activation of purinergic receptors in cells and / or nearby cells, resulting in the regulation of cellular function. Most cells have the ability to release nucleotides, which generally occurs via regulated exocytosis (Praetorius, HA; Leipziger, J. (1 March 2010) Ann Rev Physiology 7 2(1): 377-393). The released nucleotides can then be hydrolyzed extracellularly by a variety of membrane-bound enzymes called ectonucleotidases.

[0006] Ectonucleotides catalyze the conversion of ATP to adenosine, an endogenous regulator that affects multiple systems, including the immune system, cardiovascular system, central nervous system, and respiratory system. Adenosine also promotes fibrosis in various tissues. In the first step of adenosine production, ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), also known as CD39 (cluster of differentiation 39), hydrolyzes ATP to ADP, which then hydrolyzes ADP to AMP. In the next step, AMP is converted to adenosine by 5'-nucleotidase, ecto (NT5E or 5NT), also known as CD73 (cluster of differentiation 73). is converted to

[0007] The enzymatic activities of CD39 and CD73 play strategic roles in calibrating the duration, magnitude, and chemical nature of purinergic signals sent to various cells (e.g., immune cells). Alterations in these enzyme activities can alter the course or define the outcome of several pathophysiological events, including cancer, autoimmune diseases, infectious diseases, atherosclerosis, and ischemia-reperfusion injury, suggesting that these exogenous enzymes represent novel therapeutic targets for managing various disorders.

[0008] Inhibition of CD73 with monoclonal antibodies, siRNA, or small molecules slows tumor growth and metastasis (Stagg, J. (2010) PNAS USA 107:1547-52). For example, anti-CD73 Antibody therapy has been shown to inhibit breast cancer growth and metastasis in animal models (Stagg, J. (26 Jan 2010) PNAS USA,107(4):1547-52). Furthermore, the use of antibodies that specifically bind to CD73 has been evaluated for the treatment of bleeding disorders (e.g., hemophilia) (U.S. Patent No. 9,090,697). In recent years, several attempts have been made to develop therapeutically useful CD73 small molecule inhibitors. For example, Bhattarai et al. ((2015) J Med Chem 58:6248- 63) have investigated derivatives and analogues of α,β-methylene-ADP (AOPCP), one of the most metabolically stable, potent and selective CD73 inhibitors known to date, and purine CD73 derivatives have been reported in the patent literature (WO 2015 / 164573). However, the development of small molecules has been hindered, for example, by their poor metabolic stability. Summary of the Invention [Problem to be solved by the invention]

[0009] Given the role of CD73 in cancer and a variety of other diseases, disorders, and conditions, and the lack of CD73 inhibitors available to medical professionals, there is a need for new CD73 inhibitors, and related compositions and methods. [Means for solving the problem]

[0010] (Summary of the Invention) The present invention relates to compounds that modulate the conversion of AMP to adenosine by 5'-nucleotidase, ecto (NT5E or 5NT; also known as CD73), and compositions (e.g., pharmaceutical compositions) containing said compounds. Such compounds, methods for their synthesis, and compositions are described in detail below.

[0011] The present invention also relates to the use of such compounds and compositions for the treatment and / or prevention of a variety of diseases, disorders, and conditions mediated in whole or in part by CD73. CD73 inhibitors are relevant to the treatment of a variety of disorders, including cancer, fibrosis, neurological and neurodegenerative disorders (e.g., depression and Parkinson's disease), cerebral and cardiac ischemic diseases, immune-related disorders, and disorders with an inflammatory component. [See, e.g., Sorrentino et al (2013) OncoImmunol, 2:e22448, doi: 10.4161 / onci.22448; and Regateiro et al. (2012) Clin. Exp. Immunol, 171:1-7.

[0013] In specific embodiments, the compounds described herein act to inhibit the immunosuppressive and / or anti-inflammatory activity of CD73 and are useful as therapeutic or prophylactic therapies where such inhibition is desirable. Unless otherwise specified, when uses of compounds of the invention are described herein, it should be understood that such compounds may be in the form of a composition (e.g., a pharmaceutical composition).

[0012] As used herein, the terms "CD73 inhibitor," "CD73 blocker," "adenosine 5'-nucleotidase ecto-inhibitor," "NT5E inhibitor," "NT5E inhibitor," and other related terms accepted in the art refer to compounds that can directly or indirectly modulate the CD73 receptor in in vitro assays, in vivo models, and / or other means to demonstrate therapeutic efficacy. The terms also refer to compounds that demonstrate at least some therapeutic benefit in human subjects.

[0013] Although the compounds of the invention are believed to exert their activity through the inhibition of CD73, a precise understanding of the compound's underlying mechanism of action is not required to practice the invention. For example, the compounds may also exert their activity, at least in part, through modulation (e.g., inhibition) of other components of the purinergic signaling pathway (e.g., CD39). The purinergic signaling pathway involves (primarily) the synthesis, release, action, and regulation of ATP and its extracellular breakdown product, adenosine. It consists of transporters, enzymes, and receptors involved in extracellular inactivation (Sperlagh, B. et al. (Dec 2012) NeuropsychopharmacologiaHungarica 14(4):231-38). Because inhibition of CD73 results in a decrease in adenosine, CD73 inhibitors can be used to treat diseases or disorders mediated by adenosine and its action on adenosine receptors, including A1, A2A, A2B, and A3 [Yegutkin, GG (May 2008) Biochimic a Biophysica Acta 1783(5):673-94).

[0014] For purposes of this disclosure, the purinergic signaling process can be described as including the following components: First, purinergic receptors (P1, P2X, and P2Y) are membrane receptors that mediate various physiological functions (e.g., relaxation of intestinal smooth muscle) in response to the release of ATP or adenosine; generally, all cells have the ability to release nucleotides into the extracellular environment, often via regulated exocytosis. Second, nucleoside transporters (NTs) are membrane transport proteins that transport nucleoside substrates (e.g., adenosine) across the cell membrane; the extracellular concentration of adenosine may be regulated by NTs, possibly in the form of a feedback loop connecting receptor signaling and transporter function. As previously mentioned, ectonucleotidases (CD73 and CD39) hydrolyze nucleotides released into the extracellular environment and include additional components. Another component of the purinergic signaling process involves pannexins; in particular, the pannexin-1 channel (PANX1) is an essential component of the P2X / P2Y purinergic signaling pathway and makes an important contribution to pathophysiological ATP release.

[0015] In one specific embodiment, the present invention provides a compound of formula (I): [ka] or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein each R 1 is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted Aryl, and -C(R 2 R 2 )-OC(O)-OR 3 Independently selected from the group consisting of Or two R's 1 groups optionally joined to form a 5- to 7-membered ring; each R 2 is independently selected from the group consisting of H and optionally substituted C1-C6 alkyl; each R 3 is independently selected from the group consisting of H, C1-C6 alkyl, and optionally substituted aryl; R 5 is selected from the group consisting of H and optionally substituted C1-C6 alkyl; X is O, CH 2 and S; A is selected from the group consisting of [ka] each of which is selected from the group consisting of 1 to 5 R 6 Optionally substituted with a substituent, where the subscript n is an integer from 0 to 3, and Z is CH, CHR 6 , N.R. 6 , and from O each R 6 are independently selected from the group consisting of H, CH3, OH, CN, F, optionally substituted C1-C6 alkyl, and OC(O)-C1-C6 alkyl; optionally, two R on adjacent ring vertices 6 The groups are joined together to form at least one ring vertex. Het forms a 5- or 6-membered ring containing one heteroatom; [ka] where the wavy line indicates the point of attachment to the remainder of the compound, and R a are H, NH2, and NHR 7 , NHC(O)R 7 , N.R. 7 R 7 , R 7 , O.H., S.R. 7 , and OR 7 mosquito R is selected from the group consisting of b is H, halogen, NH2, NHR 7 , N.R. 7 R 7 , R 7 , OH, and OR 7 each R c and R d is H, halogen, haloalkyl, N H2, NHR 7 , N.R. 7 R 7 , R 7 , OH, and OR 7 each R is independently selected from the group consisting of e and R f is independently selected from the group consisting of H, halogen, and optionally substituted C1-C6 alkyl; and each R 7 is an optionally substituted C1-C 10 two R independently selected from the group consisting of alkyl, optionally substituted C3-C7 cycloalkyl, optionally substituted 4- to 7-membered cycloheteroalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl, and optionally attached to a nitrogen atom; 7 The groups are joined together to form a 4- to 7-membered heterocyclic ring.

[0016] Compounds excluded from the above are those in which the combination of X, A and Het is: [ka] [where R 8 is H or two R 8groups combine to form an acetonide; and (i)R c and R e is hydrogen and R a -OEt, -OCH2Ph, -SCH2P h, -NH2, methylamino, ethylamino, dimethylamino, diethylamino, N-methylamino ethyl-N-ethylamino, phenylamino, benzylamino, 2-phenylethylamino, N-benzyl-N-ethylamino, dibenzylamino, 4-aminobenzylamino, 4-chlorobenzylamino, 4-nitrobenzylamino, or 4-sulfamoylbenzylamino; or (ii)R c is hydrogen and R a is -NH2 and R e is bromo, chloro, aminomethyl, or thioethyl; or (iii)R c is hydrogen and R a is benzylamino and R e is bromo] It is a compound that gives

[0017] In some embodiments, the present invention contemplates a method of treating or preventing cancer in a subject (e.g., a human), comprising administering to the subject a therapeutically effective amount of at least one CD73 inhibitor described herein. The present invention includes methods of treating or preventing cancer in a subject by administering to the subject an amount of a CD73 inhibitor effective to reverse or halt the progression of CD73-mediated immunosuppression. In some embodiments, CD73-mediated immunosuppression is mediated by antigen-presenting cells (APCs).

[0018] Examples of cancers that can be treated using the compounds and compositions described herein include, but are not limited to, cancers of the prostate, colorectum, pancreas, cervix, stomach, endometrium, brain, liver, bladder, ovary, testis, head, neck, skin (including melanoma and basal carcinoma), mesothelial lining, white blood cells (including lymphoma and leukemia), esophagus, breast, muscle, connective tissue, lung (including small cell lung cancer and non-small cell carcinoma), adrenal gland, thyroid, kidney, or bone; glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma, choriocarcinoma, basal cell carcinoma of the skin, and testicular seminoma. In some embodiments of the present invention, the cancer is melanoma, colon cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, leukemia, brain tumor, lymphoma, sarcoma, ovarian cancer, or Kaposi's sarcoma. The compounds and compositions of the present invention Cancers that are candidates for compositional treatment are discussed further below.

[0019] The present invention contemplates methods of treating a subject undergoing bone marrow transplantation or peripheral blood stem cell transplantation by administering a therapeutically effective amount of a CD73 inhibitor sufficient to increase delayed-type hypersensitivity responses to tumor antigens, delay the time to recurrence of malignant tumors after transplantation, increase recurrence-free survival after transplantation, and / or increase long-term survival after transplantation.

[0020] In certain embodiments, the present invention contemplates a method for treating or preventing an infectious disorder (e.g., a viral infection) in a subject (e.g., a human), comprising administering to the subject a therapeutically effective amount of at least one CD73 inhibitor (e.g., a novel inhibitor of the present invention). In some embodiments, the infectious disorder is a viral infection (e.g., a chronic viral infection), a bacterial infection, a fungal infection, or a parasitic infection. In certain embodiments, the viral infection is human immunodeficiency virus or cytomegalovirus.

[0021] In yet another embodiment, the present invention contemplates methods of treating and / or preventing immune-related diseases, disorders, and conditions; diseases having an inflammatory component; and disorders related to the above, using at least one CD73 inhibitor of the present invention. Examples of immune-related diseases, disorders, and conditions are described below.

[0022] Other diseases, disorders, and conditions that can be treated or prevented, in whole or in part, by modulation of CD73 activity are potential indications for the CD73 inhibitor compounds of the present invention.

[0023] The present invention further contemplates the use of the CD73 inhibitors described herein in combination with one or more additional agents, even if said one or more additional agents have any CD73 modulating activity. The CD73 inhibitor and the one or more additional agents may be administered simultaneously and / or via separate mechanisms of action. In some embodiments, such agents include radiation (e.g., localized or systemic radiation therapy) and / or other therapeutic modalities of a non-pharmacological nature. When combination therapy is used, the CD73 inhibitor and the one or more additional agents may be in the form of a single composition or multiple compositions, and the therapeutic modalities may be administered simultaneously, sequentially, or via other regimens. As an example, the present invention contemplates a therapeutic regimen in which a radiation phase is followed by a chemotherapy phase. The combination therapy can have additive or synergistic effects. Other advantages of combination therapy are described below.

[0024] In some embodiments, the present invention further includes the use of a CD73 inhibitor described herein in combination with a bone marrow transplant, a peripheral blood stem cell transplant, or other type of transplant therapy.

[0025] In certain embodiments, the present invention contemplates the use of an inhibitor of CD73 function described herein in combination with an immune checkpoint inhibitor. Blockade of immune checkpoints, which leads to the amplification of antigen-specific T cell responses, has been shown to be a promising approach in human cancer treatment. Examples of immune checkpoints (ligands and receptors), some of which are selectively upregulated in various types of tumor cells and are candidates for blockade, include PD1 (programmed cell death protein 1); PDL1 (PD1 ligand); BTLA (B and T lymphocyte attenuator); CTLA4 (cytotoxic T lymphocyte-associated antigen 4); TIM3 (T cell membrane protein 3); LAG3 (lymphocyte activation gene 3); A2aR (adenosine A2a receptor A2aR); and killer inhibitory receptors. Immune checkpoint inhibitors and combination therapies therewith are discussed in detail elsewhere herein.

[0026] In another embodiment, the present invention provides a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of at least one CD73 inhibitor and at least one chemotherapeutic agent, including, but not limited to, an alkylating agent (e.g., nitrite, nitrite-like compound ... aldehyde mustards, such as chlorambucil, cyclophosphamide, isofamide, mechlorethamine, melphalan, and uracil mustard; aziridines, such as thiotepa; methanesulfonate esters, such as busulfan; nucleoside analogs (e.g., gemcitabine); nitrosoureas, such as carmustine and lomustine; topoisomerase 1 inhibitors (e.g., irinotecan); platinum complexes, such as cisplatin and carboplatin; bioreductive alkylating agents, such as mitomycin, procarbazine, dacarbazine, and altretamine; DNA strand breakers. (e.g., bleomycin); topoisomerase II inhibitors (e.g., amsacrine, dactinomycin, daunorubicin, idarubicin, mitoxantrone, doxorubicin, etoposide, and teniposide); DNA minor groove binders (e.g., plicamidine); antimetabolites (e.g., methotrexate and trimetrexate); pyrimidine antagonists, e.g., fluorouracil, fluorodeoxyuridine, CB3717, azacitidine, cytarabine, and floxuridine; purine antagonists, e.g., mercaptopurine, 6-thioguanine, fludarabine, rabin, pentostatin; asparginase; and ribonucleotide reductase inhibitors, such as hydroxyurea; tubulin-interacting agents (e.g., vincristine, estramustine, vinblastine, docetaxel, epothilone derivatives, and paclitaxel); hormonal agents (e.g., estrogens; conjugated estrogens; ethinyl estradiol; diethylstilbestrol; chlortrianisene; idenestrol; progestins, such as hydroxyprogesterone caproate, medroxyprogesterone, and megestrol; and androgens, such as testosterone, testosterone propionate, fluoxymesterone, and methyltestosterone; corticosteroids (e.g., prednisone, dexamethasone, methylprednisolone, and prednisolone); luteinizing hormone-releasing agents or gonadotropin-releasing hormone antagonists (e.g., leuprolide acetate and goserelin acetate); and antihormonal agents (e.g., antiandrogens such as tamoxifen, flutamide, and antiadrenergic agents such as mitotane and aminoglutethimide).The present invention also contemplates the use of CD73 inhibitors in combination with other agents known in the art (eg, arsenic trioxide) and other chemotherapeutic agents that may be developed in the future.

[0027] In some embodiments of the methods of treating cancer, administration of a therapeutically effective amount of a CD73 inhibitor in combination with at least one chemotherapeutic agent results in a cancer survival rate that is greater than the cancer survival rate observed by administering either agent alone. In further embodiments of the methods of treating cancer, administration of a therapeutically effective amount of a CD73 inhibitor in combination with at least one chemotherapeutic agent results in a reduction in tumor size or a delay in tumor growth that is greater than the reduction in tumor size or a delay in tumor growth observed by administering either agent alone.

[0028] In further embodiments, the present invention contemplates a method for treating or preventing cancer in a subject, comprising administering to the subject therapeutically effective amounts of at least one CD73 inhibitor and at least one signal transduction inhibitor (STI). In certain embodiments, the at least one STI is selected from the group consisting of a bcr / abl kinase inhibitor, an epidermal growth factor (EGF) receptor inhibitor, a her-2 / neu receptor inhibitor, and a farnesyltransferase inhibitor (FTI). Other candidate STI agents are described elsewhere herein.

[0029] The present invention also contemplates a method of enhancing tumor cell rejection in a subject, comprising administering a CD73 inhibitor in combination with at least one chemotherapeutic agent and / or radiation therapy, wherein the resulting tumor cell rejection is greater than the rejection obtained by administering either the CD73 inhibitor, the chemotherapeutic agent, or radiation therapy alone.

[0030] In a further embodiment, the present invention provides a method for treating a patient comprising administering to the patient a therapeutically effective amount of at least one CD73 inhibitor and at least one immunomodulatory agent other than a CD73 inhibitor. The present invention provides a method for treating cancer in a subject.

[0031] The present invention contemplates embodiments that include methods of treating or preventing an infectious disorder (e.g., a viral infection) in a subject (e.g., a human) comprising administering to the subject a therapeutically effective amount of at least one CD73 inhibitor and a therapeutically effective amount of an anti-infective agent, e.g., one or more antibacterial agents.

[0032] In further embodiments, the treatment of infectious diseases is carried out by co-administering a therapeutically effective amount of a CD73 inhibitor of the present invention with a vaccine. In some embodiments, the vaccine is an antiviral vaccine, including, for example, an anti-HIV vaccine. In another embodiment, the vaccine is effective against tuberculosis or malaria. In yet another embodiment, the vaccine is a tumor vaccine (e.g., a vaccine effective against melanoma); the tumor vaccine may comprise genetically modified tumor cells or cell lines, including genetically modified tumor cells or cell lines transfected to express granulocyte-macrophage colony-stimulating factor (GM-CSF). In certain embodiments, the vaccine comprises one or more immunogenic peptides and / or dendritic cells.

[0033] In certain embodiments of treating an infection by administering a CD73 inhibitor and at least one additional therapeutic agent, the status of the infection observed after administration of both the CD73 inhibitor and the additional therapeutic agent is improved relative to the status of the same infection observed after administration of either agent alone. In some embodiments, the status of the infection observed can be a decrease in viral load, an increase in CD4+ T cell count, a decrease in opportunistic infections, increased survival, eradication of chronic infection, or a combination thereof. [Brief explanation of the drawings]

[0034] [Figure 1] Figure 1 shows a simplified representation of extracellular purinergic signaling. DETAILED DESCRIPTION OF THE INVENTION

[0035] (Detailed Description of the Invention) Before the present invention is further described, it is also to be understood that the invention is not limited to the particular embodiments described herein and that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0036] When a range of values ​​is provided, each intervening value, to the tenth of the unit of the lower limit (unless otherwise specified), between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are encompassed within the invention, subject to any specific excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0037] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any element. Accordingly, this statement is intended to serve as a premise for the use of exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements or the use of a "negative" limitation.

[0038] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Further, the publication dates provided may be different from the actual publication dates, which may may need to be independently verified.

[0039] Overview The number of subjects diagnosed with cancer and the number of cancer-related deaths continue to increase. Traditional treatment approaches, including chemotherapy and radiation therapy, are generally becoming less effective and less patient-tolerable as cancers (e.g., tumors) evolve to evade such treatments. Recent experimental evidence indicates that CD73 inhibitors are an important new therapeutic approach for the treatment of cancer (e.g., breast cancer).

[0040] Promising data also support a role for inhibitors of CD73 function to inhibit the anti-inflammatory and / or immunosuppressive activities of CD73, and thus CD73 inhibitors may be useful, for example, in treating immunosuppressive diseases (e.g., HIV and AIDS). Inhibitors of CD73 may also be an important therapeutic strategy for patients with neurological or neuropsychiatric diseases or disorders, such as depression.

[0041] In particular, the present invention relates to small molecule compounds that have CD73 inhibitory activity, as well as compositions thereof, and methods of using said compounds and compositions for the treatment and prevention of the diseases, disorders, and conditions described herein. definition

[0042] Unless otherwise specified, the following terms are intended to have the meanings indicated below. Other terms are defined elsewhere herein.

[0043] The term "alkyl," by itself or as part of another substituent, means, unless otherwise specified, a straight or branched chain hydrocarbon group having the specified number of carbon atoms (i.e., C1-C8 means 1 to 8 carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

[0044] The term "cycloalkyl" refers to a hydrocarbon ring having the indicated number of ring atoms (e.g., C3-C6 cycloalkyl) that is fully saturated or has one or fewer double bonds between the ring vertices. "Cycloalkyl" also refers to bicyclic and polycyclic hydrocarbon rings, such as, for example, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, and the like.

[0045] The term "cycloheteroalkyl" refers to a cycloalkyl ring having the indicated number of ring vertices (or members) and having 1 to 5 heteroatoms selected from N, O, and S (which replace 1 to 5 carbon vertices), where the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. Cycloheteroalkyls may be monocyclic, bicyclic, or polycyclic ring systems. Non-limiting examples of cycloheteroalkyl groups include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine, and the like. A cycloheteroalkyl group can be attached to the remainder of the molecule through a ring carbon or heteroatom. When "optionally substituted" is used to describe either the term "cycloheteroalkyl" or "cycloheteroalkyl-alkyl," it means that the cycloheteroalkyl or alkyl portion is optionally substituted as defined below for the alkyl portion. For example, an optionally substituted cycloheteroalkyl-alkyl group is one in which the cycloheteroalkyl and alkyl portions are optionally substituted as defined below for the alkyl substituent. Either or both may be optionally substituted.

[0046] As used herein, a wavy line "" crossing a single bond, double bond, or triple bond in any chemical structure depicted herein is [ka] " represents the point of attachment of a single, double, or triple bond to the rest of the molecule. Additionally, bonds extending to the center of a ring (e.g., a phenyl ring) are meant to indicate attachment at any of the available ring vertices. One of ordinary skill in the art will understand that multiple substituents shown as attached to a ring may occupy any ring vertex that provides a stable compound or is otherwise sterically compatible. In the case of divalent moieties, the representation is meant to include either orientation (forward or reverse). For example, the group "-C(O)NH-" is meant to include the bond in either the -C(O)NH- or -NHC(O)- orientation; similarly, "-O-CHCH-" is meant to include both -O-CHCH- and -CHCH-O-.

[0047] The terms "alkoxy," "alkylamino," and "alkylthio" (or thioalkoxy) are used in their ordinary sense to refer to alkyl groups attached to the remainder of the molecule through an oxygen atom, an amino group, or a sulfur atom, respectively. Furthermore, for dialkylamino groups, the alkyl portions may be the same or different and may form a 3- to 7-membered ring together with the nitrogen atom to which each is attached. Thus, dialkylamino or -NR a R b A group represented as: is meant to include piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl and the like.

[0048] The terms "arylalkyl" and "heteroarylalkyl" are used in their conventional sense, referring to an aryl or heteroaryl group attached to the remainder of the molecule via a C1-C4 alkylene linker. An exemplary embodiment of "arylalkyl" is phenylmethyl (or benzyl). Similarly, an exemplary embodiment of "heteroarylalkyl" is, for example, 3-pyridylpropyl. When "optionally substituted" is used to describe either the term "arylalkyl" or "heteroarylalkyl," this refers to a group in which the aryl or heteroaryl moiety is optionally substituted as defined below, and means that the alkyl moiety is optionally substituted as defined below.

[0049] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "C1-C4 haloalkyl" is meant to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0050] The term "aryl," unless otherwise specified, means a polyunsaturated, typically aromatic hydrocarbon group which may be fused or covalently linked, monocyclic or polycyclic (up to three rings). Non-limiting examples of aryl groups include phenyl, naphthyl, and biphenyl.

[0051] The term "heteroaryl" refers to an aryl group (or ring) containing 1 to 5 heteroatoms selected from N, O, and S, where the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of heteroaryl groups include pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, quinoxalinyl, and quinazolinyl. , cinnolinyl, phthalazinyl, benzotriazinyl, purinyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzisoxazolyl, isobenzofuryl, isoindolyl, indolizinyl, benzotriazinyl, thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridine, benzothiaxolyl, benzofuranyl, benzothienyl, indolyl, quinolyl, isoquinolyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furyl, thienyl, and the like. Heteroaryl ring substituents can be selected from the group of acceptable substituents described below.

[0052] In some embodiments, the above terms (e.g., "alkyl," "aryl," and "heteroaryl") are optionally substituted. Selected substituents for each type of radical are provided below.

[0053] Optional substituents for alkyl groups (including groups often referred to as alkylene, alkenyl, alkynyl, and cycloalkyl) include halogen, -OR', -NR'R'', -SR ... -SiR'R"R''', -OC(O)R', -C(O)R', -CO2R', -CON R'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)2R', -NH-C(NH2)=NH, -NR'C(NH2)= NH, -NH-C(NH2)=NR', -S(O)R', -S(O)2R, -S(O)2N R', R", and R'" each independently represent hydrogen, unsubstituted C1-C8 alkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted C1-C8 alkyl, C1-C8 alkoxy, or C1-C8 thioalkoxy group, or unsubstituted aryl-C1-C4 alkyl group. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to include 1-pyrrolidinyl and 4-morpholinyl.

[0054] Similarly, the optional substituents for the aryl and heteroaryl groups vary and generally include -halogen, -OR', -OC(O)R', -NR'R'', -SR', -R', -CN, -NO2, -CO2R', -CONR'R'', -C(O)R', -OC(O)NR'R'', -NR''C(O)R', -NR''C(O)2R', -NR'-C(O)NR''R''', - NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2)=NR' , -S(O)R', -S(O)2R', -S(O)2NR'R", -NR'S(O)2R" , -N3, perfluoro(C1-C4)alkoxy, and perfluoro(C1-C4)alkoxy and R', R", and R"' are selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3 independently selected from C-C cycloalkyl, C-C alkenyl, and C-C alkynyl Other suitable substituents include each of the above aryl substituents attached to a ring atom by an alkylene tether of 1 to 4 carbon atoms.

[0055] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be of the formula -TC(O)-(CH) qIt may be substituted with a substituent of -U-, where T and U are independently -NH-, -O-, -CH2-, or a single bond, and q is an integer of 0 to 2. Two of the substituents on adjacent atoms of the aryl or heteroaryl ring are of the formula -A-(CH2) r -B-, where A and B are independently -CH2-, -O-, - NH-, -S-, -S(O)-, -S(O)2-, -S(O)2NR', or a single bond, where r is an integer from 1 to 3. One of the single bonds in the new ring thus formed may optionally be replaced with a double bond. Alternatively, the aryl or heteroaryl ring may be replaced with a double bond on an adjacent atom. Two of the substituents have the formula -(CH2) s -X-(CH2) t where s and t are independently integers of 0 to 3, and X is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. The substituent R' in -NR'- and -S(O)2NR'- is selected from hydrogen or unsubstituted C1 to C6 alkyl.

[0056] As used herein, the term "heteroatom" is meant to include oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).

[0057] The term "pharmaceutically acceptable salts" is intended to include salts of active compounds prepared with relatively non-toxic acids or bases, depending on the particular substituents found on the compounds described herein. When a compound of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the desired base, either pure or in a suitable inert solvent. Examples of salts derived from pharmaceutically acceptable inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc, and the like. Salts derived from pharmaceutically acceptable organic bases include primary, secondary, and tertiary amines, including substituted amines, cyclic amines, naturally occurring amines, etc., such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc. When a compound of the present invention contains a relatively basic functional group, an acid addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphate, dihydrogenphosphate, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid, or phosphorous acid, as well as salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Also included are salts of amino acids such as alginate, and salts of organic acids such as glucuronic acid or galacturonic acid (see, e.g., Berge, SM, et al., "Ph armaceutical salts”, Journal ofPharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

[0058] The neutral forms of the compounds can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents, but the salts are otherwise equivalent to the parent form of the compound for purposes of the present invention.

[0059] In addition to salt forms, the present invention provides compounds in prodrug form. Prodrugs of the compounds described herein are compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Furthermore, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.

[0060] Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to the unsolvated forms and are encompassed within the scope of the present invention. It is intended that certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

[0061] Certain compounds of the present invention have asymmetric carbon atoms (optical centers) or double bonds. Racemates, diastereoisomers, geometric isomers, positional isomers, and individual isomers (e.g., separate enantiomers) are all intended to be encompassed within the scope of the present invention. When a stereochemical depiction is shown, it is meant to refer to a compound in which one isomer is present and the other isomer is substantially absent. "Substantially free of other isomers" refers to a ratio of at least 80 / 20, more preferably 90 / 10, or 95 / 5, or greater, of the two isomers. In some embodiments, one isomer is present in an amount of at least 99%.

[0062] The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. Unnatural proportions of an isotope may be defined as a range from the amount found in nature to the amount that constitutes 100% of the atom in question. For example, the compounds may contain unnatural proportions of atomic isotopes, such as tritium ( 3 H), iodine-125( 125 I), or carbon-14 ( 14 Radioactive isotopes such as C, or deuterium ( 2 H) or carbon 13 ( 13 C) etc. Non-radioactive isotopes of the compounds of the present invention may be incorporated. Such isotopic variations may provide additional utilities to those described elsewhere in this application. For example, isotopic variants of the compounds of the present invention may find additional utility, including but not limited to, as diagnostic and / or imaging reagents or as cytotoxic / radiotoxic therapeutic agents. Furthermore, isotopic variants of the compounds of the present invention may have altered pharmacokinetic and pharmacodynamic properties that contribute to improved safety, tolerability, or efficacy during treatment. All isotopic variations of the compounds of the present invention, whether radioactive or non-radioactive, are intended to be encompassed within the scope of the present invention.

[0063] The terms "patient" and "subject" are used interchangeably to refer to a human or non-human animal (e.g., a mammal).

[0064] The terms "administration," "administering," and the like, when applied to, e.g., a subject, cell, tissue, organ, or biological fluid, refer to the contact of, e.g., an inhibitor of CD73, a pharmaceutical composition comprising same, or a diagnostic agent, with a subject, cell, tissue, organ, or biological fluid. Administration in the context of a cell includes contact of a reagent with the cell (e.g., in vitro or ex vivo), as well as contact of a reagent with a fluid when the fluid is in contact with the cell.

[0065] The terms "treat," "treating," "treatment," and the like refer to a course of action (e.g., administration of CD73 or a pharmaceutical composition comprising same) initiated after a disease, disorder, or condition has been diagnosed, observed, etc., to temporarily or permanently eliminate, reduce, inhibit, alleviate, or ameliorate at least one of the underlying causes of the disease, disorder, or condition afflicting a subject, or at least one of the symptoms associated with the disease, disorder, or condition afflicting a subject. Treatment thus includes inhibiting active disease (e.g., preventing the progression or further progression of the disease, disorder, or condition or its associated clinical symptoms).

[0066] As used herein, the term "in need of treatment" refers to a judgment made by a physician or other treating practitioner that a subject needs or would benefit from treatment, based on a variety of factors within the physician's or treating practitioner's area of ​​expertise.

[0067] The terms "prevent," "preventing," "prevention," and the like, in the context of a subject who is predisposed to a particular disease, disorder, or condition, refer to a reduction in a subject's risk of developing the disease, disorder, condition, etc. "CD73 inhibitor" refers to a course of action (e.g., administration of an inhibitor of CD73 or a pharmaceutical composition containing same) that is initiated in such a way (e.g., prior to the onset of a disease, disorder, condition, or its symptoms) to temporarily or permanently prevent, suppress, arrest, or reduce, or delay the onset of, a disease, disorder, or condition (as determined by the absence of clinical symptoms). In some cases, the term also refers to slowing the progression of a disease, disorder, or condition, or inhibiting its progression to an adverse or unwanted state.

[0068] As used herein, the term "in need of prophylaxis" refers to a judgment made by a physician or other treating practitioner that a subject needs or would benefit from prophylactic treatment, based on a variety of factors within the physician's or treating practitioner's area of ​​expertise.

[0069] The phrase "therapeutically effective amount" refers to the administration of an agent to a subject, alone or as part of a pharmaceutical composition, in an amount that, when administered to a subject, can have any detectable positive effect on any symptom, aspect, or characteristic of a disease, disorder, or condition, either in a single dose or as part of a series of doses. A therapeutically effective amount can be ascertained by measuring the relevant physiological effect and can be adjusted in conjunction with dosing regimens, diagnostic analyses of the subject's condition, and the like. By way of example, measuring serum levels of a CD73 inhibitor (or its metabolite, etc.) at a particular time after administration can indicate whether a therapeutically effective amount has been used.

[0070] The term "in an amount sufficient to cause a change" means that there is a detectable difference between the level of an indicator measured before administration of a particular therapy (e.g., baseline level) and after administration. An indicator includes any objective parameter (e.g., serum concentration) or subjective parameter (e.g., subject's sense of well-being).

[0071] The term "small molecule" refers to a compound having a molecular weight of less than about 10 kDa, less than about 2 kDa, or less than about 1 kDa. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, and synthetic molecules. Therapeutically, small molecules may be more permeable to cells, less susceptible to degradation, and less likely to elicit an immune response than larger molecules.

[0072] The term "ligand" refers to a peptide, polypeptide, membrane-associated molecule, or membrane-bound molecule, or complex thereof, that can act as, for example, an agonist or antagonist of a receptor. Ligands encompass natural and synthetic ligands, such as cytokines, cytokine variants, analogs, muteins, and antibody-derived binding compositions, as well as small molecules. The term also encompasses agents that are neither agonists nor antagonists but can bind to a receptor without significantly affecting its biological properties, such as signal transduction or adhesion. Furthermore, the term encompasses membrane-bound ligands that have been modified, for example, by chemical or recombinant methods, to form soluble versions of the membrane-bound ligand. A ligand or receptor may be entirely intracellular, i.e., it may reside in the cytosol, nucleus, or some other intracellular compartment. A complex of a ligand and a receptor is called a "ligand-receptor complex."

[0073] The terms "inhibitor" and "antagonist" or "activator" and "agonist" refer to inhibitory or activating molecules, respectively, for the activation of, e.g., a ligand, receptor, cofactor, gene, cell, tissue, or organ. Inhibitors are molecules that reduce, block, prevent, delay activation, inactivate, desensitize, or downregulate, e.g., a gene, protein, ligand, receptor, or cell. Activators are molecules that increase, activate, promote, enhance activation, sensitize, or upregulate, e.g., a gene, protein, ligand, receptor, or cell. An "agonist" is a molecule that pre-regulates a target. An inhibitor can also be defined as a molecule that reduces, blocks, or inactivates constitutive activity. An "agonist" is a molecule that interacts with a target to cause or promote increased activation of the target. An "antagonist" is a molecule that opposes the action of an agonist. An antagonist prevents, reduces, inhibits, or neutralizes the activity of an agonist, and an antagonist can also prevent, inhibit, or reduce the constitutive activity of a target (e.g., a target receptor) even in the absence of an identified agonist.

[0074] The terms "modulate," "modulation," and the like refer to the ability of a molecule (e.g., an activator or inhibitor) to directly or indirectly increase or decrease the function or activity of CD73. Modulators may act alone or may use cofactors, such as proteins, metal ions, or small molecules. Examples of modulators include small molecule compounds and other bioorganic molecules. Numerous libraries of small molecule compounds (e.g., combinatorial libraries) are commercially available and can serve as a starting point for identifying modulators. One skilled in the art can develop one or more assays (e.g., biochemical or cell-based assays) that can screen such compound libraries to identify one or more compounds with desired properties. A skilled medicinal chemist can then optimize such one or more compounds, for example, by synthesizing and evaluating analogs and derivatives thereof. Synthetic and / or molecular modeling studies can also be used to identify activators.

[0075] The "activity" of a molecule describes or refers to the binding of the molecule to a ligand or receptor; catalytic activity; the ability to stimulate gene expression or cell signaling, differentiation or maturation; antigenic activity; modulation of the activity of other molecules, etc. The term "proliferative activity" includes activity that promotes, is necessary for, or is specifically associated with normal cell division, as well as cancer, tumors, dysplasia, cell transformation, metastasis, and angiogenesis.

[0076] As used herein, "equivalent," "equivalent activity," "equivalent activity to," "equivalent effect," "equivalent effect to," and the like are relative terms that can be viewed quantitatively and / or qualitatively. The meaning of these terms often depends on the context in which they are used. As an example, two agents that activate a receptor may be considered to have comparable effects from a qualitative perspective, but if one agent can only achieve 20% of the activity of the other agent as measured in an art-recognized assay (e.g., a dose-response assay) or in an art-recognized animal model, the two agents may not be considered to have equivalent effects from a quantitative perspective. When comparing one result to another (e.g., comparing one result to a reference standard), "equivalent" often (but not always) means that the one result deviates from the reference standard by less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 7%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In specific embodiments, a result is equivalent to a reference standard if it deviates from the reference standard by less than 15%, less than 10%, or less than 5%. For example, and without limitation, activity or effect can refer to efficacy, stability, solubility, or immunogenicity.

[0077] "Substantially pure" indicates that a component constitutes more than about 50% of the total content of the composition, and typically constitutes more than about 60% of the total content of the composition. More typically, "substantially pure" refers to a composition in which the component of interest constitutes at least 75%, at least 85%, at least 90%, or more of the total composition. In some cases, the component of interest will constitute more than about 90%, or even more than about 95%, of the total content of the composition.

[0078] When referring to a ligand / receptor, antibody / antigen, or other binding pair, the term "specifically binds" is used. " or "selectively binds" refers to a binding reaction that determines the presence of a protein in a heterogeneous population of other biological substances. Thus, under specified conditions, a particular ligand binds to a specific receptor and does not bind in significant amounts to other proteins present in a sample. A binding composition derived from the antigen-binding site of an antibody of the contemplated method binds to its antigen, or a variant or mutein thereof, with an affinity that is at least 2-fold, at least 10-fold, at least 20-fold, or at least 100-fold greater than the affinity of any other antibody or binding composition derived therefrom. In certain embodiments, the antibody has an affinity of about 1, as measured, for example, by Scatchard analysis (Munsen, et al. 1980 Analyt. Biochem. 107:220-239). 0 9 It has an affinity greater than liters / mole.

[0079] For example, the term "response" of a cell, tissue, organ, or organism encompasses a change in biochemical or physiological behavior, such as concentration, density, adhesion, or migration within a biological compartment, gene expression rate, or state of differentiation, where said change correlates with activation, stimulation, or treatment, or with internal mechanisms such as genetic programming. In certain contexts, terms such as "activation," "stimulation," and the like refer to cellular activation regulated by internal mechanisms and external or environmental factors, whereas terms such as "inhibition," "downregulation," and the like refer to the opposite effect.

[0080] The terms "polypeptide," "peptide," and "protein," used interchangeably herein, refer to polymeric forms of amino acids of any length, including genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified polypeptide backbones. These terms include, but are not limited to, fusion proteins with heterologous amino acid sequences, fusion proteins with heterologous and homologous leader sequences with or without an N-terminal methionine residue, and immunologically labeled proteins.

[0081] As used herein, the terms "variants" and "homologs" are used interchangeably to refer to amino acid sequences or DNA sequences similar to a reference amino acid or nucleic acid sequence, respectively. The terms encompass naturally occurring and non-naturally occurring variants. Naturally occurring variants include homologs (polypeptides and nucleic acids that differ in amino acid or nucleotide sequence, respectively, from one species to another) and allelic variants (polypeptides and nucleic acids that differ in amino acid or nucleotide sequence, respectively, from one individual to another within a species). Thus, variants and homologs encompass naturally occurring DNA sequences and the proteins encoded thereby, as well as their isoforms and splice variants of proteins or genes. The terms also encompass nucleic acid sequences that have one or more base changes from a naturally occurring DNA sequence but that, due to the degeneracy of the genetic code, are translated into an amino acid sequence corresponding to the naturally occurring protein. Non-naturally occurring variants and homologs include polypeptides and nucleic acids, respectively, containing altered amino acid or nucleotide sequences, where the sequence changes are artificially introduced (e.g., mutant proteins). For example, the changes are generated in a laboratory by human intervention ("the hand of man"). Thus, non-naturally occurring variants and homologs may refer to those that differ from naturally occurring sequences by one or more conservative substitutions and / or tags and / or conjugates.

[0082] As used herein, the term "mutein" refers to a recombinant protein that has been broadly mutated. These proteins typically have single or multiple amino acid substitutions and are often derived from cloned genes that have been subjected to site-directed or random mutagenesis or complete mutagenesis, or from genes that are completely synthetic.

[0083] The terms "DNA," "nucleic acid," "nucleic acid molecule," "polynucleotide," and the like refer to polymeric forms of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, primers, and the like.

[0084] 5'-Nucleotidase, Ecto and its Inhibition Human CD73 (also known as 5'-nucleotidase, ecto; NT5E; or 5NT) is a 574 amino acid residue protein (accession number AAH6593). Eukaryotic CD73 functions as a noncovalent homodimer with two structural domains, the N- and C-terminal domains connected by a hinge region, which allows the enzyme to undergo large domain movements and switch between open and closed conformations (Knapp, K. et al. (2012) Structure 20:2161-73).

[0085] As used herein, the terms "CD73 inhibitor," "CD73 blocker," "adenosine by 5'-nucleotidase ecto-inhibitor," "NT5E inhibitor," "5NT inhibitor," and all other relevant art-accepted terms refer to compounds that can directly or indirectly modulate the CD73 receptor in in vitro assays, in vivo models, and / or other means of demonstrating therapeutic efficacy. The term also refers to compounds that demonstrate at least some therapeutic efficacy in human subjects. CD73 inhibitors can be competitive, noncompetitive, or irreversible CD73 inhibitors. A "competitive CD73 inhibitor" is a compound that reversibly inhibits CD73 enzyme activity at the catalytic site; a "noncompetitive CD73 inhibitor" is a compound that reversibly inhibits CD73 enzyme activity at a non-catalytic site; and an "irreversible CD73 inhibitor" is a compound that irreversibly eliminates CD73 enzyme activity by forming a covalent bond with the enzyme (or other stable means of inhibiting enzyme function).

[0086] CD73 inhibitors can modulate purinergic signaling, a type of extracellular signaling mediated by purine nucleotides and nucleosides, such as ATP and adenosine. Purinergic signaling involves the activation of purinergic receptors in cells and / or nearby cells, resulting in the regulation of cellular function. The enzymatic activity of CD73 plays a strategic role in calibrating the duration, strength, and chemical nature of purinergic signals sent to various cells (e.g., immune cells). Alterations in these enzymatic activities can determine the outcome of several pathophysiological events, including cancer, autoimmune and inflammatory diseases, infections, atherosclerosis, and ischemia-reperfusion injury, suggesting that these extracellular enzymes represent novel therapeutic targets for managing various disorders.

[0087] Studies using tissues that overexpress CD73 and using CD73 knockout mice have provided evidence that CD73 inhibitors have potential utility against melanoma, lung cancer, prostate cancer, and breast cancer (see, e.g., Sadej R. (2006) Melanoma Res 16:213-22). (See, e.g., ). Higher expression levels of CD73 are associated with tumor angiogenesis, invasiveness, resistance to chemotherapy, and metastasis, and therefore CD73 inhibitors can be used to control tumor progression and metastasis. Other potential utilities are described elsewhere herein.

[0088] As noted above, although the compounds of the invention are believed to exert their activity through inhibition of CD73, a precise understanding of their mechanism of action is not necessary to practice the invention. For example, the compounds may also exert their activity, at least in part, through modulation (e.g., inhibition) of other components of the purinergic signaling pathway (e.g., CD39). The purinergic signaling system consists of transporters, enzymes, and receptors involved in (primarily) the synthesis, release, action, and extracellular inactivation of ATP and its extracellular breakdown product, adenosine (Sperlagh, B. et al. (Dec 2012) Neuropsychopharmacologia Hungarica 14(4):23 1-38). Figure 1 shows a simplified representation of extracellular purinergic signaling (see, for example, North RA (Oct 2002) Physiological Reviews 82(4):1013-67). As such, there are several potential opportunities for modulation of signal transduction processes. However, as will be apparent to those skilled in the art, some of these opportunities are more amenable than others.

[0089] Identification of CD73 inhibitors with desirable properties The present invention relates, in part, to the identification of inhibitors of CD73 that have at least one therapeutically relevant property or characteristic. Candidate inhibitors can be identified, for example, using art-recognized assays or models, examples of which will be apparent to those skilled in the art. Assays used to determine the CD73 inhibitory activity of the compounds described herein are described in the Experimental Section.

[0090] Once identified, candidate inhibitors can be further evaluated using techniques that provide data on inhibitor characteristics (e.g., pharmacokinetic parameters). Comparison of candidate inhibitors with reference standards (which may be "best-in-class" current inhibitors) indicates the potential of such candidates.

[0091] CD73 inhibitors that can serve as reference or benchmark compounds include those described by Bhattarai et al. ((2015) J Med Chem 58:6248-63) described α,β-methylene-ADP(A O PCP) and its derivatives and analogs, and the purine CD73 derivatives reported in PCT Publication No. 2015 / 164573. Other reference compounds subsequently identified by one of skill in the art can also be used to assess the viability of candidate CD73 inhibitors.

[0092] Compounds of the Invention Formula (I): [ka] or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein: Each R 1 is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted aryl -C(R 2 R 2 )-OC(O)-OR 3 are independently selected from the group consisting of , or two R 1 groups optionally combined to form a 5- to 7-membered ring; Each R 2 are independently selected from the group consisting of H and optionally substituted C1-C6 alkyl be; Each R 3 is selected from the group consisting of H, C1-C6 alkyl, and optionally substituted aryl Independently selected; R 5 is selected from the group consisting of H and optionally substituted C1-C6 alkyl; X is selected from the group consisting of O, CH2, and S; A is the following: [ka] each of which is selected from the group consisting of 1 to 5 R 6 optionally substituted with a substituent; where the subscript n is an integer between 0 and 3; Z is CH2, CHR 6 , N.R. 6 selected from the group consisting of: Each R 6 are independently selected from the group consisting of H, CH3, OH, CN, F, optionally substituted C1-C6 alkyl, and OC(O)-C1-C6; and optionally, two R on adjacent ring vertices are 6 The groups are joined together to form a ring with at least one heteroatom as a ring vertex. forming a 5- or 6-membered ring; and Het is [ka] Selected from the group consisting of: where the wavy line indicates the point of attachment to the remainder of the compound, R a are H, NH2, and NHR 7 , NHC(O)R 7 , N.R. 7 R 7 , R 7 , O.H., S.R. 7 , and OR 7 selected from the group consisting of: R b is H, halogen, NH2, NHR 7 , N.R. 7 R 7 , R 7 , OH, and OR 7 A group consisting of Selected from; R c and R d is H, halogen, haloalkyl, NH2, NHR 7 , N.R. 7 R 7 , R 7 , O.H. , OR 7 , S.R.7 , SO2R 7 , -X 1 -NH2, -X 1 -NHR 7 , -X 1 -NR 7 R 7 , -X 1 -OH, -X 1 -OR 7 , -X 1 -SR 7 , and -X 1 -SO2R 7 Independently selected from the group consisting of be; R e and R f are independently selected from the group consisting of H, halogen, and optionally substituted C1-C6 alkyl; and each X 1 is a C1-C4 alkylene, and Each R 7 is an optionally substituted C1-C 10 Alkyl, optionally substituted C-C 10 Alkenyl, optionally substituted C-C 10 alkynyl, optionally substituted C3-C7 cycloalkyl, optionally substituted C3-C7 cycloalkylC1-C4 alkyl, optionally substituted 4-7 membered cycloheteroalkyl, optionally substituted 4-7 membered cycloheteroalkyl C1-C4 alkyl, optionally substituted aryl, optionally substituted aryl C1-C4 alkyl, optionally substituted aryl C2-C4 alkenyl, optionally substituted aryl C2-C4 alkynyl, optionally substituted heteroaryl, optionally substituted heteroaryl C1-C4 alkyl, optionally substituted heteroaryl C1-C4 alkenyl, optionally substituted heteroaryl C2-C4 alkynyl, and optionally two R 7 The groups are linked together and optionally Forming a 4- to 7-membered heterocyclic ring fused to an aryl ring; However, the compound may be such that the combination of X, A, and Het is: [ka] [where R g is H or two R g groups combine to form an acetonide; and (i)R c and R e is hydrogen and R a -OEt, -OCH2Ph, -SCH2Ph, -NH2, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl -N-ethylamino, phenylamino, benzylamino, 2-phenylethylamino, N-benzyl-N-ethylamino, dibenzylamino, 4-aminobenzylamino, 4-chlorobenzylamino, 4-nitrobenzylamino, or 4-sulfamoylbenzylamino; or (ii)R c is hydrogen and R a is -NH2 and R e is bromo, chloro, aminomethyl, or thioethyl; or (iii)R c is hydrogen and R a is benzylamino and Re is bromo.

[0093] In the above formula, the term "optionally substituted" is used in connection with alkyl, cycloalkyl, cycloheteroalkyl, aryl, and heteroaryl groups. Within each of these groups, some selected optional substituents are as follows: Alkyl groups, halogens, -OR', -NR'R", -SR', -SiR'R"R"', -OC(O)R', -C(O)R', -CO2R', -CONR'R", -OC(O)N R'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)2R', -CN, and -NO2. R', R", and R"' are each independently hydrogen, unsubstituted C1-C4 alkyl, or C1-C4 haloalkyl. When R' and R" are attached to the same nitrogen atom, or when R" and R"' are attached to the same nitrogen atom, they form a bond to that nitrogen atom. When combined with the alkyl group, they form a 3-, 4-, 5-, 6-, or 7-membered ring. For example, -NR'R" is It is meant to include 1-pyrrolidinyl and 4-morpholinyl. Cycloalkyl and cycloheteroalkyl groups: Selected substituents listed above for "alkyl groups" are also useful for cycloalkyl and cycloheteroalkyl groups. Additionally, each of the cycloalkyl and cycloheteroalkyl groups can be optionally substituted with oxo (=O). Alkyl groups and heteroaryl groups: -halogen, -OR', -OC(O)R', -NR'R", -R', -CN, -NO2, -CO2R', CONR'R", C(O)R', -OC(O)NR'R", -NR"C(O)R', -NR"C(O)2R', -NR'-C( O)NR”R”’, -S(O)2R’, S(O)2NR’R”, NR’S(O)2R”, and and perfluoro(C1-C4) alkyl, where R', R'', and R'' are hydrogen, C1 independently selected from C-C alkyl, C-C haloalkyl, and C-C cycloalkyl will be done.

[0094] In one selected group of embodiments, compounds of formula (I) are provided wherein A is of the formula [ka] (This is 1 to 5 R 6 (optionally substituted with).

[0095] In another selected group of embodiments, compounds of formula (I) are provided wherein A is [ka] having a formula selected from

[0096] In some selected embodiments, any one of a1 through a16 is combined with any one of b1 through b9 to provide selected embodiments of Formula (I). For example, provided herein are compounds of Formula (I) having the following Het-A- combinations: a1 / b1; a1 / b2; a1 / b3; a1 / b4; a1 / b5; a1 / b6; a1 / b7; a1 / b8; a1 / b9; a2 / b1; a2 / b2; a2 / b3; a2 / b4; a2 / b5; a2 / b6; a2 / b7; a2 / b8; a2 / b9; a3 / b1; a3 / b2 ;a3 / b3;a3 / b4;a3 / b5;a3 / b6;a3 / b7;a3 / b8;a3 / b9;a4 / b1;a4 / b2;a4 / b3;a4 / b4;a4 / b5;a4 / b6;a4 / b7;a4 / b8;a4 / b9;a5 / b1;a5 / b2;a5 / b3;a5 / b4;a5 / b5;a5 / b6;a5 / b7;a5 / b8;a5 / b9;a6 / b1;a6 / b2;a 6 / b3;a6 / b4;a6 / b5;a6 / b6;a6 / b7;a6 / b8;a6 / b9;a7 / b1;a7 / b2;a7 / b3;a7 / b4;a7 / b5;a7 / b6;a7 / b7;a7 / b8;a7 / b9;a8 / b1;a8 / b2;a8 / b3;a8 / b4;a8 / b5;a8 / b6;a8 / b7;a8 / b8;a8 / b9;a9 / b1;a9 / b2; a9 / b3;a9 / b4;a9 / b5;a9 / b6;a9 / b7;a9 / b8;a9 / b9;a10 / b1;a10 / b2;a10 / b3;a10 / b4;a10 / b5;a10 / b6;a10 / b7;a10 / b8;a10 / b9;a11 / b1;a11 / b2;a11 / b3;a11 / b4;a11 / b5;a11 / b6;a11 / b7;a11 / b8;a 11 / b9;a12 / b1;a12 / b2;a12 / b3;a12 / b4;a12 / b5;a12 / b6;a12 / b7;a12 / b8;a12 / b9;a13 / b1;a13 / b2;a13 / b3;a13 / b4;a13 / b5;a13 / b6;a13 / b7;a13 / b8;a13 / b9;a14 / b1;a14 / b2;a14 / b3;a14 / b4;a 14 / b5;a14 / b6;a14 / b7;a14 / b8;a14 / b9;a15 / b1;a15 / b2;a15 / b3;a15 / b4;a15 / b5;a15 / b6;a15 / b 7;a15 / b8;a15 / b9;a16 / b1;a16 / b2;a16 / b3;a16 / b4;a16 / b5;a16 / b6;a16 / b7;a16 / b8;or a16 / b9.

[0097] In yet other selected embodiments, compounds of Formula (I) are provided wherein Het is of the formula: [ka] It has. In some selected embodiments, R c is other than H.

[0098] In yet other selected embodiments, compounds of formula (I) are provided which have one of the following subformulas: [ka] Here, each R g is independently selected from the group consisting of H and C(O)-C1-C6 alkyl. In still other selected embodiments of the above subformula, X is oxygen. In other selected embodiments of the above subformula, X is oxygen and R e is hydrogen. In still other selected embodiments of the genus, X is oxygen and R e is hydrogen, and each R g is hydrogen.

[0099] In another group of selected embodiments, compounds of Formula (I) are provided wherein Het is selected from: [ka] where R a , R c , and R e has the meaning given in relation to formula (I) above. In some further selected embodiments, R 5 is H, X is O, and each R 1 is H. In yet another selected embodiment, R 5 is H, X is O, and each R 1 is H and R e is H, and R a NH2, NHR 7 , and N(R 7 In yet another selected embodiment, R 5 is H and X is O Each R 1 is H and R e is H and R c is other than H, and R a is NHR 7 is .

[0100] Yet another selected embodiment of Formula (I) is a compound having a subformula selected from the following: [ka] where R 7 and R c has the meaning provided in connection with Formula (I) and selected embodiments described herein.

[0101] Also in one group of embodiments, the compound of the formula: [ka] or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein: Each R 1 is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted aryl -C(R 2 R 2 )-OC(O)-OR 3 are independently selected from the group consisting of , or two R 1 groups optionally combined to form a 5- to 7-membered ring; Each R 2 are independently selected from the group consisting of H and optionally substituted C1-C6 alkyl be; Each R 3 is selected from the group consisting of H, C1-C6 alkyl, and optionally substituted aryl Independently selected; R 5 is selected from the group consisting of H and optionally substituted C1-C6 alkyl; X is O; A is, [ka] is selected from the group consisting of Het is [ka] Selected from the group consisting of: where the wavy line indicates the point of attachment to the remainder of the compound, R a are H, NH2, and NHR 7 , NHC(O)R 7 , N.R. 7 R 7 , R 7 , O.H., S.R. 7 , and OR 7 selected from the group consisting of: R b is H, halogen, NH2, NHR 7 , N.R. 7 R 7 , R 7 , OH, and OR 7 A group consisting of Selected from; R c and R d is H, halogen, haloalkyl, NH2, NHR 7 , N.R. 7 R 7 , R 7 , O.H. , OR 7 , S.R. 7 , SO2R 7 , -X 1 -NH2, -X 1 -NHR 7 , -X 1 -NR 7 R 7 , -X 1 -OH, -X 1 -OR 7 , -X 1 -SR 7 , and -X 1 -SO2R 7 Independently selected from the group consisting of be; R e and R f are independently selected from the group consisting of H, halogen, and optionally substituted C1-C6 alkyl; and each X 1is a C1-C4 alkylene, and Each R 7 is an optionally substituted C1-C 10 Alkyl, optionally substituted C-C 10 Alkenyl, optionally substituted C-C 10 alkynyl, optionally substituted C3-C7 cycloalkyl, optionally substituted C3-C7 cycloalkylC1-C4 alkyl, optionally substituted 4-7 membered cycloheteroalkyl, optionally substituted 4-7 membered cycloheteroalkyl C1-C4 alkyl, optionally substituted aryl, optionally substituted aryl C1-C4 alkyl, optionally substituted aryl C2-C4 alkenyl, optionally substituted aryl C2-C4 alkynyl, optionally substituted heteroaryl, optionally substituted heteroaryl C1-C4 alkyl, optionally substituted heteroaryl C1-C4 alkenyl, optionally substituted heteroaryl C2-C4 alkynyl, and optionally two R 7 The groups are linked together and optionally It forms a 4- to 7-membered heterocyclic ring fused to an aryl ring.

[0102] In one selected group of embodiments, compounds of formula (IVa) are those in which A is [ka] It is what it is.

[0103] In another selected group of embodiments, compounds of formula (IVa) are those in which Het is [ka] is selected from the group consisting of:

[0104] In yet another selected group of embodiments, the compound has the formula: [ka] or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

[0105] In one selected group of embodiments, the compound of formula (IVb) is R a However, NH2, NHR 7 , N.R. 7 R 7 , S.R. 7 and OR 7 One selected from the group consisting of: In one group of embodiments, the compound of formula (Ib) is R c But halogen, R 7 , OR 7 , S R 7 , SO2R 7 , -X 1 -NH2, -X 1 -NHR 7 , -X 1 -NR 7 R 7 , -X 1 -OH, -X 1 -OR 7 , -X 1 -SR 7 , and -X 1 -SO2R 7 is selected from the group consisting of:

[0106] In yet another group of embodiments, the compounds of formula (IVb) are R e is H do.

[0107] Synthesis method In general, the compounds provided herein can be prepared by conventional methods such as those described in the examples below.

[0108] Modifications to enhance inhibitor properties It is often beneficial, and sometimes essential, to improve one or more physical properties of the therapeutic modalities disclosed herein and / or the manner in which they are administered. Improvements in physical properties include, for example, methods to increase water solubility, bioavailability, serum half-life, and / or therapeutic half-life; and / or methods to modulate biological activity.

[0109] Modifications known in the art include PEGylation, Fc fusion, and albumin fusion. While generally associated with large molecular entities (e.g., polypeptides), such modifications have recently been evaluated with certain small molecules. See, for example, Chiang, M. et al. (J. Am. Chem. Soc., 2014 , 136(9):3370-73) described small molecule agonists of the adenosine 2a receptor conjugated to immunoglobulin Fc domains. The small molecule-Fc conjugates retained strong F receptor and adenosine 2a receptor interactions and exhibited superior properties compared to unconjugated small molecules. Covalent attachment of PEG molecules to small molecule therapeutics has also been described (Li, W. et al., Progress in P Polymer Science, 2013 38:421-44).

[0110] Therapeutic and prophylactic uses The present invention contemplates the use of the CD73 inhibitors described herein in the treatment or prevention of a wide range of diseases, disorders, and / or conditions, and / or symptoms thereof. While particular uses are described in detail below, it should be understood that the present invention is not limited thereto. Additionally, while general categories of certain diseases, disorders, and conditions are set forth below, some diseases, disorders, and conditions may be members of more than one category, and others may not be members of any of the disclosed categories.

[0111] Tumor-related disordersAccording to the present invention, CD73 inhibitors can be used to treat or prevent proliferative conditions or disorders, including cancer, for example, uterine cancer, cervical cancer, breast cancer, prostate cancer, testicular cancer, cancer of the gastrointestinal tract (e.g., esophageal cancer, oropharyngeal cancer, stomach cancer, small intestine or large intestine cancer, colon, rectum cancer), kidney cancer, renal cell carcinoma, bladder cancer, bone cancer, bone marrow cancer, skin cancer, head or neck cancer, liver cancer, gallbladder cancer, heart cancer, lung cancer, pancreatic cancer, salivary gland cancer, adrenal gland cancer, thyroid cancer, brain cancer (e.g., glioma), ganglionic cancer, cancer of the central nervous system (CNS) and peripheral nervous system (PNS), and cancer of the hematopoietic and immune systems (e.g., spleen or thymus). The present invention also provides methods for treating or preventing other cancer-related diseases, disorders, or conditions, including, for example, immunogenic tumors, non-immunogenic tumors, dormant tumors, virus-induced cancers (e.g., epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, and papillomavirus), adenocarcinoma, lymphoma, carcinoma, melanoma, leukemia, myeloma, sarcoma, teratocarcinoma, chemically-induced cancer, metastasis, and angiogenesis. The present invention contemplates reducing tolerance to tumor cells or cancer cell antigens, for example, by modulating the activity of regulatory T cells and / or CD8+ T cells (see, e.g., Ramirez-Montagut, et al. (2003) Oncogene 22:3180-87; and (See Sawaya, et al. (2003) New Engl. J. Med. 349:1501-09). In certain embodiments, The tumor or cancer is colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, glioblastoma, or leukemia. The use of the term cancer-related diseases, disorders, and conditions is intended to broadly refer to conditions that are directly or indirectly related to cancer, and includes pre-cancerous conditions such as angiogenesis and dysplasia.

[0112] In certain embodiments, the cancer may be metastatic or at risk of becoming metastatic, or may be present in diffuse tissues, including cancers of the blood or bone marrow (e.g., leukemia). In some further embodiments, the compounds of the present invention may be used to overcome T-cell tolerance.

[0113] In some embodiments, the present invention provides methods of treating a proliferative condition, cancer, tumor, or precancerous condition with a CD73 inhibitor and at least one additional therapeutic or diagnostic agent, examples of which are described elsewhere herein.

[0114] Immune-related disorders and disorders with inflammatory components As used herein, the terms "immune disease," "immune condition," "immune disorder," "inflammatory disease," "inflammatory condition," "inflammatory disorder," and the like are meant to broadly encompass any immune-based condition (e.g., autoimmune disease) or disorder with an inflammatory component that can be treated by the CD73 inhibitors described herein to provide some therapeutic benefit. Such conditions are often closely intertwined with other diseases, disorders, and conditions. By way of example, "immune condition" refers to proliferative conditions such as cancer, tumors, and angiogenesis, including infections (acute and chronic), tumors, and cancers that resist eradication by the immune system.

[0115] The CD73 inhibitors of the present invention can be used to increase or enhance immune responses; improve vaccinations, including increasing vaccine efficacy; and increase inflammation. Immunodeficiency disorders, immunosuppressive therapy, acute and / or chronic infections, and age-related immune deficiencies can be treated using the compounds disclosed herein. CD73 inhibitors can also be used to stimulate the immune system of patients suffering from iatrogenically induced immunosuppression, including patients who have undergone bone marrow transplantation, chemotherapy, or radiation therapy.

[0116] In certain embodiments of the present disclosure, CD73 inhibitors are used to enhance or potentiate immune responses to antigens by providing adjuvant activity. In certain embodiments, at least one antigen or vaccine is administered to a subject in combination with at least one CD73 inhibitor of the present invention to prolong the immune response to the antigen or vaccine. Therapeutic compositions comprising at least one antigenic substance or vaccine component, including but not limited to viruses, bacteria, and fungi, or portions thereof, proteins, peptides, tumor-specific antigens, and nucleic acid vaccines, can also be used in combination with at least one CD73 inhibitor of the present invention.

[0117] Microbial-associated disorders The present invention contemplates the use of the CD73 inhibitors described herein in the treatment and / or prevention of any viral, bacterial, fungal, parasitic, or other infectious disease, disorder, or condition in which treatment with a CD73 inhibitor would be beneficial by inhibiting the immunosuppressive and anti-inflammatory activities of CD73. Examples of such diseases and disorders include HIV and AIDS, staphylococcal and streptococcal infections (e.g., Staphylococcus aureus and oral Streptococcus), Leishmania, Toxoplasma, Trichomonas, Giardia, Candida albicans, Bacillus anthracis, and Pseudomonas aeruginosa. The compounds of the present invention can be used to treat sepsis, reduce or inhibit bacterial growth, and reduce or inhibit proinflammatory cytokines.

[0118] CNS-Related and Neurological Disorders Inhibition of CD73 may also be an important therapeutic strategy for patients with neurological, neuropsychiatric, neurodegenerative, or other diseases, disorders, and conditions that have central nervous system involvement, including disorders associated with impaired cognitive and motor function. Examples include Parkinson's disease, extrapyramidal syndrome (EPS), dystonia, atonia, tardive dyskinesia, restless legs syndrome (RLS), epilepsy, periodic limb movements during sleep (PLMS), attention deficit disorder, depression, anxiety, dementia, Alzheimer's disease, Huntington's disease, multiple sclerosis, cerebral ischemia, hemorrhagic stroke, subarachnoid hemorrhage, and traumatic brain injury.

[0119] Other disorders Embodiments of the present invention contemplate administering a CD73 inhibitor described herein to a subject for the treatment or prevention of any other disorder that can benefit from at least some level of CD73 inhibition. Such diseases, disorders, and conditions include, for example, cardiovascular (e.g., cardiac ischemia), gastrointestinal (e.g., Crohn's disease), metabolic (e.g., diabetes), hepatic (e.g., liver fibrosis, NASH, and NAFLD), pulmonary (e.g., COPD and asthma), ocular (e.g., diabetic retinopathy), and renal (e.g., renal failure) disorders.

[0120] In some embodiments, the CD73 inhibitors of the present invention can be used to inhibit statin-induced adenosine production or reduce or lower the rise in blood glucose caused by statins in subjects taking statins (e.g., lovastatin and pravastatin).

[0121] Pharmaceutical Composition The CD73 inhibitors of the present invention may be in the form of compositions suitable for administration to a subject. Generally, such compositions are "pharmaceutical compositions" comprising a CD73 inhibitor and one or more pharmaceutically or physiologically acceptable diluents, carriers, or excipients. In certain embodiments, the CD73 inhibitor is present in a therapeutically acceptable amount. Pharmaceutical compositions can be used in the methods of the present invention. Thus, for example, pharmaceutical compositions can be administered to a subject ex vivo or in vivo to practice the therapeutic and prophylactic methods and uses described herein.

[0122] The pharmaceutical compositions of the present invention can be formulated to be compatible with the intended method or route of administration. Exemplary routes of administration are described herein. Furthermore, the pharmaceutical compositions can be used in combination with other therapeutically active agents or compounds described herein to treat or prevent the diseases, disorders, and conditions contemplated by the present invention.

[0123] Pharmaceutical compositions containing an active ingredient (e.g., an inhibitor of CD73 function) may be in a form suitable for oral use, such as tablets, capsules, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, hard or soft capsules, or syrups, solutions, microbeads, or elixirs. Pharmaceutical compositions intended for oral use can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents, such as sweeteners, flavoring agents, coloring agents, and preservatives, to provide pharmaceutically elegant and palatable preparations. Tablets, capsules, and the like contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients suitable for the manufacture of tablets. These excipients may include, for example, diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrating agents such as cornstarch or alginic acid; binders such as starch, gelatin, or acacia; and lubricants such as magnesium stearate, stearic acid, or talc.

[0124] Tablets, capsules, and the like suitable for oral administration may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action. For example, a time-delay material such as glyceryl monostearate or glyceryl distearate can be used. They may also be coated by techniques known in the art to form osmotic therapeutic tablets for controlled release. Additional agents include polyesters to control delivery of the administered composition. Biodegradable or biocompatible particulate or polymeric materials include polyamine acids, hydrogels, polyvinylpyrrolidone, polyanhydrides, polyglycolic acid, ethylene-vinyl acetate, methylcellulose, carboxymethylcellulose, protamine sulfate, or lactide / glycolide copolymers, polylactide / glycolide copolymers, or ethylene-vinyl acetate copolymers. For example, oral agents can be encapsulated in microcapsules prepared by coacervation techniques or interfacial polymerization, hydroxymethylcellulose or gelatin-microcapsules, or poly(methyl methacrylate) microcapsules, or in colloidal drug delivery systems. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, microbeads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Methods for preparing the above formulations will be apparent to those skilled in the art.

[0125] Formulations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin, or microcrystalline cellulose, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin, or olive oil.

[0126] Aqueous suspensions contain the active substance in admixture with excipients suitable for their manufacture. Such excipients may be suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum arabic; dispersing or wetting agents, such as natural phosphatides (e.g., lecithin), or condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), or condensation products of ethylene oxide with long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyethylene sorbitan monooleate). Aqueous suspensions may also contain one or more preservatives.

[0127] Oily suspensions can be formulated by suspending the active ingredient in vegetable oils, such as peanut oil, olive oil, sesame oil, or coconut oil, or mineral oils such as liquid paraffin.Oily suspensions can contain thickening agents, such as beeswax, hard paraffin, or cetyl alcohol.Sweeteners and flavoring agents, such as those mentioned above, can be added to provide a palatable oral preparation.

[0128] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above.

[0129] The pharmaceutical composition of the present invention may also be in the form of an oil-in-water emulsion.The oily phase may be a vegetable oil, such as olive oil or peanut oil, or a mineral oil, such as liquid paraffin, or a mixture thereof.Suitable emulsifiers may be natural gums, such as gum arabic or gum tragacanth; natural phospholipids, such as soybean, lecithin, and esters or partial esters derived from fatty acids; hexitol anhydrides, such as sorbitan monooleate; and condensation products of partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate.

[0130] Pharmaceutical compositions typically contain a therapeutically effective amount of a CD73 inhibitor contemplated by the present invention and one or more pharmaceutically and physiologically acceptable formulations. Suitable pharmaceutically and physiologically acceptable diluents, carriers, or excipients include, but are not limited to, antioxidants. The vehicle may include, but is not limited to, agents (e.g., ascorbic acid and sodium bisulfate), preservatives (e.g., benzyl alcohol, methylparaben, ethyl or n-propyl, p-hydroxybenzoate), emulsifiers, suspending agents, dispersing agents, solvents, fillers, extenders, surfactants, buffers, vehicles, diluents, and / or adjuvants. For example, a suitable vehicle may be saline or citrate-buffered saline, possibly supplemented with other substances common in pharmaceutical compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary excipients. Those skilled in the art will readily recognize various buffers that can be used in the pharmaceutical compositions and dosage forms contemplated herein. Typical buffering agents include, but are not limited to, pharmaceutically acceptable weak acids, weak bases, or mixtures thereof. By way of example, buffering components may be water-soluble substances such as phosphoric acid, tartaric acid, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, and salts thereof. Acceptable buffers include, for example, Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), and N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS).

[0131] After the pharmaceutical composition is formulated, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. Such formulations can be stored in a ready-to-use form, a lyophilized form that must be reconstituted before use, a liquid form that must be diluted before use, or other acceptable form. In some embodiments, the pharmaceutical composition is provided in a single-use container (e.g., a single-use vial, an ampoule, a syringe, or an autoinjector (similar to EpiPen®)), while a multi-use container (e.g., a multi-use vial) is provided in other embodiments.

[0132] The formulation may also include a carrier to protect the composition against rapid degradation or elimination from the body, such as a controlled release formulation, including liposomes, hydrogels, prodrugs, and microencapsulated delivery systems. For example, a time delay material such as glyceryl monostearate or glyceryl stearate alone or in combination with a wax may be employed. Any drug delivery device can be used to deliver the CD73 inhibitor, including implants (e.g., implantable pumps) and catheter systems, slow infusion pumps, and devices, all of which are well known to those skilled in the art.

[0133] Depot injections, typically administered subcutaneously or intramuscularly, can also be used to release the CD73 inhibitors disclosed herein over a defined period of time. Depot injections are typically solid or oily and generally contain at least one of the formulation components described herein. Those skilled in the art are familiar with the possible formulations and uses of depot injections.

[0134] The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. Such suspensions can be prepared according to known techniques using suitable dispersing or wetting agents and suspending agents as described above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Acceptable diluents, solvents, and dispersion media that can be used include water, Ringer's solution, isotonic sodium chloride solution, CremophorEL® (BASF, Parsippany, NJ), or phosphate buffer. Examples of suitable solvents include phosphate-buffered saline (PBS), ethanol, polyols (e.g., glyceryl, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In addition, sterile, fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any bland fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectable solutions. To delay absorption, Prolonged absorption of certain injectable formulations can be achieved by including an agent that enhances absorption, such as aluminum monostearate or gelatin.

[0135] The present invention contemplates the administration of a CD73 inhibitor in the form of a suppository for rectal administration. Suppositories can be prepared by mixing a drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, thereby dissolving in the rectum and releasing the drug. Such materials include, but are not limited to, cocoa butter and polyethylene glycol.

[0136] The CD73 inhibitors contemplated by the present invention may be in the form of any other suitable pharmaceutical composition (eg, a nasal or inhalation spray) now known or developed in the future.

[0137] Route of administration The present invention contemplates administering CD73 inhibitors and compositions thereof by any suitable method. Suitable routes of administration include oral, parenteral (e.g., intramuscular, intravenous, subcutaneous (e.g., injection or implantation), intraperitoneal, intracapsular, intraarticular, intraperitoneal, intracerebral (intraparenchymal, intracerebroventricular), intranasal, intravaginal, sublingual, intraocular, rectal, topical (e.g., transdermal), buccal, and inhalation). Depot injections, generally administered subcutaneously or intramuscularly, may also be utilized to release the CD73 inhibitors disclosed herein over a defined period of time.

[0138] Certain embodiments of the present invention contemplate oral administration.

[0139] Combination therapy The present invention contemplates the use of CD73 inhibitors in combination with one or more active therapeutic agents (e.g., chemotherapeutic agents) or other prophylactic or therapeutic methods (e.g., radiation). In such combination therapies, the various active agents often have different, complementary mechanisms of action. Such combination therapies can be particularly advantageous by allowing for dose reduction of one or more agents, thereby reducing or eliminating adverse effects associated with one or more agents. Furthermore, such combination therapies can have a synergistic therapeutic or prophylactic effect against the underlying disease, disorder, or condition.

[0140] As used herein, "combination" is meant to include therapies that may be administered separately, e.g., formulated separately for separate administration (e.g., as may be provided in a kit), and therapies that are administered together in a single formulation (e.g., "co-formulation").

[0141] In certain embodiments, the CD73 inhibitors are administered or applied sequentially, e.g., when one agent is administered before one or more other agents. In other embodiments, the CD73 inhibitors are administered simultaneously, e.g., when two or more agents are administered simultaneously or near simultaneously, the two or more agents may be present in two or more separate formulations or may be combined in a single formulation (i.e., co-formulation). Regardless of whether two or more agents are administered sequentially or simultaneously, they are considered to be administered in combination for purposes of the present invention.

[0142] The CD73 inhibitors of the present invention can be used in combination with at least one other (active) agent in any manner appropriate to the circumstances. In one embodiment, treatment with at least one active agent and at least one CD73 inhibitor of the present invention is maintained for a period of time. In another embodiment, treatment with at least one active agent is reduced or discontinued (e.g., if the subject is stable), while treatment with a CD73 inhibitor of the present invention is maintained at a constant dosage regimen. In a further embodiment, treatment with at least one active agent is reduced or discontinued (e.g., if the subject is stable), while treatment with a CD73 inhibitor of the present invention is maintained at a constant dosage regimen. In a further embodiment, treatment with at least one active agent is maintained and treatment with a CD73 inhibitor of the present invention is reduced or discontinued (e.g., if the subject is stable), while treatment with a CD73 inhibitor of the present invention is reduced (e.g., lower dose, less frequent administration, or shorter treatment regimen). In a further embodiment, treatment with at least one active agent is maintained and treatment with a CD73 inhibitor of the present invention is reduced or discontinued (e.g., lower dose, less frequent administration, or shorter treatment regimen). In a further embodiment, treatment with at least one active agent and treatment with a CD73 inhibitor of the present invention are reduced or discontinued (e.g., lower dose, less frequent administration, or shorter treatment regimen).

[0143] Cancer-related diseases The present invention provides methods for treating and / or preventing a proliferative condition, cancer, tumor, or precancerous disease, disorder, or condition using a CD73 inhibitor and at least one additional therapeutic or diagnostic agent.

[0144] In certain embodiments, the present invention provides a method for tumor suppression of tumor growth, comprising administering a CD73 inhibitor described herein in combination with a signal transduction inhibitor (STI) to achieve additive or synergistic suppression of tumor growth. As used herein, the term "signal transduction inhibitor" refers to an agent that selectively inhibits one or more steps in a signal transduction pathway. Signal transduction inhibitors (STIs) of the present invention include: (i) bcr / abl kinase inhibitors (e.g., Gleevec); (ii) epidermal growth factor (EGF) receptor inhibitors, including kinase inhibitors and antibodies; (iii) her-2 / neu receptor inhibitors (e.g., Herceptin); (iv) inhibitors of Akt family kinases or Akt pathways (e.g., rapamycin); (v) cell cycle kinase inhibitors (e.g., flavopiridol); and (vi) phosphatidylinositol kinase inhibitors. Agents involved in immune regulation may also be used in combination with the CD73 inhibitors described herein to suppress tumor growth in cancer patients.

[0145] Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, metholedopa, and uredopa; ethyleneimines and methylameramines such as altretamine, triethylenemelamine, trimethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustards such as methylameramine, ... for example, chlorambucil, chlornaphazine, cholinephosphamide, estramustine, ifosfamide, mechlorethamine, mephloretaine oxide hydrochloride, melphalan, novembitine, phenesterine, prednimustine, troposphamide, uracil mustard; nitrosoureas, for example, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, for example, aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, Calicheamicin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex , zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, Antiadrenal drugs such as cyclosporine, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as furoic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine ;Razoxane;Sizofiran;Spirogermanium;Tenuazonic acid;Triaziquone;2,2',2''-Trichlorotriethylamine;Urethane;Vindesine;Dacarbazine;Mannomustine;Mitobronitol;Mitolactol;Pipobroman;Gacytosine;Arabinoside (Ara-C);Cyclophosphamide;Thiotepa;Taxoids, e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum and platinum coordination complexes such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT11; topoisomerase inhibitors; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0146] Chemotherapeutic agents also include antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens (e.g., tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxin, keoxifene, onapristone, and toremifene); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. In certain embodiments, the combination therapy includes the administration of hormones or related hormonal agents.

[0147] Additional therapeutic methods that may be used in combination with CD73 inhibitors include radiation therapy, monoclonal antibodies against tumor antigens, monoclonal antibody-toxin conjugates, T-cell adjuvants, bone marrow transplantation, or antigen-presenting cells (e.g., dendritic cell therapy).

[0148] Immune checkpoint inhibitors The present invention contemplates the use of inhibitors of CD73 function described herein in combination with immune checkpoint inhibitors.

[0149] The vast array of genetic and epigenetic alterations characteristic of all cancers provides the immune system with a diverse set of antigens that it can use to distinguish tumor cells from their normal counterparts. In the case of T cells, the ultimate strength (e.g., level of cytokine production or proliferation) and quality (e.g., type of immune response generated, such as the pattern of cytokine production) of the response initiated by antigen recognition by the T cell receptor (TCR) are regulated by the balance between costimulatory and inhibitory signals (immune checkpoints). Under normal physiological conditions, immune checkpoints are crucial for preventing autoimmunity (i.e., maintaining self-tolerance) and protecting tissues from damage when the immune system is responding to pathogen infection. Expression of immune checkpoint proteins can be dysregulated by tumors as an important immune resistance mechanism.

[0150] Examples of immune checkpoints (ligands and receptors), some of which are selectively upregulated in various types of tumor cells that are candidates for blockade, include PD1 (programmed cell death protein 1); PDL1 (PD1 ligand); BTLA (B lymphocyte and T lymphocyte receptor 1); These include the killer inhibitory receptors (KIRs), which can be divided into two classes based on their structural characteristics: 1) killer cell immunoglobulin-like receptors (KIRs) and 2) C-type lectin receptors (members of the type II transmembrane receptor family). Other less well-defined immune checkpoints have been described, including both receptors (e.g., the 2B4 (also known as CD244) receptor) and ligands (e.g., certain B7 family inhibitory ligands, such as B7-H3 (also known as CD276) and B7-H4 (also known as B7-S1, B7x, and VCTN1)). [See Pardoll, (April 2012) Nature Rev. Cancer 12:252-64].

[0151] The present invention contemplates the use of inhibitors of CD73 function described herein in combination with inhibitors of the immune checkpoint receptors and ligands described above, as well as those yet to be described. Certain modulators of immune checkpoints are currently available, while others are in later stages of development. Illustratively, ipilimumab (YERVOY; Bristol-Myers Squibb), a fully humanized CTLA4 monoclonal antibody, became the first immune checkpoint inhibitor to receive regulatory approval in the United States when it was approved for the treatment of melanoma in 2011. Fusion proteins comprising CTLA4 and an antibody (CTLA4-Ig; abatcept (ORENCIA; Bristol-Myers Squibb)) have been shown to be effective against CTLA4-related leukemia (MEDs), including leukemia-associated leukemia (MEDs), leukemia-associated leukemia (MEDs), and leukemia-associated leukemia-associated leukemia (MEDs). These fusion proteins, including CTLA4 and an antibody (CTLA4-Ig; abatcept (ORENCIA; Bristol-Myers Squibb)), are also useful. It has been used to treat rheumatoid arthritis, and other fusion proteins have been shown to be effective in kidney transplant patients infected with Epstein-Barr virus. PD1 antibodies are in development (e.g., nivolumab (Bristol-Myers Squibb)) and lambloblastoma (Lambrov). Antibodies against PDL1 (e.g., lambrolizumab (Merck)) and anti-PDL1 antibodies (e.g., MPDL3280A (Roche)) are also being evaluated. Nivolumab has shown promise in patients with melanoma, lung, and kidney cancer. is shown.

[0152] The present invention includes pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0153] Metabolic and cardiovascular diseases The present invention provides methods for treating and / or preventing certain cardiovascular and / or metabolic related diseases, disorders, and conditions, and disorders associated therewith, using a CD73 inhibitor and at least one additional therapeutic or diagnostic agent.

[0154] Examples of therapeutic agents useful in combination therapy for the treatment of hypercholesterolemia (and atherosclerosis) include statins, which inhibit the enzymatic synthesis of cholesterol (e.g., Crestor, Lecol, Lipitor, Mevacor, Pravachol, and Zocor); bile acid resins, which sequester cholesterol and prevent its absorption (e.g., Cholestetide, Locholest, Prevalite, Kestlan, and Welchol); ezetimibe (Zetia), which blocks cholesterol absorption; fibric acids (e.g., Trichol), which lower triglycerides and modestly increase HDL; niacin (e.g., Niacol), which modestly lowers LDL cholesterol and triglycerides; and / or combinations of the above (e.g., Vitorin (ezetimibe and simvastatin)). Alternative cholesterol treatments that can be used in combination with the CD73 inhibitors described herein include various supplements and herbs (e.g., garlic, policosanol, and guggul).

[0155] The present invention includes pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0156] Immune-related disorders and disorders with an inflammatory component The present invention provides methods for treating and / or preventing immune-related diseases, disorders, and conditions; and diseases, disorders, and conditions having an inflammatory component, using a CD73 inhibitor and at least one additional therapeutic or diagnostic agent.

[0157] Examples of therapeutic agents useful in combination therapy will be specific to the underlying disease, disorder, or condition, and will be known to those of skill in the art.

[0158] microbial disease The present invention provides methods for treating and / or preventing viral, bacterial, fungal, and parasitic diseases, disorders, and conditions, and disorders related thereto, using a CD73 inhibitor and at least one additional therapeutic or diagnostic agent (e.g., one or more additional therapeutic or diagnostic agents (e.g., one or more other antiviral agents and / or one or more agents not related to viral treatment)).

[0159] Such combination therapies include antiviral agents that target various viral life cycle stages and have different mechanisms of action, including, but not limited to: inhibitors of viral uncoating (e.g., amantadine and rimantidine); reverse transcriptase inhibitors (e.g., acyclovir, zidovudine, and lamivudine); agents that target integrase; agents that block transcription factor binding to viral DNA; agents that affect translation (e.g., antisense molecules) (e.g., formylcene); agents that modulate translation / ribozyme function; protease inhibitors; viral assembly modulators (e.g., rifampicin); antiretroviral agents, such as nucleoside analog reverse transcriptase inhibitors (e.g., azidothymidine (AZT), dd1, ddC, 3TC, d4T); non-nucleoside reverse transcriptase inhibitors (e.g., efavirenz, nevirapine); nucleotide analog reverse transcriptase inhibitors; and agents that prevent viral particle release (e.g., zanamivir and oseltamivir). Treatment and / or prevention of certain viral infections (eg, HIV) often involves a group (a "cocktail") of antiviral agents.

[0160] Other antiviral agents contemplated for use in combination with CD73 inhibitors include, but are not limited to, abacavir, adefovir, amantadine, amprenavir, Ampligen, arbidol, atazanavir, atripla, boceprevirertet, cidofovir, combivir, darunavir, delavirdine, zidabicin, docosanol, edoxudine, emtricitabine, enfuvirtide, entecavir, famciclovir, fosamprenavir, foscarnet, fosfonet, http: / / en.wikipedia.org / wiki / Fusion_inhibitor ganciclovir, ibuprofen Basitabine, Immunovir, idoxuridine, imiquimod, indinavir, inosine, various interferons (e.g., peginterferon alfa-2a), lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfinavir, nexavir, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, ribavirin, ritonavir, pyramidine, saquinavir, stavudine, telaprevir, tenofovir, tipranavir, trifluridine, trizivir, tromantadine, truvada, valacyclovir, valganciclovir, vicriviroc, vidarabine, viramidine, and zalaytabine.

[0161] The present invention contemplates the use of inhibitors of CD73 function described herein in combination with antiparasitic agents. Such agents include, but are not limited to, thiabendazole, pyrantel pamoate, mebendazole, praziquantel, niclosamide, bithionol, oxamniquine, metrifonate, ivermectin, albendazole, eflornithine, melarsoprol, pentamidine, benznidazole, nifurtimox, and nitroimidazole. Those skilled in the art will recognize other agents that may be useful in treating parasitic diseases.

[0162] Embodiments of the present invention contemplate the use of CD73 inhibitors described herein in combination with agents useful for the treatment or prevention of bacterial disorders. Antibacterial agents can be classified in various ways, including based on mechanism of action, chemical structure, and spectrum of activity. Examples of antibacterial agents include those that target bacterial cell walls (e.g., cephalosporins and penicillins). ) or cell membranes (e.g., polymyxins) or interfere with essential bacterial enzymes (e.g., sulfonamides, rifamycins, and quinolines). Most antibacterial agents that target protein synthesis (e.g., tetracyclines and macrolides) are bacteriostatic, while agents such as aminoglycosides are bactericidal. Another means of classifying antibacterial agents is based on their target specificity; "narrow spectrum" agents target specific types of bacteria (e.g., gram-positive bacteria such as streptococci), while "broad spectrum" agents have activity against a wider range of bacteria. Those skilled in the art will recognize the types of antibacterial agents appropriate for use in specific bacterial infections.

[0163] Embodiments of the present invention contemplate the use of CD73 inhibitors described herein in combination with agents useful for the treatment or prevention of fungal diseases. Antifungal agents include polyenes (e.g., amphotericin, nystatin, and pimaricin); azoles (e.g., fluconazole, itraconazole, and ketoconazole); allylamines (e.g., naftifine and terbinafine), and morpholines (e.g., amorolfine); and antimetabolites (e.g., 5-fluorocytosine).

[0164] The present invention includes pharmaceutically acceptable salts, acids, or derivatives of the agents (and members of classes of agents) listed above.

[0165] Administration The CD73 inhibitors of the present invention can be administered to a subject in an amount that depends, for example, on the goal of administration (e.g., the desired degree of resolution); the age, weight, sex, and health and physical condition of the subject to whom the formulation is being administered; the route of administration; and the nature of the disease, disorder, condition, or symptoms thereof. The dosage regimen can also take into account the existence, nature, and extent of adverse effects associated with the administered agent. Effective dosages and dosage regimens can be readily determined, for example, from safety and dose escalation studies, in vivo studies (e.g., animal models), and other methods known to those skilled in the art.

[0166] Generally, dosing parameters dictate that the dosage be less than that which may be irreversibly toxic to the subject (the maximum tolerated dose (MTD)), and no less than that required to produce a measurable effect in the subject, such amount being determined, for example, by pharmacokinetic and pharmacodynamic parameters relevant to ADME, taking into account the route of administration and other factors.

[0167] The effective dose (ED) is the dose or amount of a drug that produces a therapeutic response or desired effect in a proportion of subjects who take it. 50 ED is the dose or amount of an agent that produces a therapeutic response or desired effect in 50% of the population to which it is administered. 50 The ED is commonly used as a measure of the reasonable expectation of a drug's effectiveness, but is not necessarily the dose that a physician would consider appropriate given all relevant factors. Therefore, in some situations, the effective dose may be calculated as the ED 50 and in other situations the effective dose is greater than the calculated ED 50 and in other situations the effective dose is less than the calculated ED 50 is the same as:

[0168] Furthermore, an effective amount of a CD73 inhibitor of the present invention can be an amount that, when administered one or more times to a subject, produces the desired result relative to a healthy subject. For example, in a subject experiencing a particular disorder, an effective dose is one that improves a diagnostic parameter, measure, marker, etc. of that disorder by at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, with 100% being defined as the diagnostic parameter, measure, marker, etc. exhibited by a normal subject.

[0169] In certain embodiments, CD73 inhibitors contemplated by the present invention can be administered (e.g., orally) to a subject at dosage levels of about 0.01 mg / kg to about 50 mg / kg body weight, or about 1 mg / kg to about 25 mg / kg body weight per day, one or more times per day to achieve the desired therapeutic effect.

[0170] For oral administration, the compositions can be provided in the form of tablets, capsules and the like containing 1.0 to 1000 milligrams of the active ingredient, particularly containing 1.0, 3.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the active ingredient.

[0171] In certain embodiments, the desired dose of CD73 inhibitor is contained in a "unit dosage form." The phrase "unit dosage form" refers to physically discrete units, each unit containing a predetermined amount of a CD73 inhibitor, alone or in combination with one or more additional agents, in an amount sufficient to produce a desired effect. It will be understood that the parameters of the unit dosage form will depend on the particular agent and the effect to be achieved. kit

[0172] The present invention also contemplates kits comprising CD73 inhibitors and pharmaceutical compositions thereof. Kits are generally in the form of physical structures housing various components, as described below, that can be utilized, for example, in practicing the methods described above.

[0173] The kit may include one or more CD73 inhibitors disclosed herein (e.g., provided in a sterile container), which may be in the form of a pharmaceutical composition suitable for administration to a subject. The CD73 inhibitor may be provided in a ready-to-use form (e.g., a tablet or capsule) or in a form that requires reconstitution or dilution before administration (e.g., a powder). If the CD73 inhibitor is in a form that requires reconstitution or dilution by the user, the kit may also include a diluent (e.g., sterile water), buffer, pharmaceutically acceptable excipient, etc., packaged with or separately from the CD73 inhibitor. If combination therapy is intended, the kit may contain several agents separately or already combined in the kit. Each component of the kit may be enclosed in an individual container, or all of the various containers may be in a single package. The kits of the present invention may be designed for conditions (e.g., refrigeration or freezing) necessary to properly maintain the components contained therein.

[0174] The kit may include a label or package insert containing identification of the components therein and instructions for their use (e.g., administration parameters of the active ingredients, clinical pharmacology, e.g., pharmacokinetics and pharmacodynamics, adverse effects, contraindications, etc.). The label or package insert may include manufacturer information, such as lot number and expiration date. The label or package insert may, for example, be incorporated into the physical structure that houses the component, may be contained separately within the physical structure, or may be affixed to a component of the kit (e.g., an ampoule, tube, or vial).

[0175] The label or package insert may further include computer readable media such as a disk (e.g., hard disk, card, memory disk), an optical disk such as a CD-ROM or DVD-ROM / RAM, an electronic storage medium such as a DVD, MP3, next-generation tape, or RAM and ROM, or a hybrid thereof, e.g., a magnetic / optical storage medium, flash media, or memory-type cards; in some embodiments, no actual instructions are present in the kit, but means are provided for obtaining instructions from a remote source, e.g., via the internet. [Example]

[0176] Example The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments have been performed or that they are all that may be performed. It should be understood that exemplary descriptions written in the present tense have not necessarily been performed, and that the descriptions may be made to generate data of the described nature, etc. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for.

[0177] Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius (°C), and pressure is at or near atmospheric. Standard abbreviations are used: wt = wild type; bp = base pairs; kb = kilobases. nt = nucleotide; aa = amino acid; s or sec = second; min = minute; h or hr = hour; ng = nanogram; μg = microgram; mg = milligram; g = gram; kg = kilogram; dl or dL = deciliter; μl or μL = microliter; ml or mL = milliliter; l or L = liter; μM = micromolar; mM = millimolar; M = molar; kDa = kilodalton; im = intramuscular; ip = intraperitoneal; SC or SQ = subcutaneous; QD = daily; BID = twice a day; QW = weekly; QM = monthly; HPLC = high performance liquid chromatography; BW = body weight; U = unit; ns = not statistically significant; PBS = phosphate buffered saline; IHC = immunohistochemistry; DMEM = Dulbecco's modified Eagle's medium; EDTA = ethylenediaminetetraacetic acid.

[0178] LC: Agilent 1100 series; Mass spectrometer: Agilent G6120BA, single quadrupole type; L C-MS method: Agilent Zorbax Eclipse Plus C18, 4.6×100mm, 3.5μM, 3 5 °C, 1.5 mL / min flow rate, 2.5 min gradient from 0% to 100% B with a 0.5 min wash at 100% B; A = 0.1% formic acid / 5% acetonitrile / 94.9% water; B = 0.1% formic acid / 5% water / 94.9% acetonitrile. Flash column: ISCO Rf + Reverse-phase HPLC: ISCO-EZ; Column: Kinetex 5 μm EVO C18 100A; 250 × 21.2 mm (Phenomenex) [Example 1] Synthesis of [({[(2R,3S,4R,5R)-5-[6-(cyclopentylamino)-2-chloro-9H-purin-9-yl]-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0179] Step a: A mixture of 2,6-dichloropurine riboside (321 mg, 1 mmol), cyclopentylamine (103 μL, 1.05 mmol, 1.05 equiv.), and triethylamine (146 μL, 1.05 mmol, 1.05 equiv.) in absolute EtOH (3 mL) was stirred at 60° C. overnight. The reaction mixture was evaporated, and the crude product was used in the next step without further purification. 15 H 21 ESIMS of ClNO4 [M+H] + , Calculated value 370.8, Measured value 370.2.

[0180] Step b: The product from step a (370 mg, 1 mmol) was dissolved in trimethyl phosphate (5 mL) and cooled to 0 °C (ice bath), followed by the dropwise addition of a cold solution of methylenebis(phosphonic acid dichloride) (1.25 g, 5 mmol, 5 equiv.) in trimethyl phosphate (2 mL). The reaction mixture was stirred at 0 °C for 3 h, then carefully quenched with 0.5 M triethylammonium bicarbonate solution (7 mL) and stirred at 0 °C for 15 min, then at room temperature for 2 h. The reaction mixture was purified by reverse-phase HPLC (C18 column, 0-30% gradient of acetonitrile and water with 0.1% TFA) to give the product (181 mg) as a white solid in 28% yield: 1 HNMR(400 MHz, DMSO) δ 8.45 - 8.32 (m, 2H), 5.85 (d, J = 5.5 Hz, 1H), 4.5 5 - 4.36 (m, 2H), 4.23 - 4.07 (m, 4H), 2.26(t, J = 20.5 Hz, 2H), 2.04 - 1.85 (m , 2H), 1.77 - 1.46 (m, 6H). C 16 H 25 ESIMS [M+H] of ClN5O9P2 + , Calculated value 528.8, Measured value 528.1. [Example 2] Synthesis of (((((2R,3S,4R,5R)-5-(6-((4-(tert-butyl)benzyl)amino)-2-chloro-9H-purin-9-yl)(3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0181] The title compound was synthesized in a similar manner to Example 1, using 4-tert-butylbenzylamine instead of cyclopentylamine: 1 H NMR(400 MHz, DMSO-d6) δ 8.91 (t, J = 6.3 Hz, 1H), 8.43 (s, 1H), 7.33(d, J= 8.2 Hz, 2H), 7.26 (d, J = 8.2 Hz, 2H), 5.86 (d, J = 5.8 Hz, 1H), 4.68 - 4.56(m, 2H), 4.52 (t, J = 5.4 Hz, 1H), 4. 23 - 4.03 (m, 4H), 2.26 (t, J = 20.5 Hz,2H), 1.25 (s, 9H). C 22 H 31 ESI of ClN5O9P2 MS [M+H] + , Calculated value 606.1, Measured value 606.2. [Example 3] Synthesis of (((((2R,3S,4R,5R)-5-(2-chloro-6-(isopropylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0182] The title compound was synthesized in a similar manner to Example 1, using isopropylbenzylamine instead of cyclopentylamine: 1H NMR(400 MHz, DMSO-d6) δ 8.40 (s, 1H), 8.23 ​​(d, J = 8.1 Hz, 1H), 5.85(d, J = 5.9Hz, 1H), 4.51 (t, J = 5.5 Hz, 1H), 4.3 6 (s, 1H), 4.24 - 4.03 (m, 4H), 2.25 (t, J= 20.5 Hz, 2H), 1.21 (dd, J = 6.6, 2. 0 Hz, 5H). C 14 H 22 ESIMS [M+H] of ClN5O9P2 + , Calculated value 502.1, Measured value 502.1. [Example 4] Synthesis of (((((2R,3S,4R,5R)-5-(2-chloro-6-(cyclopropylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0183] The title compound was synthesized in a similar manner to Example 1, using cyclopropylamine instead of cyclopentylamine: 1 H NMR (400 MHz, DMSO-d6) δ8.54 (s, 1H), 8.42 (s, 1H), 5.86 (d, J = 5.8 Hz, 1H), 4.52 (t, J= 5.4 Hz, 1H),4.28 - 4.03 (m, 4H), 2.97 (s, 1H), 2.25 (t, J = 20.5 Hz, 2H),0.75 (s, 2H), 0.64 (s, 3H).C 14 H 20 ClN5 ESI MS of O9P2 [M+H] + ,Calculated value 500.1, Measured value 500.1. [Example 5] Synthesis of (((((2R,3S,4R,5R)-5-(2-chloro-6-(neopentylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0184] The title compound was synthesized in a similar manner to Example 1, using neopentylamine instead of cyclopentylamine: 1 H NMR (400MHz, DMSO-d6) δ 8.42 (s, 1H), 8.32 (t, J = 6.4 Hz, 1H), 5.85 (d, J =5.7 Hz, 1H),4.52 (t, J = 5.4 Hz, 1H), 4.31 - 4.04 (m, 4H), 3.82 (d, J = 7.0 Hz, 1H), 3.42 -3.17 (m, 2H), 2.26 (t, J = 20.5 Hz,2H), 0.91 (s, 9H). 16 H 26 ESIMS of ClN5O9P2 [M+H] + , Calculated value 530.1, Measured value 530.2. [Example 6] Synthesis of (((((2R,3S,4R,5R)-5-(2-chloro-6-(isopropyl(methyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0185] The title compound was synthesized in a similar manner to Example 1, using N-methylisopropylamine instead of cyclopentylamine: 1H NMR(400 MHz, DMSO-d6) δ 8.42 (s, 1H), 5.88 (d, J = 5.9 Hz, 1H), 4.50(t, J = 5.4Hz, 1H), 4.22 - 4.17 (m, 1H), 4.11 (d, C 15 H 24 ESIMS of ClN5O9P2 [M+H] + , Calculated value 516.1, Measured value 516.1. [Example 7] Synthesis of (((((2R,3S,4R,5R)-5-(6-((3,5-bis(trifluoromethyl)benzyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0186] The title compound was synthesized in a similar manner to Example 1, except that in step a, 6-chloropurine riboside and 3,5-bis(trifluoromethyl)benzylamine were used: 1 H NMR (400 MHz, DMSO-d6) δ 8.67 (s,1H), 8.47 (s, 1H), 8.26 (s, 1H), 8.07(s, 2H), 7.99 (s, 1H), 5.94 (d, J = 5.7 Hz, 1H), 4.88(s, 2H), 4.61 (t, J = 5.4Hz, 1H), 4 .23 (t, J = 4.2 Hz, 1H), 4.20 - 4.04 (m,3H), 2.25 (t, J = 20.5 Hz, 2H). C 20 H 20 ESIMS of F6N5O9P2 [M-H] - , Calculated value 650.1, Actual value 650.2. [Example 8] Synthesis of (((((2R,3S,4R,5R)-5-(6-((4-bromobenzyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0187] The title compound was synthesized in a similar manner to Example 1, except using 6-chloropurine riboside and the corresponding amine in step a: 1 H NMR(400 MHz, DMSO-d6) δ 8.60 (s, 1H), 8.44 (s, 1H), 8.24 (s, 1H), 7.49(d, J =8.3 Hz, 2H), 7.29 (d, J = 8.3 Hz, 2H ), 5.94 (d, J = 5.7 Hz, 1H), 4.67 (s, 2H),4.61 (t, J = 5.3 Hz, 1H), 4.23 (t, J = 4.2 Hz, 1H), 4.19 - 4.05 (m, 3H), 2.25(t, J = 20.5 Hz, 2H).C 18 H 21 BrN5O9P2 ESI MS [M-H] - , Calculated value 592.0, Actual value 592.1. [Example 9] Synthesis of (((((2R,3S,4R,5R)-5-(6-((4-(tert-butyl)benzyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0188] The title compound was synthesized in a similar manner to Example 1, except using 6-chloropurine riboside and the corresponding amine in step a:1 H NMR (400 MHz, DMSO-d6) δ8.62 (s, 1H), 8.45 (s, 1H), 8.26 (s, 1H), 7.37 - 7.22(m, 4H), 5.94 (d, J = 5.7Hz, 1H), 4. 67 (s, 2H), 4.60 (t, J = 5.4 Hz, 1H), 4.23(t, J = 4.1 Hz, 1H), 4.20 - 4.05 (m, 3H), 2.25 (t, J = 20.5 Hz, 2H), 1.24 (s,9H).C 22 H 30 ESIMS [M-H] of N5O9P2 - , total Calculated value 570.1, measured value 570.3. [Example 10] Synthesis of (((((2R,3S,4R,5R)-5-(6-(([1,1'-biphenyl]-4-ylmethyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0189] The title compound was synthesized in a similar manner to Example 1, except that in step a, 6-chloropurine riboside and the corresponding amine were used: H NMR (400 MHz, DMSO-d6) δ 8.67 (s, 1H) , 8.46 (s, 1H), 8.27 (s, 1H), 7.66 - 7.57(m, 4H), 7.49 - 7.40 (m, 4H), 7.37 - 7 .30 (m, 1H), 5.95 (d, J = 5.7 Hz, 1H), 4.76(s, 2H), 4.61 (t, J = 5.3 Hz, 1H), 4 .24 (t, J = 4.1 Hz, 1H), 4.20 - 4.06 (m,3H), 2.25 (t, J = 20.5 Hz, 2H). C24 H 26 ESIMS [M-H] of N5O9P2 - , Calculated value 590.1, Measured value 590.2. [Example 11] Synthesis of (((((2R,3S,4R,5R)-3,4-dihydroxy-5-(6-((4-(trifluoromethyl)benzyl)amino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0190] The title compound was synthesized in a similar manner to Example 1, except using 6-chloropurine riboside and the corresponding amine in step a: 1 H NMR(400 MHz, DMSO-d6) δ 8.68 (s, 1H), 8.46 (s, 1H), 8.25 (s, 1H), 7.67(d, J =8.1 Hz, 2H), 7.54 (d, J = 8.1 Hz, 2H ), 5.95 (d, J = 5.8 Hz, 1H), 4.79 (s, 2H),4.61 (t, J = 5.3 Hz, 1H), 4.24 (t, J = 4.1 Hz, 1H), 4.20 - 4.06 (m, 3H), 2.25(t, J = 20.5 Hz, 2H).C 19 H 21 F3N5O9P2 ESI MS [M-H] - , Calculated value 582.1, Measured value 582.2. [Example 12] ((((2R,3S,4R,5R)-3,4-dihydroxy-5-(6-((4-methylbenzyl)amino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl Synthesis of (hydroxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0191] The title compound was synthesized in a similar manner to Example 1, except using 6-chloropurine riboside and the corresponding amine in step a: 1 H NMR(400 MHz, DMSO-d6) δ 8.61 (s, 1H), 8.45 (s, 1H), 8.26 (s, 1H), 7.22(d, J =7.8 Hz, 2H), 7.10 (d, J = 7.8 Hz, 2H ), 5.94 (d, J = 5.7 Hz, 1H), 4.67 (s, 2H),4.60 (t, J = 5.4 Hz, 1H), 4.23 (t, J = 4.2 Hz, 1H), 4.19 - 4.04 (m, 3H), 2.31 -2.18 (m, 5H). C 19 H 24 ESIMS of N5O9P2 [MH] - , Calculated value 528.1, Measured value 528.2. [Example 13] Synthesis of (((((2R,3S,4R,5R)-5-(6-((3,5-dichlorobenzyl)amino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0192] The title compound was synthesized in a similar manner to Example 1, except using 6-chloropurine riboside and the corresponding amine in step a: 1 H NMR(400 MHz, DMSO-d6) δ 8.61 (s, 1H), 8.46 (s, 1H), 8.26 (s, 1H), 7.48(t, J =2.0 Hz, 1H), 7.39 (s, 2H), 5.95 (d, J = 5.7 Hz, 1H), 4.70 (s, 2H), 4.61 (t, J =5.4 Hz, 1H), 4.24 (t, J = 4.2 Hz, 1H) ), 4.20 - 4.05 (m, 3H), 2.26 (t, J = 20.5Hz, 2H). C 18 H 20 ESIMS of Cl2N5O9P2 [M- H] - , Calculated value 582.1, Measured value 582.2. [Example 14] Synthesis of (((((2R,3S,4R,5R)-5-(6-(benzylamino)-2-methyl-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0193] Step a: To a nitrogen-purged reaction mixture of the iodo derivative (1.03 g, 1.7 mmol) and tetramethyltin (470 μL, 3.34 mmol) in NMP (10 mL), Pd(PPh3)4 (196 mg, 0.17 mmol, 10 mol%) was added and the reaction mixture was heated at 120 °C overnight. LCMS showed the formation of the product. It was cooled to room temperature, diluted with water, extracted with ethyl acetate, dried (MgSO4), filtered, and concentrated. The residue was flash-dried. The product (1 g) was obtained by column purification. 24 H 27 ESIMS of N5O7 [M+H] + , calculated value 498. 2, Actual value 498.3.

[0194] Step b: To a solution of the acetate derivative from step a (1 g, 2.01 mmol) in methanol (5 mL) was added K2CO3 (276 mg, 2 mmol), and the reaction mixture was stirred at room temperature for 1 h. It was then diluted with dichloromethane and filtered through a pad of silica. The filtrate was concentrated and purified by flash column (ISCO, 40 g column, 0-20% methanol in dichloromethane, 20 min) to give the compound as an off-white solid (450 mg, 60%). 18 H 21 ESI MS of N5O4 [M+H] + ,Calculated value 372.2, Measured value 372.2.

[0195] Step c: The product from step b (150 mg, 0.4 mmol) was dissolved in trimethyl phosphate (3 mL), cooled to 0° C. (ice bath), and then dissolved in trimethyl phosphate (1 mL). An ice-cold solution of methylenebis(phosphonic acid dichloride) (504 mg, 2 mmol, 5 equiv.) was added dropwise. The reaction mixture was stirred at 0°C for 3 h, then carefully quenched with 0.5 M triethylammonium bicarbonate solution (8 mL) and stirred at 0°C for 15 min, then at room temperature for 2 h. The reaction mixture was purified by reverse-phase HPLC (C18 column, 0-30% gradient of acetonitrile and water with 0.1% TFA) to give the product as a white solid: 1 HNMR (400 MHz, DMSO-d6) δ 8.48 - 8.32(m, 2H), 7.38 - 7.18 (m, 5H),5.92 (d, J = 6.0 Hz , 1H), 4.71 (s, 2H), 4.55 (t, J = 5.5 Hz,1H), 4.19 - 3.98 (m, 4H), 2,44 (s, 3H) , 2.23 (t, J = 20.5 Hz, 2H).C 19 H 25 ESIMS [M-H] of N5O9P2 - , Calculated value 528.1, Measured value 528.2. [Example 15] Synthesis of (((((2R,3S,4R,5R)-5-(6-(benzylamino)-2-vinyl-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0196] Step a: N in 1,2-dimethoxyethane:HO (9:1, 10 mL) 6 A mixture of 2-benzyl-2-chloropurine riboside (783 mg, 2 mmol), vinylboronic acid pinacol ester (462 mg, 3 mmol, 1.5 equiv.), KCO (828 mg, 6 mmol, 3 equiv.), and Pd(PPh) was stirred at 85 °C under N for 1 day. The solution was cooled to room temperature, diluted with EtOAc (100 mL), and washed with H2O (50 mL). The organic layer was separated, dried over MgSO4, filtered, and the solvent was evaporated to give a yellow solid. The crude product was washed with MTBE (50 mL) and used for the next step (550 mg, 72%).

[0197] Step b: The title compound was synthesized in the same manner as in Example 1. 1 H NMR (400 MHz, DMSO-d6) δ8.39 (s, 1H), 7.38 (d, J = 7.0 Hz, 2H),7.29 (t, J = 7.6 Hz, 2H), 7.25 - 7.15 (m, 1H), 6.64 (dd, J = 17.2, 10.4 Hz, 1H),6.39 (dd, J = 17.2, 2.4 Hz, 1H),5.94 (d, J = 6.0 Hz, 1H), 5.55 (d, J = 10.5Hz, 1H), 4.73 (s, 2H), 4.63 (t, J =5.5 Hz, 1H), 4.28 - 4.00 (m, 4H), 2.25 (t, J =20.4 Hz, 2H). 20 H 26ESIM of N5O9P2 S [M+H] + , Calculated value 542.1, Measured value 542.2.

[0198] Step c: The product from step b (40 mg, 0.06 mmol) was dissolved in MeOH (10 mL), purged with N2, and 10% Pd / C (50% wet, 30 mg) was added to the reaction mixture. The mixture was stirred vigorously under H2 (balloon) for 2 hours, filtered, and the product was purified by RP18 HPLC. (H2O+0.1% TFA / acetonitrile+0.1% TFA) to obtain a white A solid was obtained (14 mg, 35%): 1 H NMR (400 MHz, DMSO-d6) δ 8.52 - 8.18(m, 2H), 7.33 - 7.27 (m, 2H), 7.27 - 7.18 (m, 2H),7.15 (t, J = 7.2 Hz, 1H),5.86 (d, J = 6.0 Hz, 1H), 4.64 (s, 2H), 4.55 (t, J =5.5 Hz, 1H), 4.19 - 3.98 (m, 4H),2.70 - 2.61 (m, 2H), 2.16 (t, J = 20.5 Hz,2H), 1.16 (t, J = 7.6Hz, 3H).C 20 H 27 N ESI MS [M+H] of 5O9P2 + , Calculated value 544.1, Measured value 544.2. [Example 16] ((((2R,3S,4R,5R)-5-(2-allyl-6-(benzylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)meth Synthesis of (hydroxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0199] The title compound was synthesized in the same manner as in Example 15.1 H NMR (400 MHz, DMSO-d6) δ 8.46(s, 1H), 8.37 (s, 1H), 7.41 - 7.34 (m,2H), 7.30 (t, J = 7.5 Hz, 2H), 7.25 - 7 .18 (m, 1H), 6.17 - 6.03 (m, 1H), 5.92 (d,J = 6.0 Hz, 1H), 5.28 - 5.00 (m, 2H), ESI MS[M+H] of 4.70 (s, 2H), 4.60 (t, J = 5.6 Hz, 1H),4.27 - 4.02 (m, 4H), 3.49 (d, J = 6.8Hz, 2H), 2.24 (t, J = 20.5 Hz, 2H). + , calcdC 21 H 28 ESIMS of N5O9P2 [M+H] + , Calculated value 556.1, Measured value 556.3. [Example 17] Synthesis of (((((2R,3S,4R,5R)-5-(6-(benzylamino)-2-propyl-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0200] The title compound was synthesized in a similar manner to Example 15: 1 H NMR (400 MHz, DMSO-d6) δ 8.40(s, 2H), 8.29 (s, 1H), 7.29 (d, J = 7.6Hz, 2H), 7.23 (t, J = 7.5 Hz, 2H), 7.1 5 (t, J = 7.2 Hz, 1H), 5.86 (d, J = 6.0 Hz,1H), 4.64 (s, 2H), 4.54 (t, J = 5.5 Hz, 1H), 4.19 - 3.94 (m, 4H), 2.71 - 2.55(m, 2H), 2.17 (t, J = 20.5 Hz, 2H), 1. 66 (q, J = 7.4 Hz, 2H), 0.93 - 0.70 (m,3H). C 21 H 30 ESIMS of N5O9P2 [M+H] + , total Calculated value 558.1, measured value 558.2. [Example 18] Synthesis of [({[(2R,3S,4R,5R)-5-[6-(benzylamino)-2-methoxy-9H-purin-9-yl]-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0201] Step a: The known riboside (250 mg, 0.64 mmol) was dissolved in 25% NaOMe in MeOH solution (2 mL) and stirred at 60 °C overnight. The reaction mixture was concentrated under reduced pressure, and the residue was then diluted with H2O (15 mL) and acetic acid until neutral pH was reached. Filtration afforded the product. The product was collected (white solid, 180 mg, 73%). 18 H 22 ESIMS of N5O5 [M+H] + , calculation Value 388.4, actual value 388.1.

[0202] Step b: Using a method similar to that in Example 1, the title compound was obtained as a white solid (37 mg, 14%): 1 H NMR (400 MHz, DMSO)δ 8.48 (s, 1H), 8.20 (s, 1H), 7.37 - 7.17 (m, 5H), 5.82 (d, J = 5.9 Hz, 1H),4.64(d, J = 5.0 Hz, 3H), 4.28 - 4.00 (m, 4 H), 3.80 (s, 3H), 2.23 (t, J = 20.5 Hz,2H).C 19 H 26 N5O 10 ESIMS of P2 [M+H] + , total Calculated value 546.4, measured value 546.1. [Example 19] Synthesis of [({[(2R,3S,4R,5R)-5-[6-(benzylamino)-2-(methylamino)-9H-purin-9-yl]-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0203] Step a: A known riboside (250 mg, 0.64 mmol) in HO solution (2 mL) The mixture was dissolved in 40% MeNH2 and stirred at 60 °C overnight. The reaction mixture was then concentrated under reduced pressure. The residue was diluted with H2O (15 mL). The product was collected by filtration (white solid, 21 0mg, 85%). C 18 H 23 ESI MS of N6O4 [M+H] + ,Calculated value 387.4, Measured value 387.3.

[0204] Step b: Using a method similar to that in Example 1, the title compound was obtained as a white solid (38 mg, 15%): 1 H NMR (400 MHz,DMSO) δ 8.08 (s, 1H), 7.42 - 7.19 (m, 5H), 5.79 (d, J = 6.1 Hz, 1H), 4.75 -4.45 (m, 3H),4.24 - 4.02 (m, 4H), 2.81 (s, 3H), 2 .22 (t, J = 20.4 Hz, 2H). C 19 H 26 ESIMS of N6O9P2 [M-H] -, Calculated value 543.4, Measured value 54 3.2. [Example 20] Synthesis of (((((2R,3S,4R,5R)-5-(6-(benzylamino)-2-(dimethylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0205] The title compound was synthesized in a similar manner to Example 19, except using dimethylamine in step a: 1 HNMR (400 MHz, DMSO-d6)δ 8.15 (s, 1H), 8.09 (s, 1H), 7.36 (d, J = 7. 2 Hz, 2H), 7.29 (t, J = 7.5 Hz, 2H), 7.21(t, J = 7.2 Hz, 1H), 5.81 (d, J = 5.5 Hz, 1H), 4.68 - 4.57 (m, 3H), 4.26 - 4.20(m, 1H), 4.20 - 4.00 (m, 3H), 3.06 (s, 6H), 2.24 (t, J = 20.4 Hz, 2H).C 20 H 29 ESIMS of N6O9P2 [M+H] + , calculated value 559.1, Actual value: 559.2. [Example 21] Synthesis of (((((2R,3S,4R,5R)-5-(6-(benzylamino)-2-(pyrrolidin-1-yl)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0206] The title compound was synthesized in a similar manner to Example 19, except using pyrrolidine in step a: 1 HNMR (400 MHz, DMSO-d6)δ 8.15 (s, 2H), 7.42 - 7.14 (m, 5H), 5.82 ( d, J = 5.5 Hz, 1H), 4.71 - 4.51 (m, 3H),4.26 (t, J = 4.3 Hz, 1H), 4.21 - 4.00 ( C 22 H 31 N6O9P2 ESI MS [M+H] + , Calculated value 585.1, Actual value 585.2. [Example 22] Synthesis of (((((2R,3S,4R,5R)-5-(6-(benzylamino)-2-(piperidin-1-yl)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0207] The title compound was synthesized in a similar manner to Example 19, except using piperidine in step a: 1 HNMR (400 MHz, DMSO-d6)δ 8.20 (s, 1H), 8.10 (s, 1H), 7.38 - 7.33 ( m, 2H), 7.33 - 7.25 (m, 2H), 7.25 - 7.16(m, 1H), 5.81 (d, J = 5.6 Hz, 1H), 4.66 C23 H 31 ESIMS of N6O9P2 [MH ] - , Calculated value 597.2, Measured value 597.3. [Example 23] Synthesis of (((((2R,3S,4R,5R)-5-(6-(benzylamino)-2-morpholino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0208] The title compound was synthesized in a similar manner to Example 19, except using morpholine in step a: 1 HNMR (400 MHz, DMSO-d6)δ 8.24 - 8.02 (m, 2H), 7.37 - 7.17 (m, 5H), C 22 H 29 NO 10 ESIMS of P2 [MH] - , Calculated value 599.2, Actual measurement Value 599.3 [Example 24] Synthesis of (((((2R,3S,4R,5R)-5-(6-(benzylamino)-2-(isopropylthio)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0209] Step a: A solution of compound X (5 g, 10.2 mmol) in MeOH (60 ml) was treated with ammonia gas at −20° C. for 10 minutes. The mixture was then warmed to room temperature and stirred until the reaction was complete. Nitrogen was then bubbled through the reaction to remove excess ammonia gas. The mixture was concentrated and purified by preparative HPLC to give the desired product (750 mg, 20%).

[0210] Step b: The product from step a (0.36 g, 1 mmol), benzylamine (0.115 mL, 1.05 mmol, 1.05 equiv.), and EtN (0.15 mL, 1.1 mmol, 1.1 equiv.) in absolute EtOH (3.3 mL) were stirred at 70 °C for 4 h. The reaction mixture was cooled to room temperature, concentrated and used without further purification.

[0211] Step c: The product from step b was dissolved in trimethyl phosphate (5 mL), cooled to 0 °C (ice bath), and then a cold solution of methylenebis(phosphonic acid dichloride) (1.2 g, 15 mmol, 5 equiv.) in trimethyl phosphate (3 mL) was added dropwise. The reaction mixture was stirred at 0 °C for 3 h and then carefully quenched with 0.5 M triethylammonium bicarbonate solution (6 mL). The mixture was quenched and stirred at 0° C. for 15 minutes, then at room temperature for 2 hours. The reaction mixture was purified by reverse-phase HPLC (C18 column, 0-40% gradient of acetonitrile and water with 0.1% TFA) to give the product as a white solid in 6% yield (38 mg): 1 H NMR (400 MHz, DMSO-d6) δ8.53 (s, 1H), 8.27 (s, 1H), 7.37 - 7.17(m, 5H), 5.84 (d, J = 5.8 Hz, 1H), 4.65 (s, 2H), 4.56 (t, J = 5.5 Hz, 1H),4.24 - 4.17 (m, 1H), 4.17 - 4.01 (m, 3H) , 3.82 - 3.71 (m, 1H), 2.24 (t, J = 20.5Hz, 2H), 1.28 (d, J = 6.8 Hz, 6H).. C2 1H29 ESI MS of N5O9P2S [M+H] + ,Calculated value 590.1, Measured value 590.2 [Example 25] Synthesis of (((((2R,3S,4R,5R)-5-(6-(benzylamino)-2-(isopropylsulfonyl)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0212] Step a: The product from Step a of Example 24 (4.5 g, 12.5 mmol) in methylene chloride (50 mL) was treated portionwise with m-CPBA (2.2 g, 38.2 mmol). The reaction was stirred at room temperature until the reaction was complete. The mixture was diluted with methylene chloride (200 mL), washed twice with aqueous NaHSO3, dried over Na2SO4, and concentrated. The residue was analyzed. Purification by preparative HPLC gave the desired product as a white solid (780 mg, 16%).

[0213] Step b: The product from step a (0.393 g, 1 mmol), benzylamine (0.115 mL, 1.05 mmol, 1.05 equiv.), and EtN (0.15 mL, 1.1 mmol, 1.1 equiv.) in absolute EtOH (3.3 mL) were stirred at 70 °C for 4 h. The reaction mixture was cooled to room temperature, concentrated and used without further purification.

[0214] Step c: The product from step b was dissolved in trimethyl phosphate (4 mL) and cooled to 0 °C (ice bath), followed by the dropwise addition of a cold solution of methylenebis(phosphonic acid dichloride) (1.2 g, 5 mmol, 5 equiv.) in trimethyl phosphate (2 mL). The reaction mixture was stirred at 0 °C for 3 h, then carefully quenched with 0.5 M triethylammonium bicarbonate solution (6 mL) and stirred at 0 °C for 15 min, then at room temperature for 2 h. The reaction mixture was purified by reverse-phase HPLC (C18 column, 0-40% gradient of acetonitrile and water with 0.1% TFA) to give the product as a white solid in 22% yield (50 mg): 1 HNMR (400 MHz, DMSO-d6) δ 9.21 (t, J = 6.2 Hz, 1H), 8.66 (s, 1H),7.42- 7.15 (m, 5H), 5.97 (d, J = 6 .1 Hz, 1H), 4.74 - 4.66 (m, 2H), 4.60 (dd,J = 6.1, 5.0 Hz, 1H), 4.26 - 4.22 (m, C 21 H 29 N5O 11 ESIMS of P2S [M+H] + , calculated value 622.1, Actual value: 622.2. [Example 26] Synthesis of (((((2R,3S,4S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0215] The title compound was synthesized in a similar manner to step b of Example 1 using the corresponding alcohol: 1HNMR (400 MHz, DMSO-d6)δ 8.17 (s, 1H), 8.02 - 7.72 (m, 2H), 6.15 (d,J C 11 H 17 ESIMS [M+H] of FN5O9P2 + , Calculated value 444.0, Measured value 444.1. [Example 27] Synthesis of (((((2R,3R,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0216] The title compound was synthesized in a similar manner to step b of Example 1 using the corresponding alcohol: 1 HNMR (400 MHz, DMSO-d6)δ 8.45 (s, 1H), 8.26 (s, 1H), 7.92 (s, 2H),6.2 7 (dd, J = 17.2, 2.8 Hz, 1H), 5.50 (ddd, J= 52.5, 4.5, 2.8 Hz, 1H), 4.64 - 4.52 (m, 1H), 4.29 - 4.08 (m, 3H), 2.25 (t, J =20.4 Hz, 2H). C 11 H 16 ESI of FN5O8P2 MS [M+H] + , Calculated value 428.1, Measured value 428.1. [Example 28] Synthesis of [({[(2R,3R,4S,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0217] The title compound was synthesized in a similar manner to Example 1 using commercially available alcohol: 1 H NMR (400 MHz, DMSO-d6) δ 8.28 (d,J = 2.2 Hz, 1H), 7.92(s, 2H), 6.36 (dd, J = 1 4.3, 4.6 Hz, 1H), 5.26 (dt, J = 52.5, 4.3Hz, 1H), 4.51 (dt, J = 18.6, 4.7 Hz, 1 H), 4.19 (t, J = 6.0 Hz, 2H), 4.04 (t, J =5.0 Hz, 1H), 2.26 (t, J = 20.5 Hz, 2H ); C 11 H 15 MS of ClFN5O8P2: (ES) m / z [M−H] - Calculated value 460.1, measured value 460.1. [Example 29] Synthesis of (((((2R,3R,4S,5R)-5-(6-(benzylamino)-2-chloro-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0218] Step a: 2,6-Dichloropurine (3.6 g, 18.8 mmol) was dissolved in 90 mL of acetonitrile and treated with CsCO (7.5 g, 23 mmol, 1.2 equiv.). The mixture was stirred at room temperature for 30 min. The known bromo derivative (8.75 g, 21 mmol, 1.1 equiv.) was dissolved in 100 mL of acetonitrile and added dropwise to the mixture via an addition funnel. The mixture was stirred at room temperature overnight. The mixture was filtered over a pad of silica gel and concentrated. The residue was adsorbed onto silica and purified using column chromatography (hexane / ethyl acetate) to give the product as a white solid in 77% yield (7.72 g). 1 HNMR (400 MHz, Chloroform-d) δ 8.39 (d, J = 3.0Hz, 1H), 8.10 (ddt, J = 8.5, 3.1,0.9 Hz, 4H), 7.74 - 7.36 (m, 6H), 6.64 (dd, J= 21.8, 2.8 Hz, 1H), 5.83 - 5.69 (m,1H), 5.40 (ddd, J = 49.9, 2.8, 0.8 Hz, 1H),4.89 - 4.77 (m, 2H), 4.62 (q, J = 4. 0 Hz, 1H). C 24 H 17 ESIMS of Cl2FN4O5 [M+H] + , Calculated value 531.1, Measured value 531.1.

[0219] Step b: The product from step a (9.0 g, 17 mmol), benzylamine (3 mL, 26 mmol, 1.5 equiv.), and EtN (5 mL, 3 The resulting mixture (4 mmol, 2.0 equiv.) was stirred at 70° C. for 4 hours. The reaction mixture was then cooled to room temperature, and the product was collected by filtration and used without further purification (white solid, 8.9 g, 87%). 31 H 25 ESI MS of ClFN5O5 [M+H] + ,Calculated value 602.2, Measured value 602.0.

[0220] The above product (10.2 g, 17 mmol) and K2CO3 (7 g, 51 mmol, 3 equiv.) were dissolved in 170 mL of methanol and stirred at room temperature for 4 h. The reaction mixture was then filtered through a pad of silica gel and concentrated. The reaction mixture was purified using column chromatography (methylene chloride / methanol) to give the product as a white solid in 80% yield (5.3 g): 1 HNMR(400 MHz, DMSO-d6) δ 8.97 (t, J = 6.3 Hz, 1H), 8.31 (d, J = 2.0 Hz, 1H), 7.36 - 7.18 (m, 5H), 6.34 (dd, J=13.6, 4.7 Hz, 1H), 5.23 (dt, J = 52. 6, 4.3 Hz, 1H), 4.66 (q, J = 7.3, 5.7 Hz,2H), 4.43 (dt, J = 19.0, 4.8 Hz, 1H), 3.84 (q, J = 4.9 Hz, 1H), 3.65 (tq, J =12.0, 6.2, 5.2 Hz, 2H). ).C 17 H 18 ClFN ESI MS [M+H] of 5O3 + , Calculated value 394.1, Measured value 394.1.

[0221] Step c: The product from step b (800 mg, 2 mmol) was dissolved in trimethyl phosphate (15 mL) and cooled to 0 °C (ice bath), followed by the dropwise addition of a cold solution of methylenebis(phosphonic acid dichloride) (2.5 g, 10 mmol, 5 equiv.) in trimethyl phosphate (5 mL). The reaction mixture was stirred at 0 °C for 3 h, then carefully quenched with 0.5 M triethylammonium bicarbonate solution (15 mL) and stirred at 0 °C for 15 min, then at room temperature for 2 h. The reaction mixture was purified by reverse-phase HPLC (C18 column, 0-40% gradient of acetonitrile and water with 0.1% TFA) to give the product (290 mg) as a white solid in 22% yield: 1HNMR(400 MHz, DMSO-d6) δ 8.99 (t, J = 6.3 Hz, 1H), 8.30 (d, J = 2.2 Hz, 1H), 7.40 - 7.18 (m, 5H), 6.38 (dd, J =14.3, 4.6 Hz, 1H), 5.45 - 5.04 (m, 1 H), 4.65 (t, J = 5.5 Hz, 2H), 4.54 - 4.42(m, 1H), 4.19 (t, J = 6.1 Hz, 2H), 4.0 4 (t, J = 5.1 Hz, 1H), 2.26 (t, J = 20.5Hz, 2H).C 18 H 21 ESIMS of ClFN5O8P2 [MH ] - , Calculated value 550.8, Measured value 550.2 [Example 30] Synthesis of (((((2R,3R,4S,5R)-5-(6-(benzyl(methyl)amino)-2-chloro-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0222] The title compound was synthesized in a similar manner to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 H NMR (400MHz, DMSO-d6) as a mixture of rotamers δ 8.32 (d, J = 2.1 Hz, 1H),7.40 - 7.19 (m,5H), 6.42 (dd, J = 14.5, 4.6 Hz, 1H), 5.55 (s, 1H), 5.27 (dt, J = 52.4, 4.2 Hz,1H), 4.95 (s, 1H), 4.50 (dt, J = 18.4, C 19 H 23 ESIMS of ClFN5O8P2 [M+H] + , calculated value 566.1, actual value 566.2. Example 31a Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0223] The title compound was synthesized in a similar manner to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 H NMR (400MHz, DMSO-d6) δ 8.36 (q, J = 4.6 Hz, 1H), 8.27 (s, 1H), 6.45 (brs, 2H), 6.37 (dd, J= 14.3, 4.6 Hz, 1H), 5.25 (dt, J =52.4, 4.3 Hz, 1H), 4.50 (dt, J = 18.6, 4.6Hz, 1H), 4.19 (t, J = 5.9 Hz, 2H),4.04 (q, J = 5.2 Hz, 1H), 3.33 (brs, 1H), 2.93(d, J = 4.5 Hz, 3H), 2.26 (t, J =20.4 Hz, 2H). C 12 H 17 ESIMS of ClFN5O8P2 [M-H] - , Calculated value 474.7, Measured value 474.1. Example 31b Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-(ethylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0224] The title compound was synthesized in a similar manner to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 H NMR (400MHz, DMSO-d6) δ 8.43 (t, J = 5.7 Hz, 1H), 8.26 (s, 1H), 7.28 (brs, 2H), 6.37 (dd, J= 14.3, 4.6 Hz, 1H), 5.25 (dt, J =52.4, 4.3 Hz, 1H), 4.50 (dt, J = 18.5, 4.6Hz, 1H), 4.19 (t, J = 6.1 Hz, 2H),4.03 (q, J = 5.1 Hz, 1H), 3.87 (brs, 1H), 3.45(m, 1H), 2.27 (t, J = 20.5 Hz, 2H),1.17 (t, J = 7.2 Hz, 3H). C 13 H 19 ESIMS of ClFN5O8P2 [M+H] + , Calculated value 490.7, Measured value 490.1. [Example 32] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-(isopropylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0225] The title compound was synthesized in a similar manner to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 H NMR (400MHz, DMSO-d6) δ 8.27 (m, 2H), 6.37 (d, J = 13.9 Hz, 1H), 5.28 (brs,2H), 5.25 (d, J= 52.1 Hz, 1H), 4.98 (brs, 1H), 4.51 (d, J = 18.3 Hz, 1H), 4.35 (sept, J = 7.9Hz, 1H), 4.19 (m, 2H), 4.04 (m, 1H), 2 .26 (t, J = 20 Hz, 2H), 1.21 (dd, J = 6.6,2.1 Hz, 6H).C 14 H 21 ESIMS of ClFN5O8P2 [MH] - , Calculated value 502.7, Measured value 502.2. [Example 33] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-(cyclopropylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0226] The title compound was synthesized in a similar manner to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 H NMR (400MHz, DMSO-d6) δ 8.59 (s, 1H), 8.28 (d, J = 2.1 Hz, 1H), 6.38 (dd, J= 14.2, 4.6 Hz,1H), 5.26 (ddd, J = 52.5, 4.3, 4.3 Hz , 1H), 4.51 (dt, J = 18.5, 4.5 Hz, 1H),4.19 (t, J = 6.1 Hz, 2H), 4.03 (q, J = 5 C 14 H 18 ClFNO ESI MS of 8P2 [MH] - , Calculated value 500.03, Actual value 500.0. [Example 34] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-((cyclopropylmethyl)amino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0227] The title compound was synthesized in a similar manner to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 H NMR (400MHz, DMSO-d6) δ 8.54 (s, 1H), 8.28 (s, 1H), 6.37 (dd, J = 14.2, 4.6Hz, 1H), 5.25(ddd, J = 52.5, 4.3, 4.3 Hz, 1H), 4.54 - 4.47 (m, 1H), 4.19 (t, J = 6.3 Hz, 2H),4.05 - 4.01 (m, 1H), 3.81 - 3.74 (m, 1 H), 3.30 - 3.27 (m, 1H), 2.26 (dd, J =20.5, 20.5 Hz, 2H), 1.1 - 1.3 (m, 1H), 0 .48 - 0.37 (m, 2H), 0.28 - 0.26 (m, 2H).C 15 H 20 ESIMS of ClFN5O8P2 [M-H] - , calculation Value 514.1, actual value 514.0. [Example 35] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-(cyclopentylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0228] The title compound was synthesized in a similar manner to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 H NMR (400MHz, DMSO-d6) δ 8.41 (d, J = 7.8 Hz, 1H), 8.27 (s, 1H), 6.37 (dd, J = 14.4, 4.6 Hz,1H), 5.25 (dt, J = 52.4, 4.3 Hz, 1H), 4.55 - 4.37 (m, 2H), 4.19 (t, J = 6.1 Hz,2H), 4.03 (q, J = 5.1 Hz, 1H), 2.26(t, J = 20.5 Hz, 2H), 1.93 (s, 2H), 1.64 (d,J = 62.5 Hz, 6H).C 16 H 23 ClFN5O8P2 ESI MS [M+H] + , Calculated value 530.1, Actual value 530.2. [Example 36] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-(((S)-tetrahydrofuran-3-yl)amino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0229] The title compound was synthesized in a similar manner to Example 29 using the product of step a of Example 29 and the corresponding amine:1 H NMR (400MHz, DMSO-d6) δ 8.66 (d, J = 6.2 Hz, 1H), 8.31 (s, 1H), 6.38 (dd, J = 14.3, 4.6 Hz,1H), 5.26 (ddd, J = 52.4, 4.2, 2.4Hz, 1H), 4.61 - 4.67 (m, 1H), 4.57 - 4.45 (m,1H), 4.19 (t, J = 6.1 Hz, 2H),4.04 (q, J = 5.0 Hz, 1H), 3.89 (dt, J = 15.3,7.8 Hz, 2H), 3.73 (q, J = 7.8 Hz, 1H), 3.61 (dd, J = 8.9, 4.4 Hz, 1H), 2.36 -1.99 (m, 4H).C 15 H 20 ESIMS of ClFN5O9P2 [ MH] - , Calculated value 530.04, Actual value 530.1. [Example 37] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-(((R)-tetrahydrofuran-3-yl)amino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0230] The title compound was synthesized in a similar manner to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 H NMR (400MHz, DMSO-d6) δ 8.65 (d, J = 6.8 Hz, 1H), 8.31 (s, 1H), 6.39 (dd, J = 14.2, 4.6 Hz,1H), 5.26 (ddd, J = 52.4, 4.3, 4.3Hz, 1H), 4.69 - 4.56 (m, 1H), 4.51 (dt, J =18.6, 4.6 Hz, 1H), 4.20 (t, J = 6.1Hz , 2H), 4.04 (q, J = 5.0 Hz, 1H), 3.89 (dt,J = 18.6, 7.9 Hz, 2H), 3.74 (q, J = 7 .8 Hz, 1H), 3.67 - 3.54 (m, 1H), 2.35 -1.90 (m, 4H). 15 H 20 ESIMS of ClFN5O9P2 [ MH] - , Calculated value 530.04, Actual value 530.1. [Example 38] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-((tetrahydro-2H-pyran-4-yl)amino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0231] The title compound was synthesized in a similar manner to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 H NMR (400MHz, DMSO-d6) δ 8.47 - 8.34 (m, 1H), 8.30 (s, 1H), 6.37 (dd, J = 14.1, 4.8 Hz, 1H),5.25 (ddd, J = 52.4, 4.3, 4.3 Hz,1H), 4.92 - 4.65 (m, 1H), 4.59 - 4.39 (m, 1H),4.19 (t, J = 6.2 Hz, 2H), 4.03(q, J C 16 H 22 ESIMS [M−H] of ClFN5O9P2 - , calculated value 5 44.06, actual value 544.1. [Example 39] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-(pyrrolidin-1-yl)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0232] The title compound was synthesized in a similar manner to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 H NMR (400MHz, DMSO-d6) δ 8.27 (d, J = 2.1 Hz, 1H), 6.39 (dd, J = 14.1, 4.7 Hz, 1H), 5.26 (dt, J = 52.5, 4.3 Hz, 1H), 4.50 (dt, J = 2.01 (p, J = 6.7Hz, 2H),1.92 (q, J = 6.7 Hz, 2H). C 15 H 21 ESIMS of ClFN5O8P2 [M+H] + , Calculated value 516.1, Measured value 516.1. [Example 40] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-(piperidin-1-yl)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0233] The title compound was synthesized in a similar manner to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 H NMR (400MHz, DMSO-d6) δ 8.30 (d, J = 2.2 Hz, 1H), 6.39 (dd, J = 14.3, 4.6 Hz, 1H), 5.26 (dt, J = 52.4, 4.3 Hz, 1H), 4.50 (dt, J = 18.4, 4.6 Hz, 1H), 4.19 (t, J = 6.0 Hz,2H), 4.04 (q, J = 5.0 Hz, 1H), 3.88 (m, 2H), 2.27 (t, J = 20.5 Hz, 2H), 1.64 (d, J= 31.0 Hz, 8H).C 16 H 23 ES of ClFN5O8P2 I MS [M+H] + , Calculated value 530.1, Actual value 530.2. [Example 41] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-morpholino-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0234] The title compound was synthesized in a similar manner to Example 29 using the product of step a of Example 29 and the corresponding amine: 1H NMR (400MHz, DMSO-d6) δ 8.34 (d, J = 2.1 Hz, 1H), 6.41 (dd, J = 13.9, 4.6 Hz, 1H), 5.27 (dt, J = 52.5, 4.3 Hz, 1H), 4.51 (dt, J = C 15 H 21 ESIMS of ClFN5O9P2 [M+H] + , calculated value 532.1, Actual value 532.1. [Example 42] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-(isoindolin-2-yl)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0235] The title compound was synthesized in a similar manner to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 H NMR (400MHz, DMSO-d6) δ 8.37 (d, J = 2.1 Hz, 1H), 7.48 (dt, J = 9.9, 4.7 Hz, 2H), 7.43 -7.28 (m, 2H), 6.44 (dd, J = 13.8, 4.7Hz, 1H), 5.41 (s, 2H), 5.29 (dt, J = 52.6,4.4 Hz, 1H), 4.98 (s, 2H), 4.54 (dt,J = 18.7, 4.7 Hz, 1H), 4.21 (t, J = 5.9 Hz,2H), 4.05 (q, J = 4.9 Hz, 1H), 2.27 (t , J = 20.5 Hz, 2H). C 19 H 21 ESIMS of ClFN5O8P2 [M+H] + , Calculated value 564.1, Measured value 564.1 . [Example 43] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-((4-chlorobenzyl)amino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0236] The title compound was synthesized in a similar manner to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 H NMR (400MHz, DMSO-d6) δ 9.01 (t, J = 6.2 Hz, 1H), 8.32 (d, J = 2.1 Hz, 1H), 7.64 - 7.08 (m,4H), 6.38 (dd, J = 14.3, 4.6 Hz, 1H), 5.26 (dt, J = 52.5, 4.3 Hz, 1H), 4.64 (q,J = 7.3, 5.4 Hz, 2H), 4.51 (dt, J =18.7, 4.6 Hz, 1H), 4.28 - 4.11 (m, 2H), 4.04(q, J = 5.1 Hz, 1H), 2.27 (t, J =20 .5 Hz, 2H). C 18 H 20 ESIMS of Cl2FN5O8P2 [M-H] - , Calculated value 584.0, Measured value 584.1. [Example 44] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-((4-fluorobenzyl)amino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0237] The title compound was synthesized in a similar manner to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 H NMR (400MHz, DMSO-d6) δ 9.00 (t, J = 6.3 Hz, 1H), 8.31 (d, J = 2.2 Hz, 1H), 7.52 - 7.24 (m,3H), 7.23 - 7.01 (m, 2H), 6.38 (dd, J = 14.3, 4.6 Hz, 1H), 5.26 (dt, J = 52.4,4.3 Hz, 1H), 4.72 - 4.55 (m, 2H), 4.20 (t, J = 6.0 Hz, 3H), 4.04 (q, J = 5.1 Hz,1H), 2.27 (t, J = 20.5 Hz, 2H).C 18 H 20 ESIMS of ClF2N5O8P2 [M-H] - , Calculated value 568.0, Measured value 568.2. [Example 45] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-((3-methylbenzyl)amino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0238] In a manner similar to that of Example 29, the product of step a of Example 29 and the corresponding amine were used. to synthesize the title compound as a white solid (87.1 mg; 31%): 1 H NMR (400 MHz, DMSO-d6) δ 8.96 (t, J = 6.3Hz, 1H), 8.30(d, J = 2.2 Hz, 1H), 7.20 (t, J = 7.5 Hz, 1H), 7.17 - 7.10 (m, 2H), 7.04 (d,J = 7.4 Hz, 1H), 6.38 (dd, J = 14.3, 4.6 Hz, 1H), 5.25 (dt, J = 52.4, 4.3 Hz,1H), 4.68 - 4.56 (m, 2H), 4.51 (dt, J = C 19 H 22 ESIMS of ClFN5O8P2 [M-H] - , Calculated value 564.1, Measured value 564.2. [Example 46] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-((3-fluorobenzyl)amino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0239] In a similar manner to Example 29, using the product of step a of Example 29 and the corresponding amine, the title compound was synthesized as a white solid (65.1 mg; 23%): 1 HNMR (400 MHz, DMSO-d6) δ 9.02 (t, J = 6.3Hz, 1H), 8.32 (d, J = 2.2Hz, 1H), 7.42 - 7.32 ( m, 1H), 7.17 (t, J = 9.2 Hz, 2H), 7.07 (td,J = 8.4, 2.2 Hz, 1H), 6.39 (dd, J = 14.4, 4.6 Hz, 1H), 5.26 (dt, J = 52.5, 4.3Hz, 1H), 4.74 - 4.60 (m, 2H), 4.51 (d t, J = 18.5, 4.7 Hz, 1H), 4.26 - 4.13 (m,2H), 4.04 (q, J = 5.0 Hz, 1H), 2.27 (t , J = 20.5 Hz, 2H). C 18 H 19 ESIMS of ClF2N5O8P2 [M-H] - , Calculated value 568.0, Measured value 568.2 . [Example 47] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-((3-chlorobenzyl)amino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0240] In a manner similar to that of Example 29, using the product of step a of Example 29 and the corresponding amine, the title compound was synthesized as a white solid (70.6 mg; 24%): 1 HNMR (400 MHz, DMSO-d6) δ 9.03 (t, J = 6.2Hz, 1H), 8.33 (d, J = 2.2Hz, 1H), 7.45 - 7.27 ( m, 4H), 6.39 (dd, J = 14.4, 4.6 Hz, 1H),5.26 (dt, J = 52.4, 4.2 Hz, 1H), 4.74 - C 18 H 19 ESIMS of Cl2FN5O8P2 [M-H] - , Calculated value 584.0, measured value 584.0. [Example 48] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-((2-chlorobenzyl)amino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0241] The title compound was synthesized in a similar manner to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 H NMR (400MHz, DMSO-d6) δ 8.99 (t, J = 6.1 Hz, 1H), 8.35 (s, 1H), 7.47 (dd, J = 6.0, 3.3 Hz,1H), 7.35 - 7.22 (m, 3H), 6.40 (dd, J= 14.2, 4.6 Hz, 1H), 5.27 (dt, J = 52.4,4.3 Hz, 1H), 4.73 (d, J = 5.2 Hz, 2H), 4.52 (d, J = 18.5 Hz, 1H), 4.20 (t, J = 6.2Hz, 2H), 4.05 (q, J = 5.1 Hz, 1H), 2 .27 (t, J = 20.5 Hz, 2H). C 18 H 20 ESIMS [M+H] of Cl2FN5O8P2 + , Calculated value 586.0, Actual measurement Value 586.1. [Example 49] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-((2-chlorobenzyl)(methyl)amino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0242] The title compound was synthesized in a similar manner to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 H NMR (400MHz, DMSO-d6) δ 8.32 (d, J = 37.7 Hz, 1 H), 7.55 - 7.46 (m, 1 H), 7.31(bs, 2 H),7.15 (bs, 1 H), 6.41 (d, J = 14.4 Hz, 1 H), 5.61 (bs, 1 H), 5.26 (d, J = 52.6 Hz,1 H), 5.00 (b, 1 H), 4.49 (bs, 1 H),4.17 (bs, 2 H), 4.03 (bs, 1 H), 3.70 (bs, 1H), 3.18 (bs, 2 H), 2.25 (t, J = 20. 4 Hz, 2 H). C 19 H 24 ESIMS of Cl2N5O9P2 [M+H] + , Calculated value 600.0, Measured value 600.1. [Example 50] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-((pyridin-4-ylmethyl)amino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0243] The title compound was synthesized in a similar manner to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 H NMR (400MHz, DMSO-d6) δ 9.13 (s, 1H), 8.66 (d, J = 5.7 Hz, 2H), 8.37 (s,1H), 7.65 (d, J =5.6 Hz, 2H), 6.40 (dd, J = 14.0, 4.6 H z, 1H), 5.40 - 5.08 (m, 1H), 4.80 (d, J =6.1 Hz, 2H), 4.53 (d, J = 18.3 Hz, 1H) , 4.19 (s, 2H), 4.04 (d, J = 5.2 Hz, 1H),2.25 (t, J = 20.4 Hz, 2H). C 17 H 20 ClFN ESI MS [M+H] of 6O8P2 + , Calculated value 553.1, Measured value 553.2. [Example 51] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-(phenethylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0244] The title compound was synthesized in a similar manner to Example 29 using the product of step a of Example 29 and the corresponding amine: 1 H NMR (400MHz, DMSO-d6) δ 8.50 (t, J = 5.7 Hz, 1H), 8.27 (s, 1H), 7.38 - 7.13 (m, 5H), 6.37(dd, J = 14.4, 4.7 Hz, 1H), 5.25 (dt, J = 52.4, 4.2 Hz, 1H), 4.51 (dt, J = 18.5,4.6 Hz, 1H), 4.19 (t, J = 6.1 Hz, 2H), C 19 H 23 ESIMS of ClFN5O8P2 [M+H] + , Calculated value 566.1, Measured value 566.1. [Example 52] Synthesis of (((((2R,3R,4S,5R)-5-(6-(benzylamino)-2-methyl-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0245] The title compound was synthesized in a similar manner to Example 29, using 6-chloro-2-methylpurine instead of 2,6-dichloropurine: 1 H NMR(400 MHz, DMSO-d6) δ 8.51 (s, 1H), 8.23 ​​(s, 1H), 7.44 - 7.19 (m,5H),6.44 (dd, J = 15.0, 4.6 Hz, 1H), 5.41 - 5.13 (m, 1H), 4.72 (s, 2H), 4.53 (dd, J =18.4, 4.7 Hz, 1H), 4.19 (t, J = 6.1 Hz , 2H), 4.04 (t, J = 5.1 Hz, 1H), 2.46 (s,3H), 2.26 (t, J = 20.5 Hz, 2H). 19 H2 ESI MS [M+H] of 4FN5O8P2 + , Calculated value 532.1, Actual value 532.2. [Example 53] Synthesis of (((((2R,3R,4S,5R)-5-(6-(cyclopentylamino)-2-methyl-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0246] The title compound was synthesized in a similar manner to Example 29, using 6-chloro-2-methylpurine instead of 2,6-dichloropurine and cyclopentylamine instead of benzylamine: 1 HNMR(400 MHz, DMSO-d6) δ 8.56 (s, 1H), 8.30 (s, 1H), 6.45 (dd, J = 14.4, 4.6 Hz, 1H), 5.25 (dt, J = 52.5,4.3 Hz, 1H), 4.53 (dt, J = 18.3, 4.5 Hz, 1H), 4.20 (t, J = 6.1 Hz, 2H), 4.04(q, J = 5.0 Hz, 1H), 2.26 (t, J = 20.5 Hz,2H), 1.98 (s, 2H), 1.82 - 1.46 (m, 6H).C 17 H 26 ESI MS [M+H] of FN5O8P2 + , calculation Value 510.1, actual value 510.2. [Example 54] Synthesis of (((((2R,3R,4S,5R)-5-(6-(benzylamino)-2-(trifluoromethyl)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0247] The title compound was synthesized in a similar manner to Example 29, using 6-chloro-2-trifluoromethylpurine instead of 2,6-dichloropurine: 1 HNMR (400 MHz, DMSO-d6) δ 9.11 (d, J = 6.3 Hz, 1 H), 8.49 (d, J =2.1Hz, 1 H), 7.39 - 7.35 (m, 2 H), 7 .34 - 7.27 (m, 2 H), 7.25 - 7.20 (m, 1 H),6.48 (dd, J = 14.0, 4.7 Hz, 1 H), 5.3 0 (dt, J = 52.4, 4.3 Hz, 1 H), 5.20 (bs, 1H), 4.70 (t, J = 5.7 Hz, 1 H), 4.56 ( dt, J = 18.6, 4.7 Hz, 1 H), 4.21 (t, J =6.2 Hz, 2 H), 4.06 (q, J = 5.1 Hz, 1 H) , 2.26 (t, J = 20.5 Hz, 2 H).C 19 H 21 ESIMS [M+H] of F4N5O8P2 + , Calculated value 586.1, Actual measurement Value 586.2. [Example 55] Synthesis of (((((2R,3R,4S,5R)-5-(6-(cyclopentylamino)-2-(trifluoromethyl)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0248] The title compound was synthesized in a similar manner to Example 29, using 6-chloro-2-trifluoromethylpurine instead of 2,6-dichloropurine and cyclopentylamine instead of benzylamine: 1HNMR (400 MHz, DMSO-d6) δ 8.59 - 8.40 (m, 2 H), 6.47 (dd, J = 13.9,4.7 Hz, 1 H), 5.30 (dt, J =52.4, 4.3 Hz, 1 H), 5.11 (bs, 1 H), 4 .52 (dd, J = 28.1, 14.1 Hz, 2 H), 4.21 (t,J = 6.0 Hz, 2 H), 4.06 (q, J = 5.2 Hz , 1 H), 2.26 (t, J = 20.4 Hz, 2 H), 2.08 -1.90 (m, 2 H), 1.80 - 1.50 (m, 6 H). C 17 H 23 ESIMS [M+H] of F4N5O8P2 + , Calculated value 564.2, Measured value 564.1. [Example 56] Synthesis of (((((2R,3R,4S,5R)-5-(6-(benzylamino)-2-phenyl-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0249] Step a: The product of step b(1) of Example 29 (750 mg, 1.25 mmol), phenylboronic acid (229 mg, 1.88 mmol), and potassium carbonate (518 mg, 3.75 mmol) were suspended in 3:1 THF:HO (10.3 mL). The mixture was degassed with N sparging for 10 minutes. Pd(PPh) (144 mg, 0.13 mmol) was then added, and the resulting mixture was degassed for an additional 5 minutes, then sealed and heated to 80 °C overnight. After cooling to room temperature, the reaction was diluted with EtOAc and washed with water and brine. The organic layer was dried over MgSO, filtered, and concentrated under reduced pressure. This crude material was used directly in step b. The reaction mixture consisted of a mixture of mono- and di-debenzoylated products.

[0250] Step b: The product from step a was dissolved in methanol (12.5 mL) and potassium carbonate (518 mg, 3.75 mmol) was added. The resulting suspension was stirred at room temperature overnight and then partitioned between EtOAc and water. The organic layer was washed with brine, then dried (MgSO4), and Concentration under reduced pressure was carried out and the desired product was isolated by column chromatography (SiO2, MeOH and and CH2Cl2 (0-10% gradient) as a white solid (41 mg, 8%, 2 steps). 23 H 22 ESIMS of FN5O3 [M+H] + , Calculated value 436.2, Measured value 436.3.

[0251] Step c: Using a method similar to that in Example 1, the title compound was obtained as a white solid: 1 H NMR (400 MHz, DMSO-d6)δ 8.58 (s,1H), 8.44 - 8.32 (m, 2H), 8.29 (d, J = 2.4 Hz, 1H), 7.40 - 7.50 (m, 5H), 7.31 (dd, J =8.3, 6.9 Hz, 2H), 7.24 - 7.15 (m, 1H),6.59 (dd, J = 15.4, 4.6 Hz, 1H), 5.30 (dt,J = 52.4, 4.1 Hz, 1H), 4.82 (s, 2H), 4.69 - 4.48 (m, 1H), 4.22 (d, J = 6.6 Hz,2H), 4.08 (q, J = 5.1 Hz, 1H), 2.27 (t , J = 20.5 Hz, 2H). C 24 H 26 ESIMS [M-H] of FN5O8P2 - , Calculated value 592.1, Measured value 592.2. [Example 57] Synthesis of (((((2R,3R,4S,5R)-5-(2-benzyl-6-(benzylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0252] Step a: The product of Step b(1) of Example 29 (391 mg, 0.659 mmol), potassium benzyltrifluoroborate (391 mg, 1.98 mmol), and cesium carbonate (1.07 g, 3.30 mmol) were suspended in 20:1 THF:HO (6.5 mL). The mixture was degassed by sparging with N for 10 minutes. Pd(PPh)Cl (96 mg, 0.132 mmol) was then added, and the resulting mixture was degassed for an additional 5 minutes, then sealed and heated at 80 °C for 48 hours. After cooling to room temperature, the reaction was diluted with EtOAc and washed with water and brine. The organic layer was dried over MgSO, filtered, and concentrated under reduced pressure. The desired The product was purified by column chromatography (SiO2, EtOAc / hexane) Obtained as a brown solid (174 mg, 40%).

[0253] Step b: The product from step a (174 mg, 0.265 mmol) was dissolved in methanol (2.65 mL) and potassium carbonate (110 mg, 3.75 mmol) was added. The resulting suspension was stirred at room temperature for 1.5 h and then partitioned between EtOAc and water. The organic layer was washed with brine, then dried (MgSO4) and concentrated under reduced pressure. The desired product was obtained by filtration. After column chromatography (SiO2, 0-10% gradient of MeOH and CH2Cl2), the white C was obtained as a solid (102 mg, 86%). 24 H 24 ESIMS of FN5O3 [M+H] + , calculated value 450 .2, actual value 450.3.

[0254] Step c: Using a method similar to that in Example 1, the title compound was obtained as a white solid: 1 H NMR (400 MHz, DMSO-d6)δ 8.54 (s,1H), 8.22 (s, 1H), 7.61 - 6.94 (m, 10H), 6.44 (dd, J = 15.1, 4.6 Hz, 1H), 5.23 (dt, J =52.4, 4.1 Hz, 1H), 4.82 - 4.40 (m, 3H) , 4.18 (t, J = 6.5 Hz, 2H), 4.03 (dd, J =10.9, 5.9 Hz, 3H), 2.26 (t, J = 20.5 H z, 2H). C 25 H 28 ESIMS [M-H] of FN5O8P2 - , Calculated value 606.1, Measured value 606.3. [Example 58] Synthesis of (((((2R,3R,4S,5R)-5-(6-(cyclopentylamino)-2-(piperidin-1-ylmethyl)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0255] Step a: The product of Step a of Example 35 (10.0 g, 17.24 mmol), phenylvinylboronic acid (3.83 g, 25.86 mmol), and sodium carbonate (5.44 mg, 51.72 mmol) were suspended in 3:1 THF:HO (100 mL). The mixture was degassed with N2 sparge for 10 min. Then Pd(PPh3)4 (1.99 g, 1.72 mmol) was added and the resulting mixture was degassed for an additional 5 min and then heated to reflux overnight. After cooling to room temperature, the reaction was diluted with EtOAc and washed with water and brine. The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The desired product was isolated by column chromatography. After filtration (SiO2, 5% to 50% EtOAc / hexane), a colorless solid (8.06 g, 72%).

[0256] Step b: The product from step a (8.0 mL) in 2:1 THF:HO (127.5 mL) To a suspension of 2,6-lutidine (2.80 mL, 24.1 mmol), potassium osmate dihydrate (100 mg, 0.30 mmol) was added. The resulting thick suspension was stirred overnight at room temperature and then partitioned between EtOAc and water. The organic layer was washed successively with water and brine, dried over MgSO4, and concentrated under reduced pressure. The title compound was purified by column chromatography. After filtration (SiO2, EtOAc / hexanes) an off-white oil (6.74 g, 97 %). C 30 H 28 ESI MS [M+H] of FN5O6 + ,Calculated value 574.2, Measured value 574.4.

[0257] Step c: 1) To a solution of the product of step b (500 mg, 0.87 mmol) in dichloroethane (4.5 mL) was added piperidine (104 μL, 1.05 mmol) followed by sodium triacetoxyborohydride (223 mg, 1.05 mmol) in one portion. The reaction was stirred at room temperature overnight and then partitioned between EtOAc and water. The organic layer was washed with brine. , dried over MgSO4, and concentrated under reduced pressure to give the title compound, which could be further purified. I used it without any problems. 35 H 39 ESI MS of FN6O5 [M+H] + ,Calculated value 643.3, Measured value 643.3.

[0258] Step c:2) The above crude product was dissolved in methanol (8.7 mL) and potassium carbonate (362 mg, 2.62 mmol) was added. The resulting suspension was stirred at room temperature overnight and then partitioned between EtOAc and water. The organic layer was washed with brine, then dried (MgSO4), and reduced The desired product was purified by column chromatography (SiO, MeOH and Obtained as a white solid after 0-100% gradient of CH2Cl2 (151 mg, 40%, 2 steps). 21 H 31 ESIMS of FN6O3 [M+H] + , Calculated value 435.2, Measured value 435.3.

[0259] Step d: Using a method similar to that in Example 1, the title compound was obtained as a white solid: 1 H NMR (400 MHz, DMSO-d6)δ 9.40 (s,1H), 8.58 - 8.03 (m, 2H), 6.45 (dd, J = 14.1, 4.8 Hz, 1H), 5.25 (dt, J = 52.5, 4.3 Hz,1H), 4.59 (d, J = 16.3 Hz, 2H), 4.40 (s , 1H), 4.20 (t, J = 6.1 Hz, 2H), 4.04 (q, J= 5.1 Hz, 1H), 3.61 (s, 1H), 3.08 (s , 2H), 2.24 (t, J = 20.4 Hz, 2H), 2.06 -1.35 (m, 10H). C 22 H 35 ESIMS of FN6O8P2 [ MH] - , Calculated value 591.2, Measured value 591.3. [Example 59] Synthesis of (((((2R,3R,4S,5R)-5-(6-(cyclopentylamino)-2-(methoxymethyl)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0260] Step a: To a solution of the product of Step b, Example 58 (1.0 g, 1.74 mmol) in dichloroethane (20 mL) was added sodium triacetoxyborohydride (443 mg, 2.09 mmol) in one portion. The reaction was stirred at room temperature overnight and then partitioned between EtOAc and water. The organic layer was washed with brine, dried over MgSO4, and concentrated under reduced pressure to give the title compound. A mixture was obtained which was used without further purification. 30 H 30 ESIMS of FN5O6 [M+H] + , Calculated value 576.2, measured value 576.3.

[0261] Step b: 1) To a solution of the product of step a in dichloromethane (10 mL) at 0 °C was added TsCl (436 mg, 2.29 mmol) and triethylamine (400 μL, 2.87 mmol). The reaction was allowed to warm to room temperature and stirred overnight. The reaction was diluted with EtOAc and washed with saturated NaHCO3, 10% citric acid, water, and brine. The organic layer was dried over MgSO4. Drying and concentration under reduced pressure gave the crude title compound (1.20 g, 94%, 2 steps) which was used directly in the next step.

[0262] Step b:2) To a flask containing the crude tosylate salt (700 mg, 0.959 mmol) and potassium carbonate (662 mg, 4.8 mmol) was added methanol (10 mL). The resulting suspension was stirred overnight, then diluted with EtOAc and washed with water and brine. The organic layer was dried over MgSO4 and concentrated under reduced pressure. The title compound (85 mg, 23%) was obtained from the column. The compound was obtained after column chromatography (SiO2, 0-15% gradient of MeOH and CH2Cl2). 17 H 24 ESIMS of FN5O4 [M+H] + , Calculated value 382.2, Measured value 382.3.

[0263] Step c: Using a method similar to that in Example 1, the title compound was obtained as a white solid: 1 H NMR (400 MHz, DMSO-d6)δ 8.24 (s,1H), 6.46 (dd, J = 14.9, 4.6 Hz, 2H), 5.24 (dt , J = 52.5, 4.2 Hz, 1H), 4.54 (dt, J =18.3, 4.4 Hz, 2H), 4.40 (s, 2H), 4.20 (t, C 18 H 28 ESIMS [MH] of FN5O9P2 - , calculated value 5 38.1, actual value 538.2. [Example 60] Synthesis of (((((2R,3R,4S,5R)-5-(6-(cyclopentylamino)-2-(hydroxy(phenyl)methyl)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0264] Step a: To a solution of the product of Step b of Example 58 (330 mg, 0.58 mmol) in THF (6 mL) at −78° C. was added phenylmagnesium bromide (3.0 M in EtO, 0.8 6 mL) was added. The reaction was stirred at this temperature for 1 h and then quenched with saturated NaHCO3. The crude reaction mixture was partitioned between EtOAc and water. The organic layer was washed with water and brine, dried over MgSO4, and concentrated under reduced pressure. The crude material was the isomeric mono-debenzoylation product. This was used directly in step b. 29 H 30 ESIMS [M+H] of FN5O5 + , calculated value 548 .2, actual value 548.3.

[0265] Step b: The product from step a was dissolved in methanol (5.8 mL) and potassium carbonate (240 mg, 1.74 mmol) was added. The resulting suspension was stirred at room temperature overnight and then diluted with Et The organic layer was washed with brine, then dried (MgSO4), and Concentration under reduced pressure was carried out and the desired product was isolated by column chromatography (SiO2, MeOH and and CH2Cl2 (0-10% gradient) as a white solid (118 mg, 46%, 2 steps). 22 H 26 ESIMS of FN5O4 [M+H] + , Calculated value 444.2, Measured value 444.3.

[0266] Step c: Using a method similar to that in Example 1, the title compound was obtained as a white solid (1:1 mixture of diastereomers): 1 H NMR (400MHz, DMSO-d6) δ 8.78 - 7.85 (m, 4H), 7.49 (s, 4H), 7.41 - 7.08 (m,8H), 6.47(dd, J = 14.8, 4.6 Hz, 2H), 5.98 - 5.39 (m, 2H), 5.24 (dt, J = 52.4, 4.2 Hz, 1H),5.07 (s, 1H), 4.54 (d, J = 14.1 Hz, 0H ), 4.39 - 3.86 (m, 6H), 2.26 (t, J = 20.5Hz, 3H), 1.99 (d, J = 34.0 Hz, 5H), 1. 65 (d, J = 52.4 Hz, 13H). C 23 H 30 ESIMS [MH] of FN5O9P2 - , Calculated value 600.2, Measured value 60 0.3. [Example 61] Synthesis of (((((2R,3R,4S,5R)-5-(6-(benzylamino)-2-(phenylethynyl)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0267] Step a: The product of Step b(1) of Example 29 (750 mg, 1.24 mmol) was suspended in DMF (8.3 mL) and EtN (260 μL) was added, followed by phenylacetylene (205 μL) was added. The mixture was degassed by N sparging for 10 minutes. uI (24 mg) and Pd(PPh3)2Cl2 (44 mg) were added, and the resulting mixture was The reaction was heated to 0° C. overnight. After cooling to room temperature, the reaction was diluted with EtOAc and washed with 10% citric acid (aq), water, and brine. The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The desired product was isolated by column chromatography (SiO2, EtOAc / Hexane After filtration, the product was obtained as a tan oil (762 mg, 92%).

[0268] Step b: The product from step a (762 mg, 1.14 mmol) was dissolved in methanol (11.4 mL) and potassium carbonate (473 mg, 3.42 mmol) was added. The resulting suspension was stirred at room temperature overnight and then partitioned between EtOAc and water. The organic layer was washed with brine, then dried (Na2SO4) and concentrated under reduced pressure. The desired product was obtained as a colorless oil after column chromatography (SiO2, 0-10% gradient of MeOH and CH2Cl2). C 25 H 22 ESI MS of FN5O3 [M+H] + ,Calculated value 460.2, Measured value 460.2.

[0269] Step c: Using a method similar to that in Example 1, the title compound was obtained as a white solid: 1 H NMR (400 MHz, DMSO-d6) δ 8.65 (s,1H), 8.37 (d, J = 2.3Hz, 1H), 7.67 - 7.57 (m, 1H), 7.47 (td, J = 5.2, 2.1 Hz, 2H), 7.39- 7.29 (m, 4H), 7.28 - 7.16 (m, 1H),6.50 (dd, J = 15.2, 4.4 Hz, 1H), 5.28 (dt,J = 52.4, 4.1 Hz, 1H), 4.75 (s, 2H), 4.52 (d, J = 18.1 Hz, 1H), 4.20 (d, J = 6.4Hz, 2H), 4.06 (q, J = 5.0 Hz, 1H), 2 .28 (t, J = 20.5 Hz, 2H). C 26 H 26 ESIMS [M-H] of FN5O8P2 - , Calculated value 616.1, Measured value 61 6.3. [Example 62] Synthesis of (((((2R,3R,4S,5R)-5-(6-(benzylamino)-2-phenethyl-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0270] Step a: To a solution of the product of Step b of Example 61 (203 mg, 0.44 mmol) in ethanol (4.4 mL) under a nitrogen atmosphere was added palladium on activated carbon (10 wt% wet, 20 mg). The nitrogen atmosphere was replaced with hydrogen and the mixture was stirred at room temperature. After stirring overnight, the reaction was diluted with EtOAc and filtered through Celite. The filtrate was concentrated under reduced pressure to give the title compound (161 mg, 79%), which was used without further purification. 25 H 26 FN5O3 ES I MS [M+H] + , Calculated value 464.2, Actual value 464.4.

[0271] Step b: Using a method similar to that in Example 1, the title compound was obtained as a white solid: 1 1 H NMR (400 MHz, DMSO-d6)δ 8.60 -8.14 (m, 2H), 7.58 - 6.91 (m, 11H), 6.44 (d, J = 15.0 Hz, 1H), 5.22 (d, J = 52.4 Hz, 1H),4.71 (s, 2H), 4.54 (dt, J = 18.4, 4.4Hz, 1H), 4.19 (t, J = 6.2 Hz, 2H), 4.11 -3.96 (m, 1H), 3.23 - 2.83 (m, 5H), 2.2 6 (t, J = 20.5 Hz, 2H). C 26 H 30 ESIMS [M-H] of FN5O8P2 - , Calculated value 620.2, Measured value 620 .2. [Example 63] Synthesis of (((((2R,3R,4S,5R)-5-(6-(benzylamino)-2-ethynyl-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0272] Step a: The product of Step b(1) of Example 29 (2.0 g, 3.32 mmol) was suspended in DMF (7.4 mL) and diisopropylamine (2.3 mL) was added, followed by trimethylsilylacetylene (703 μL, 4.98 mL). The mixture was sparged with N2. The mixture was degassed for 10 minutes. Then, CuI (125 mg, 0.66 mmol) and Pd(PPh3)2Cl2 (233 mg, 0.033 mmol) were added, and the resulting mixture was degassed for another 5 minutes. The mixture was degassed for 1 hour, then sealed and heated to 80 °C for 36 hours. After cooling to room temperature, the reaction was diluted with EtOAc and washed with saturated NH4Cl (aq), water, and brine. The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The desired product was obtained after column chromatography (SiO2, 5% to 70% EtOAc / hexanes) as a beige solid (950 mg, 43% ) was obtained.

[0273] Step b: The product from step a (950 mg, 1.43 mmol) was dissolved in methanol (14 mL) and potassium carbonate (592 mg, 4.29 mmol) was added. The resulting suspension was stirred at room temperature overnight and then partitioned between EtOAc and water. The organic layer was washed with brine, then dried (Na2SO4) and concentrated under reduced pressure. The desired product was obtained after column chromatography (SiO2, 0-10% gradient of MeOH and CH2Cl2), affording the title compound. Obtained as a white solid (230 mg, 42%). 19 H 18 ESIMS of FN5O3 [M+H] + , calculated value 384.1, actual value 384.2.

[0274] Step c: Using a method similar to that in Example 1, the title compound was obtained as a white solid: 1 H NMR (400 MHz, DMSO-d6)δ 8.65 (s,1H), 8.36 (d, J = 2.2 Hz, 1H), 7.39 - 7.26 (m, 5H), 7.28 - 7.17 (m, 1H), 6.44 (dd, J =14.8, 4.5 Hz, 1H), 5.25 (dt, J = 52.5,4.1 Hz, 1H), 4.69 (s, 2H), 4.51 (d, J =18.1 Hz, 1H), 4.19 (d, J = 7.1 Hz, 2H), 2.27 (t, J = 20.5 Hz, 2H). C 20 H 22 ESIMS [M-H] of FN5O8P2 - , Calculated value 540.1, Measured value 5 40.2. [Example 64] Synthesis of [({[(2R,3S,4S,5R)-5-[6-(benzyloxy)-2-chloro-9H-purin-9-yl]-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0275] Step a: Under a nitrogen atmosphere, sodium hydride (90 mg, 2.26 mmol, 1.2 equiv., 60% in oil) and benzyl alcohol (10 mL) were stirred at room temperature for 15 minutes. The product of Step b(1) of Example 29 (1.00 g, 1.88 mmol) was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was directly purified by column chromatography (0-10% MeOH in dichloromethane) to give the desired product as a white solid (721 mg, 97%). 17 H 17 ESIMS of ClFN4O4 [M+H] +, Calculated value 395.1, Measured value 395.1.

[0276] Step b: The product from step a (197 mg, 0.5 mmol) was dissolved in trimethyl phosphate (2.5 mL) and cooled to 0°C. A solution of methylenebis(phosphonic acid dichloride) (624 mg, 2.5 mmol, 5 equiv.) in trimethyl phosphate (1.5 mL) was added dropwise. The reaction mixture was stirred at 0°C for 3 hours and then carefully quenched with 0.5 M triethylammonium bicarbonate solution (3.6 mL) at -20°C. The mixture was stirred at -20°C for 15 minutes, then at 0°C for 15 minutes, and then at room temperature for 15 minutes. The mixture was washed three times with ethyl acetate (10 mL). The aqueous layer was directly purified by reverse-phase HPLC (C18 column, 0-50% gradient of acetonitrile and water with 0.1% TFA) to give the desired product as a white solid (40.2 mg, 15%): 1 HNMR (400 MHz, DMSO-d6) δ 8.55 (d, J = 2.1 Hz, 1H), 7.57 - 7.51 (m,2H), 7.46 -7.35 (m, 3H), 6.49 (dd, J = 13.6, 4.7 Hz, 1H), 5.61 (s, 2H), 5.30 (dt, J = 52.4,4.4 Hz, 1H), 4.53 (dt, J = 18.6, 4.7 C 18 H 19 ESIMS of ClFN4O9P2 [M-H] - , Calculated value 551.0, Measured value 551.2. [Example 65] Synthesis of (((((2R,3R,4S,5R)-5-(6-(benzylamino)-2-chloro-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0277] Step a: 2,4-Dichloro-7H-pyrrolo[2,3-d]pyrimidine (350 mg, 1.86 mmol) was dissolved in 15 mL of acetonitrile and treated with CsCO (788 mg, 2.42 mmol, 1.3 equiv.). The mixture was stirred at room temperature for 60 min. 2-Deoxy-2-fluoro-α-D-arabinofuranosyl bromide 3,5-dibenzoate (787 mg, 1.86 mmol, 1 equiv.) was dissolved in 10 mL of acetonitrile and added dropwise to the mixture via an addition funnel. The mixture was stirred at room temperature overnight. The mixture was filtered through a pad of silica gel and concentrated. The residue was adsorbed onto silica and purified using column chromatography (hexane / ethyl acetate) to give the product as a white solid in 49% yield (480 mg).

[0278] Step b: The product from step a (480 mg, 0.9 mmol), benzylamine (97 mg, 0.9 mmol), and EtN (91 mg, 0.9 mmol) in absolute EtOH (4 mL) A mixture of 2,4-dimethyl-3,5-trimethyl-2,5-trimethyl-1 ...

[0279] Step c: The product from step b (360 mg, 0.91 mmol) was dissolved in trimethyl phosphate (4 mL) and cooled to 0 °C (ice bath), followed by the dropwise addition of a cold solution of methylenebis(phosphonic acid dichloride) (801 g, 3.2 mL, 3.5 equiv.) in trimethyl phosphate (2 mL). The reaction mixture was stirred at 0 °C for 1 h, then carefully quenched with ice-cold 0.5 M triethylammonium bicarbonate solution (11 mL) and stirred at 0 °C for 15 min, then at room temperature for 1 h. The reaction mixture was purified by reverse-phase HPLC (C18 column, 0-40% gradient of acetonitrile and water with 0.1% TFA) to give the product as a white solid: 1HNMR (400 MHz, DMSO-d6) δ 8.60 (t,J = 6.0 Hz, 1H), 7.59 - 7.13 (m,6H), 6.72 (s, 1H), 6.49 (dd, J = 15.7, 4.5 Hz, 1H), 5.45- 5.04 (m, 1H), 4.80 - 4.57 (m, 2H), C 18 H 22 ESIMS of ClFN4O8P2 [M-H] - , Calculated value 549.1, Measured value 549.2 [Example 66] ((((2R,3R,4S,5R)-5-(2-chloro-6-(cyclopentylamino) Synthesis of (9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0280] The title compound was synthesized in a similar manner to Example 65, using cyclopentylamine instead of benzylamine: 1 H NMR (400MHz, DMSO-d6) δ 7.90 (d, J = 7.2 Hz, 1H), 7.24 (s, 1H), 6.72 (d, J = 3.6 Hz, 1H),6.47 (dd, J = 15.9, 4.4 Hz, 1H), 5.15(dt, J = 52.6, 4.1 Hz, 1H), 4.52 - 4.35 (m,2H), 4.15 (q, J = 6.3, 5.3 Hz, 2H),3 .97 (q, J = 5.1 Hz, 1H), 2.23 (d, J = 20.5Hz, 1H), 1.98 (d, J = 10.6 Hz, 2H), 1 .72 (s, 2H), 1.67 - 1.45 (m, 5H).C 17 H 24 ESIMS of ClFN4O8P2 [M-H] - , calculated value 527.1, Actual value: 527.2 [Example 67] Synthesis of ((((1-(6-(benzylamino)-9H-purin-9-yl)propan-2-yl)oxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0281] The title compound was synthesized in a similar manner to step b of Example 1 using the corresponding alcohol: 1 HNMR (400 MHz, DMSO-d6)δ 8.79 (s, 1H), 8.31 (d, J = 15.8 Hz, 2H),7.46 - 7.13 (m, 5H), 4.92 - 4.62 (m, 2H), 4.49- 4.25 (m, 2H), 2.17 (td, J = 20.4,4.8 Hz, 2H), 1.14 (d, J = 6.3 Hz, 3H). 16 H 22 ESIMS of N5O6P2 [M+H] + , calculated value 442 .1, actual value 442.1. [Example 68] Synthesis of (((2-(6-(benzylamino)-9H-purin-9-yl)propoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0282] The title compound was synthesized in a similar manner to step b of Example 1 using the corresponding alcohol: 1 HNMR (400 MHz, DMSO-d6)δ 8.73 (s, 1H), 8.35 (s, 1H), 8.27 (s, 1H),7.4 1 - 7.18 (m, 5H), 4.96 - 4.82 (m, 1H), 4.72(s, 2H), 4.39 - 4.19 (m, 2H), 2.18 ( t, J = 20.5, 1.6 Hz, 2H), 1.55 (d, J = 7.0Hz, 3H).C 16 H 22 ESIMS of N5O6P2 [M+H ] + , Calculated value 442.0, Measured value 442.1. [Example 69] Synthesis of ((((((2R,3R,4S,5R)-5-(2-chloro-6-(cyclopentylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphoryl)bis(oxy)bis(methylene)diisopropyl bis(carbonate) [ka]

[0283] Methylenebisphosphonic acid (20 mg, 0.03 mmol, trifluoroacetate salt from Example 66) was dissolved in 0.5 mL of DMSO. Hunig's base (0.18 mL, 1 mmol, 30 equiv.) was added, followed by chloromethyl isopropyl carbonate (0.13 mL, 1 mmol, 30 equiv.). The reaction mixture was stirred at room temperature for 5 days. The reaction mixture was purified by reverse-phase HPLC (C18 column, 0-40% gradient of acetonitrile and water with 0.1% TFA) to give the product as a white solid in 14% yield (3.6 mg). 1 HNMR (400 MHz, DMSO-d6) δ 8.42 (d,J = 7.7 Hz, 1H), 8.25 (s, 1H), 6.37(dd, J = 1 5.2, 4.4 Hz, 1H), 5.67 - 5.43 (m, 4H), 5.24(ddt, J = 52.1, 7.7, 4.1 Hz, 1H), 4. 79 (pd, J = 6.2, 3.8 Hz, 2H), 4.57 - 4.38(m, 1H), 4.37 - 4.19 (m, 2H), 4.06 (q, C 26 H 39 ClFNO 14 ESIMS of P2 [MH] - , Calculated value 760.2, Measured value 760.3. [Example 70] Synthesis of [({[(2S,3S,4R,5R)-5-{2-chloro-6-[cyclopentyl(methyl)amino]-9H-purin-9-yl}-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0284] The title compound was synthesized in the same manner as in Example 1. 1 H NMR (400 MHz, DMSO-d6) δ 8.4 2 (s, 1H), 5.88 (d, J = 5.9 Hz, 1H), 4.53-4.46 (m, 1H), 4.19 (dd, J = 5.0, 3.1 Hz, 1H), 4.15 - 4.06 (m, 3H), 3.17 (brs,3H), 2.26 (t, J = 20.5 Hz, 2H), 1.94 - 1.53 (m, 9H). C 17 H 27 ESIMS of ClN5O9P2 [M+H] +, Calculated value 542.1, Measured value 542.2. [Example 71] Synthesis of [({1-[(2S,3S,4R,5R)-5-[6-(benzylamino)-2-chloro-9H-purin-9-yl]-3,4-dihydroxyoxolan-2-yl]ethoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0285] Step a: The alcohol (4.8 g, 11.1 mmol) was dissolved in anhydrous CHCl (100 mL) and Dess-Martin periodinane (5.6 g, 13.3 mmol, 1.2 equiv.) was added. The reaction mixture was stirred at room temperature for 3 h, then 10% NaSO (20 mL) and saturated HCl were added. The mixture was quenched with NaHCO3 (50 mL). The organic layer was separated, dried over MgSO4, filtered, and the solvent was evaporated. The crude aldehyde was purified by column chromatography (SiO2, CHCl2 →CH2Cl2:MeOH, 9:1) to give a yellow solid (4.8 g, quantitative). 20 H 21 ESIMS of ClNO4 [M+H] + , Calculated value 430.1, Measured value 430.2.

[0286] Step b: The product from step a (860 mg, 2.0 mmol) was dissolved in anhydrous THF (20 mL) and cooled to -78 °C. A 3 M solution of MeMgBr in EtO (2 mL, 6 mmol) was added. The reaction mixture was stirred at -78 °C for 10 min, then slowly warmed to room temperature and stirred at room temperature for 2 h. It was quenched with saturated NH4Cl (10 mL). The organic layer was separated, dried over MgSO4, filtered and the solvent was evaporated. It was used without any problems. 21 H 25 ESI MS of ClNO4 [M+H] + , Calculated value 446.2, Measured value 446.3 .

[0287] Step c: The phosphonylation step was carried out in the same manner as in Example 1. 1 H NMR (400 MHz, DMSO-d6) δ9.00 - 8.88 (m, 1H), 8.51(s, 1H), 7.37 - 7.17 (m, 5H), 5.81 (d, J = 7.0 Hz, 1H), 4.70 - 4.51 (m, 4H), 4.32 - 4.25(m, 1H), 3.83 (dd, J = 5.3, 2.6 Hz, 1H ), 2.22 (t, J = 20.5 Hz, 2H), 1.26 (d, J =6.4 Hz, 3H).C 19 H 25 ESIM of ClN5O9P2 S [M+H] + , Calculated value 564.1, Measured value 564.1. [Example 72] [({1-[(2S,3S,4R,5R)-5-[6-(benzylamino)-2-chloro Synthesis of [(9H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]propoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0288] The title compound was synthesized in the same manner as in Example 71. 1 H NMR (400 MHz, DMSO-d6) δ 8.97- 8.91 (m, 1H), 8.53 (s, 1H), 7.38 - 7.20(m, 5H), 5.79 (d, J = 7.4 Hz, 1H), 4 .65 (d, J = 6.3 Hz, 2H), 4.62 - 4.56 (m,1H), 4.54 - 4.46 (m, 1H), 4.34 (d, J = 5.5 Hz, 1H), 3.89 (dd, J = 6.1, 2.2 Hz,1H), 2.22 (t, J = 20.5 Hz, 2H), 1.69 (s, 1H), 1.58 (q, J = 7.1 Hz, 1H), 0.90 (t, J= 7.4 Hz, 3H).C 20 H 27 ESI of ClN5O9P2 MS [M+H] + , Calculated value 578.1, Measured value 578.2. [Example 73] Synthesis of [({[(2R,3R,4R,5R)-5-[2-chloro-6-(cyclopentylamino)-9H-purin-9-yl]-3,4-dihydroxy-4-methyloxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0289] Step a: To (β-D-ribofuranose, 2-C-methyl-1,2,3,5-tetrabenzoate (4.0 g, 6.89 mmol, 1 equiv.), and 2,6-dichloropurine (1.43 g, 7.58 mmol, 1.1 equiv.) in acetonitrile (23 mL) at 0° C. was added 1,8-diazabicyclo[5.4.0]undec-7-ene (2.58 mL, 17.23 mmol, 2.5 equiv.) followed by trimethylsilyl trifluoromethanesulfonate (5.11 mL, 28.25 mmol, 4.1 equiv.) dropwise over 5 minutes. The reaction mixture was stirred at 0° C. for 15 minutes and then heated at 65° C. for 5 hours. After cooling to room temperature, the reaction was diluted with dichloromethane and washed with saturated aqueous sodium bicarbonate (×2) and brine (×1). The organic layer was then eluted. It was dried over MgSO4, filtered, and concentrated under reduced pressure. After column chromatography (SiO2, 25% to 66% EtOAc / Hexanes), the desired product was obtained as a white solid (1.30 g, 97%).

[0290] Step b: 1) The product from step a (1.3 g, 2.01 mmol), cyclopentylamine (297 μL, 3.01 mmol, 1.5 equiv.), and triethylamine (560 μL, 4.02 mmol, 2.0 equiv.) were suspended in absolute EtOH (6.7 mL). The mixture was stirred at 70° C. for 4 h. After cooling to room temperature, the mixture was concentrated under reduced pressure, and the resulting material was used without further purification.

[0291] 2) The above product was dissolved in methanol (20 mL) and potassium carbonate (1.06 g, 7.63 mmol, 3.8 equiv.) was added. After stirring at ambient temperature for 2 h, the residue was adsorbed onto Celite and purified by column chromatography (SiO2, 0%-10% DCM / MeOH). Purification using HCl gave a colorless oil (612 mg, 79%, 2 steps).

[0292] Step c: The title compound was synthesized in the same manner as in Example 1. 1 H NMR (400 MHz, DMSO-d6) δ8.38 (dd, J = 18.2, 8.1 Hz, 1 H), 8.26(d, J = 10.0 Hz, 1 H), 5.86 (s, 1 H), 4.42 (q, J = 7.2 Hz, 1 H), 4.27 (h, J =10.6, 10.0 Hz, 2 H), 4.06 (s, 3 H), 2.28 (t, J = 20.4 Hz, 2 H), 1.93 (d, J = 16.3Hz, 2 H), 1.78 - 1.43 (m, 6 H).C 17 H 25 ESIMS of ClN5O9P2 [M-H] - , Calculated value 540.1, Measured value 540.2. [Example 74] Synthesis of ((2R,3S,4R,5R)-5-(2-chloro-6-(cyclopentylamino)-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl hydrogen ((hydroxy(methoxy)phosphoryl)methyl)phosphonate [ka]

[0293] Step a: The nucleoside (2.0 g, 5.4 mmol) was dissolved in trimethyl phosphate (30 mL) and cooled to 0 °C (ice bath), then a cold solution of methylenebis(phosphonic acid dichloride) (4.0 g, 16.2 mmol, 3 equiv.) in trimethyl phosphate (15 mL) was added dropwise. The reaction mixture was stirred at 0 °C for 2 h, then cooled to approximately -40 °C, anhydrous MeOH (30 mL) was added, and the mixture was allowed to warm slowly to room temperature. The reaction mixture was then diluted with saturated NaHCO (80 mL). The mixture was neutralized with HCl (150 mL) and diluted with water (150 mL) and EtOAc (150 mL). The organic layer was separated, dried over MgSO4, filtered, and the solvent was evaporated. The product was first purified by column chromatography. (SiO2, EtOAc → EtOAc:MeOH, 8:2), then RP18 HP Purification by LC (H2O + 0.1% TFA / acetonitrile + 0.1% TFA) gave the desired The product was obtained as a white solid in 11% yield (405 mg). 19 H 31 ESIMS of ClN5O9P2 [M+H] + , Calculated value 570.1, Measured value 570.2.

[0294] Step b: To a solution of the product from step a (75 mg, 0.13 mmol) in acetone (1 mL) was added sodium iodide (50 mg, 0.33 mmol). The solution was heated at 65°C for 6 hours. The solvent was evaporated, and the residue was dissolved in water and purified by reverse-phase HPLC (C18 column, 0-40% gradient of acetonitrile and water with 0.1% TFA) to give the product as a white solid in 62% yield (51 mg). 1 HNMR (400 MHz, DMSO-d6) δ 8.41 (d, J = 2.0 Hz, 1H), 8.36 (d, J =7.8Hz, 1H), 5.85 (d, J = 5.6 Hz, 1H), 5.0 4 (brs, 1H), 4.53 (t, J = 5.5 Hz, 1H), 4.47- 4.34 (m, 1H), 4.24 - 3.95 (m, 4H), 3.58 (d, J = 11.3 Hz, 2H), 2.37 (dd, J =20.5, 20.5 Hz, 2H), 2.07 - 1.36 (m,8H). C 17 H 26 ESIMS of ClN5O9P2 [M+H] + , Calculated value 542.8, Measured value 542.2. [Example 75] Synthesis of [(2R,3S,4R,5R)-5-{2-chloro-6-[cyclopentyl(methyl)amino]-9H-purin-9-yl}-3,4-dihydroxyoxolan-2-yl]methylphenyl[(diphenoxyphosphoryl)methyl]phosphonate [ka]

[0295] The alcohol (380 mg, 1 mmol) was dissolved in trimethyl phosphate (5 mL) and cooled to 0 °C (ice bath). A cold solution of methylenebis(phosphonic acid dichloride) (375 mg, 1.5 mmol, 1.5 equiv) in trimethyl phosphate (3 mL) was then added dropwise and the reaction mixture was stirred at 0 °C for 3 h. Solid phenol (470 mg, 5 mmol, 5 equiv) was added and, once dissolved, TEA (835 μL, 6 mmol, 6 equiv) was added dropwise. The mixture was stirred at 0 °C for 15 min and then at room temperature overnight. The product was diluted with HO (15 mL) and purified as MT The combined organic layers were dried over MgSO4, filtered, and the solvent The crude product was purified by column chromatography (SiO2, Hex → 100% EtO Ac) to give a white solid (80 mg, 10%). 1H NMR (400 MHz, DMSO-d6) δ8.38 (d, J = 4.3 Hz,1H), 7.41 - 7.33 (m, 4H), 7.32 - 7.25 (m, 2H), 7.2 5 - 7.11 (m, 9H), 5.89 (dd, J = 5.3, 3.2Hz, 1H), 5.63 (dd, J = 6.0, 4.4 Hz, 1H) , 5.47 - 5.41 (m, 1H), 4.62 - 4.54 (m, 1H),4.49 - 4.32 (m, 2H), 4.28 - 4.08 (m, 1H), 3.67 - 3.47 (m, 2H), 3.35 (s, 3H),1.90 - 1.52 (m, 8H). 35 H 39 ClN5O9P2 ESI MS [M+H] + , Calculated value 770.2, Actual value 770.3. [Example 76] Synthesis of bis(3-chlorophenyl)[({[(2R,3S,4R,5R)-5-{2-chloro-6-[cyclopentyl(methyl)amino]-9H-purin-9-yl}-3,4-dihydroxyoxolan-2-yl]methoxy}(3-chlorophenoxy)phosphoryl)methyl]phosphonate [ka]

[0296] The title compound was synthesized in the same manner as in Example 75. 1 H NMR (400 MHz, DMSO-d6) δ 8.32(s, 1H), 7.43 - 7.34 (m, 2H), 7.34 -7.05 (m, 10H), 5.88 (t, J = 4.7 Hz, 1H), C35 H 36 ESIMS of Cl4N5O9P2 [M+H] + , Calculated value 872.1, Measured value 872.2. [Example 77] Synthesis of bis(3,4-dichlorophenyl)[({[(2R,3S,4R,5R)-5-{2-chloro-6-[cyclopentyl(methyl)amino]-9H-purin-9-yl}3,4-dihydroxyoxolan-2-yl]methoxy}(3,4-dichlorophenoxy)phosphoryl)methyl]phosphonate [ka]

[0297] The title compound was synthesized in the same manner as in Example 75. 1 H NMR (400 MHz, DMSO-d6) δ 8.27(s, 1H), 7.65 - 7.57 (m, 2H), 7.52 -7.40 (m, 3H), 7.32 - 7.04 (m, 4H), 5.87 (t, J = 5.0 Hz, 1H), 5.63 (t, J = 6.1 Hz,1H), 5.43 (dd, J = 5.6, 3.7 Hz, 1H), 4 .59 - 4.35 (m, 3H), 4.30 - 4.08 (m, 2H),3.85 (t, J = 22.0 Hz, 2H), 3.32 (s, 3H) , 1.89 - 1.45 (m, 8H). C 35 H 33 ESIMS of Cl7N5O9P2 [M+H] + , Calculated value 974.0, Measured value 9 74.2. [Example 78] Synthesis of methyl 2-({[(2R,3S,4R,5R)-5-{2-chloro-6-[cyclopentyl(methyl)amino]-9H-purin-9-yl}-3,4-dihydroxyoxolan-2-yl]methoxy}[2-(methoxycarbonyl)phenoxy]phosphoryl)methyl][2-(methoxycarbonyl)phenoxy]phosphoryl}oxy)benzoate [ka]

[0298] The title compound was synthesized in the same manner as in Example 75. 1 H NMR (400 MHz, DMSO-d6) δ 8.31- 8.27 (m, 1H), 7.82 - 7.74 (m, 3H), 7.57- 7.49 (m, 2H), 7.46 - 7.39 (m, 1H), 7.36 - 7.17 (m, 6H), 5.83 (t, J = 5.9 Hz,1H), 5.60 - 5.52 (m, 1H), 5.37 (s,1H), 4.55 - 4.29 (m, 3H), 4.15 - 4.04 (m,2H), 3.81 - 3.74 (m, 2H), 3.72 - 3.65(m) , 3H), 3.32 (s, 9H), 1.88 - 1.47 (m, 8H).C 41 H 45 ClNO 15 ESI MS of P2 [M+H] + , total Calculated value 944.2, measured value 944.3. [Example 79] Synthesis of [({[(2R,3R,4S,5R)-5-(2-chloro-6-{[(1S)-1-(4-fluorophenyl)ethyl)amino]-9H-purin-9-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0299] The title compound was synthesized in the same manner as in Example 29. 1 H NMR (400 MHz, DMSO-d6) δ 8.92(d, J = 8.3 Hz, 1H), 8.28 (s, 1H), 7.45(bs, 2H), 7.11 (td, J = 9.1, 1.4 Hz, 2 H), 6.34 (dd, J = 14.3, 4.6 Hz, 1H), 5.39(bs, 1H), 5.31 - 5.12 (m, 1H), 5.14 (b s, 1H), 4.48 (dt, J = 18.5, 4.5 Hz, 1H),4.17 (s, 3H), 4.01 (d, J = 5.2 Hz, 2H), 2.24 (t, J = 20.4 Hz, 3H), 1.51 (d, J =7.0 Hz, 3H).C 19 H 23 ESIMS of ClF2N5O8P2 [M+H] + , Calculated value 584.1, Measured value 584.2. [Example 80] Synthesis of [({[(2R,3R,4S,5R)-5-(2-chloro-6-{[(1R)-1-phenylethyl]amino}-9H-purin-9-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0300] The title compound was synthesized in the same manner as in Example 29. 1 H NMR (400 MHz, DMSO-d6) δ 8.90(d, J = 8.3 Hz, 1H), 8.28 (s, 1H), 7.42(d, J = 7.6 Hz, 2H), 7.29 (t, J = 7.5 Hz, 2H), 7.19 (bs, 1H), 6.34 (dd, J = 14.8,4.4 Hz, 1H), 5.39 (bs, 1H), 5.23 (d, C 19 H 24 E of ClFN5O8P2 SI MS [M+H] + , Calculated value 566.1, Measured value 566.1. [Example 81] Synthesis of [({[(2R,3R,4S,5R)-5-(2-chloro-6-{[(1S)-1-phenylethyl]amino}-9H-purin-9-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0301] The title compound was synthesized in the same manner as in Example 29. 1 H NMR (400 MHz, DMSO-d6) δ 8.91(d, J = 8.3 Hz, 1H), 8.28 (s, 1H), 7.41(d, J = 7.5 Hz, 2H), 7.29 (t, J = 7.6 Hz, 2H), 7.20 (d, J = 7.6 Hz, 1H), 6.34 (d,J = 14.1 Hz, 1H), 5.39 (bs, 1H), 5.2 1 (d, J = 52.5 Hz, 1H), 4.47 (d, J = 18.3Hz, 2H), 4.17 (s, 2H), 4.01 (s, 1H), 2 .24 (t, J = 20.6 Hz, 2H), 1.52 (d, J = 7.1Hz, 3H). C 19 H 24 ESIMS [M +H] + , Calculated value 566.1, Measured value 566.1. [Example 82] Synthesis of [({[(2R,3R,4S,5R)-5-[6-(cyclopentylamino)-2-[hydroxy(oxan-4-yl)methyl]-9H-purin-9-yl]-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]-phosphonic acid [ka]

[0302] Step a: The product of Step b, Example 58 (1.00 g, 1.75 mmol) was dissolved in THF (9 mL) and cooled to -78°C. 4-Tetrahydropyranylmagnesium bromide (9 mL, 8.75 mmol, 0.2 M in THF) was added dropwise. The reaction mixture was allowed to warm to room temperature and stirred at room temperature for 3 hours. The reaction mixture was cooled to 0°C, methanol (50 mL) was added, and the mixture was stirred at room temperature for 14 hours. The reaction mixture was dry-loaded onto silica gel and purified by silica gel chromatography (0-10% MeOH in DCM) to give the desired product as a white solid (273 mg, 35%).

[0303] Step b: In a similar manner to Example 20, the title compound was synthesized as a white solid (44 mg; 29%). 1 HNMR (400 MHz,DMSO-d6) δ 6.60 - 6.40 (m, 1H), 5.26 (d, J = 53. 3 Hz, 1H), 4.63 - 4.39 (m, 2H), 4.30 - 4.13(m, 2H), 4.13 - 3.97 (m, 1H), 3.94 - C 23 H 35 ESIMS [MH] of FN5O9P2 - , Calculated value 606.2, Measured value 60 6.3. [Example 83] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-(cyclopentyl(methyl)amino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)methyl)phosphonic acid [ka]

[0304] This compound was obtained in the same manner as in Example 29. 1 H NMR (400 MHz, DMSO-d6) δ 8.7(brs, 2H), 8.30 (d, J = 2.1 Hz, 1H), 6.40 (dd,J = 14.3, 4.6 Hz, 1H), 6.09 (brs,1H) ), 5.25 (dt, J = 52.5, 4.3 Hz, 1H),4.53-4.43 (m, 1H), 4.23-4.14 (m, 2H), 4.09 - 3.98 (m, 1H), 2.28 (dd, J = 20.5 Hz, J=20.5 Hz, 2H), 2.5 (s, 3H), 1.96 - 1.44 (m, 9H).C 17 H 25 ESIMS of ClFN5O8P2 [M+H] + , calculated value 544.8, found: 544.2. [Example 84] Synthesis of ((2R,3R,4S,5R)-5-(2-chloro-6-(cyclopentyl(methyl)amino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl hydrogen ((hydroxy(methoxy)phosphoryl)methyl)phosphonate [ka]

[0305] Step a: 2-Chloropurine fluororiboside (579 mg, 1.5 mmol) was dissolved in trimethyl phosphate (7.5 mL) and cooled to 0°C (ice bath), and a cold solution of methylenebis(phosphonic acid dichloride) (1.87 g, 7.5 mmol, 5 equiv.) in trimethyl phosphate (4.5 mL) was added dropwise. The reaction mixture was stirred at 0°C for 3 hours, then carefully quenched with methanol (7 mL), and stirred at 0°C for 30 minutes, then at room temperature for 1 hour, and then at 40°C for 3 hours. The reaction mixture was concentrated under reduced pressure and dissolved in ethyl acetate (20 mL). The organic layer was washed with saturated aqueous NaHCO3, dried over sodium sulfate, and evaporated to dryness. The residue The material was purified by column chromatography (gradient of 0-10% methanol in dichloromethane) to give the desired product as a pale yellow solid (701 mg, 80%). 1 HNMR (400 MHz, DMSO-d6) δ 8.32 (dd, J =12.0, 2.3 Hz, 1H), 6.42 (dd, J= 15.5, 4.4 Hz, 1H ), 6.15 (t, J = 4.8 Hz, 1H), 5.43 - 5.07(m, 1H), 4.60 - 4.39 (m, 1H), 4.27 (q, J = 7.3, 5.7 Hz, 2H), 4.12 - 4.03 (dq, J =9.6, 5.3 Hz, 1H), 3.69 - 3.59 (m, 9H) , 2.96 - 2.74 (m, 2H), 2.50 (s, 3H), 2.04 -1.42 (m,9H). C 20 H 31 ESI of ClFN5O8P2 MS [M+H] + , Calculated value 586.9, Measured value 586.2.

[0306] Step b: To a solution of the product from step a (58 mg, 0.1 mmol) in acetone (1 mL) was added sodium iodide (75 mg, 0.5 mmol). The solution was heated at 60°C for 24 hours. The solvent was evaporated, and the residue was dissolved in water and purified by reverse-phase HPLC (C18 column, 0-30% gradient of acetonitrile and water with 0.1% TFA) to give the product (42 mg) as a white solid in 65% yield. 1 HNMR (400 MHz, DMSO-d6) δ 8.31 (d, J = 2.2 Hz, 1H), 6.40 (dd, J = 14.6,4.6Hz, 1H), 5.25 (dt, J = 52.4, 4.2 Hz, 1H), 4.48 (dt, J = 18.3, 4.4 Hz, 1H), 4.18(t, J = 6.1 Hz, 2H), 4.04 (m, 2H), 3. 58 (d, J = 11.2 Hz, 3H), 2.5 (s, 3H). 2.39(dd, J = 20.4 Hz, J = 20.4 Hz, 2H), 2 .00 - 1.42 (m, 9H). C 18 H 27 ESIMS of ClFN5O8P2 [M+H] + , Calculated value 558.8, Measured value 558.2 . [Example 85] Synthesis of ((2R,3R,4S,5R)-5-(2-chloro-6-(cyclopentyl(methyl)amino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl hydrogen((dimethoxyphosphoryl)methyl)phosphonate, and Synthesis of methyl hydrogen (((((2R,3R,4S,5R)-5-(2-chloro-6-(cyclopentyl(methyl)amino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(methoxy)phosphoryl)methyl)phosphonate [ka]

[0307] Step a: To a solution of the product from Example 83, Step a (150 mg, 0.26 mmol) in acetone (3 mL) was added sodium iodide (40 mg, 0.26 mmol). The solution was stirred at room temperature for 24 hours. The solvent was evaporated, and the residue was dissolved in water and purified by reverse-phase HPLC (C18 column, 0-30% gradient of acetonitrile and water containing 0.1% TFA) to give ((2R,3R,4S,5R)-5-(2-chloro-6-(cyclopentyl(methyl)amino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl hydrogen((dimethoxyphosphoryl)methyl)phosphonate as a white solid (35 mg) in 20% yield. 1 HNMR (400 MHz, DMSO-d6) δ 8.31 (d, J = 2.2 Hz, 1H), 6.41 (dd, J =14.9,4.5 Hz, 1H), 5.25 (dt, J = 52.3, 4.1 Hz, 1H), 4.53-4.43 (m, 1H), 4.24 - 4.12 (m,2H), 4.08-4.02 (m, 1H), 3.66 (d, J = 2.0 Hz, 3H), 3.63 (d, J = 2.0 Hz, 3H),2.60 (dd, J = 20.8 Hz, J = 20.8 Hz, 2H), 2.50 (s, 3H), 2.01 - 1.55 (m, 9H).C 19 H 29 ESIMS of ClFN5O8P2 [M+H] + , Calculated value 572.9, Measured value 572.3.

[0308] Methyl hydrogen (((((2R,3R,4S,5R)-5-(2-chloro-6-(cyclopentyl(methyl)amino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(methoxy)phosphoryl)methyl)phosphonate was obtained as a white solid in 30% yield (52%) as a 1:1 mixture of diastereoisomers. mg). 1HNMR(400 MHz, DMSO-d6) δ 8.34 (d, J = 2.3 Hz, 0.5H, 1 st dia), 8.30 (d, J = 2.3 Hz, 0.5H, 2 nd dia), 6.54 - 6.32 (m, 1H), 5.38 - 5.11(m, 1H), 4 .59 - 4.39 (m, 1H), 4.26 (m, 2H), 4.07 (m,1H), 3.64 (d, J = 11.3 Hz, 3H), 3.59 (d, J = 11.2, 1.5H, 1 st dia),3.59 (d, J = 11.2, 1.5H, 1 st dia), 2.69-2.53 (m, 2 H), 2.5 (s, 3H), 1.97 - 1.52 (m, 9H).C 19 H 29 ESIMS of ClFN5O8P2 [M+H] + , calculated value 5 72.9, actual value 572.2. [Example 86] Synthesis of (((((2R,3R,4S,5R)-5-(2-chloro-6-(cyclopentyl(methyl)amino)-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(methoxy)phosphoryl)methyl)phosphonic acid [ka]

[0309] This compound was obtained in a similar manner to Example 84 as a 1:1 mixture of diastereoisomers. 1 HNMR (400 MHz, DMSO-d6)δ 8.34 (d, J = 2.3 Hz, 0.5H, 1 st dia), 8.29 (d, J = 2.3 Hz, 0.5H, 2 nddia),6.52 - 6.32 (m, 1H), 6.07 (brs, 1H),5.34 - 5.14 (m, 1H), 4.56 - 4.43 (m, 1H), 4.30 - 4.21(m, 2H), 4.11 - 4.03 (m,1H), 3.63 ( d, J = 11.2 Hz, 1.5H, 1 st dia),3.63 (d, J = 11.2 Hz, 1.5H, 2 nd dia), 2.50 (s, 3 H), 2.48 - 2.34 (m, 2H), 1.92 - 1.53 (m,9H).C 18 H 27 ESIMS of ClFN5O8P2 [M+H] + , Calculated value 558.3, measured value 558.2. [Example 87] Synthesis of [({[(2R,3S,4R,5R)-5-[6-chloro-4-(cyclopentylamino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0310] Step a: 4,6-Dichloro-1H-pyrazolo[3,4-d]pyrimidine (25 g, 132 mmol) and ammonium sulfate (0.20 g, 1.5 mmol) were dissolved in 150 mL of hexamethyldisilazane. The mixture was then heated to reflux and stirred for 3 hours. The mixture was then concentrated to dryness. The solid residue was then taken up in 300 mL of acetonitrile and protected ribose (50.6 g, 159 mmol) was added. The mixture was cooled to 0°C and TMSOTf (27 mL, 145 mmol) was added dropwise. The mixture was then warmed to room temperature and stirred overnight. The mixture was then concentrated and taken up in ethyl acetate. The organic layer was washed with saturated NaHCO3 and HCl. The organic layer was dried over MgSO4, filtered and concentrated. The crude residue was purified by column chromatography. Purification using chromatography (hexanes / ethyl acetate) gave the desired compound (48 g, 108 mmol) in 82% overall yield. 1 H NMR(400 MHz, DMSO-d6) δ 8.75 (s, 1H), 6.47 (d, J = 3.2 Hz, 1H), 5.82(dd, J= 5.3, 3.2 Hz, 1H), 5.63 (t, J = 5.8 Hz, 1H), 4.47 - 4.40 (m, 1H), 4.37 - 4.30(m, 1H), 4.12 - 4.02 (m, 1H), 2.09 (s, 3H), 2.06 (s, 3H), 1.97 (s, 3H).C 16 H 16 ESIMS of Cl2N4NaO7 [M+H] + ESI MS [M+H] + , Calculated value 469.0, Measured value 469.0.

[0311] Step b: The product from step a (22 g, 49.3 mmol) was dissolved in MeOH (100 mL) and cooled to 0°C. Cyclopentylamine (5.1 g, 51.8 mmol, 1.05 equiv.) and triethylamine (7.2 mL, 51.8 mmol, 1.05 equiv.) were added and the reaction mixture was stirred at 0°C for 15 minutes and then at room temperature for 4 hours. 7M NH3 in MeOH (60 mL) was added and the reaction was stirred at room temperature for 1 day. The reaction mixture was evaporated and the solvent was removed to give the crude product. The product was used in the next step without purification. 15 H 21 ESIMS of ClNO4 [M+H] + , calculated value 370.1, actual value 370.2.

[0312] Step c: The phosphonylation step was carried out in the same manner as in Example 1. 1 H NMR (400 MHz, DMSO-d6) δ8.68 (d, J = 7.2 Hz,1H), 8.24 (s, 1H), 6.00 (d, J = 4.2 Hz, 1H), 4.49 (t, J = 4.7 Hz, 1H), 4.41 (q, J = 6.7 Hz,1H), 4.26 (t, J = 4.7 Hz, 1H), 4.15 - 4.00 (m, 2H), 3.94 - 3.84 (m, 1H), 2.16 (t,J = 20.5 Hz, 2H), 2.04 - 1.91 (m, 2H ), 1.79 - 1.45 (m, 6H). C 16 H 25 ESIMS of ClN5O9P2 [M+H] + , Calculated value 528.1, Measured value 5 28.2. [Example 88] Synthesis of [({[(2R,3S,4R,5R)-5-[4-(benzylamino)-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-3-,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0313] The title compound was synthesized in the same manner as in Example 87. 1 H NMR (400 MHz, DMSO-d6) δ 9.38- 9.18 (m, 1H), 8.35 - 8.16 (m, 1H), 7.39- 7.19 (m, 5H), 6.07 - 5.94 (m, 1H), 4.69 (d, J = 5.4 Hz, 2H), 4.58 - 4.44 (m,1H), 4.30 - 4.20 (m, 1H), 4.15 - 4.01 (m, 2H), 3.96 - 3.80 (m, 1H), 2.17 (t, J =20.9 Hz, 2H). C 18 H 22 ESIM of ClN5O9P2 S [MH] - , Calculated value 548.1, Measured value 548.1. [Example 89] Synthesis of [({[(2R,3S,4R,5R)-5-(6-chloro-4-{[(1S)-1-phenylethyl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0314] The title compound was synthesized in the same manner as in Example 87. 1 H NMR (400 MHz, DMSO-d6) δ 9.26- 8.95 (m, 1H), 8.35 - 8.17 (m, 1H), 7.48- 7.28 (m, 4H), 7.28 - 7.09 (m, 1H), 6.09 - 5.87 (m, 1H), 5.42 (q, J = 6.9 Hz,1H), 4.60 - 4.33 (m, 1H), 4.33 - 4.16 (m, 1H), 4.13 - 3.96 (m, 2H), 3.97 - 3.80(m, 1H), 2.35 - 1.95 (m, 2H), 1.62 -1.36 (m, 3H). 19 H 24 ESIMS of ClN5O9P2 [M-H] - , Calculated value 562.1, Measured value 562.2. [Example 90] Synthesis of [({[(2R,3S,4R,5R)-5-(6-chloro-4-{[(1R)-1-phenylethyl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0315] The title compound was synthesized in the same manner as in Example 87. 1H NMR (400 MHz, DMSO-d6) δ 9.16(d, J = 8.4 Hz, 1H), 8.32 (s, 1H), 7.48 -7.30 (m, 4H), 7.28 - 7.15 (m, 1H), 6 .09 - 5.79 (m, 1H), 5.47 - 5.36 (m, 1H),4.58 - 4.42 (m, 1H), 4.32 - 4.19 (m, 1H) ), 4.17 - 3.95 (m, 2H), 3.95 - 3.79 (m,1H), 2.18 (t, J = 20.8 Hz, 2H), 1.71 - 1 .37 (m, 4H). C 19 H 24 ESIMS of ClN5O9P2 [M-H] - , Calculated value 562.1, Measured value 562.2. [Example 91] Synthesis of [({[(2R,3S,4R,5R)-5-(6-chloro-4-{[(4-chlorophenyl)methyl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0316] The title compound was synthesized in the same manner as in Example 87. 1 H NMR (400 MHz, DMSO-d6) δ 9.41- 9.19 (m, 1H), 8.32 - 8.17 (m, 1H), 7.43- 7.30 (m, 4H), 6.02 (d, J = 2.9 Hz, 1H), 4.68 (d, J = 4.4 Hz, 2H), 4.56 - 4.45(m, 1H), 4.33 - 4.18 (m, 1H), 4.13 - 3.80 (m, 2H), 3.62 - 3.44 (m, 1H), 2.17(t, J = 20.4 Hz, 1H).C 18 H21 Cl2N5O9P2 ESI MS [M-H] - , Calculated value 582.0, Actual value 582.0. [Example 92] Synthesis of [({[(2R,3S,4R,5R)-5-(6-chloro-4-{[(1S)-1-(2-fluorophenyl)ethyl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0317] The title compound was synthesized in the same manner as in Example 87. 1 H NMR (400 MHz, DMSO-d6) δ 9.28- 9.15 (m, 1H), 8.33 (dd, J = 1.5, 0.7Hz, 1H), 7.43 (t, J = 7.8 Hz, 1H), 7.29 (dd, J = 7.8, 5.6 Hz, 1H), 7.23 - 7.08 (m,2H), 6.00 (d, J = 4.2 Hz, 1H), 5.65- 5.51 (m, 1H), 4.48 (t, J = 4.9 Hz, 1H),4.26 (t, J = 4.5 Hz, 1H), 4.05 (dq, J = 10.1, 5.9, 5.2 Hz, 2H), 3.88 (dt, J =11.3, 6.0 Hz, 1H), 2.29 - 2.08 (t, J = 2 0.4 Hz, 2H), 1.53 (d, J = 6.8 Hz, 3H).C 19 H 24 ESIMS of ClFN5O9P2 [M+H] + , calculated value 582.1, Actual value 582.1 [Example 93] Synthesis of [({[(2R,3S,4R,5R)-5-(6-chloro-4-{[(1R)-1-(2-fluorophenyl)ethyl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0318] The title compound was synthesized in the same manner as in Example 87. 1 H NMR (400 MHz, DMSO-d6) δ 9.23(d, J = 7.6 Hz, 1H), 8.34 (s, 1H), 7.44(t, J = 7.8 Hz, 1H), 7.30 (q, J = 7.0 Hz, 1H), 7.18 (dt, J = 9.4, 6.4 Hz, 2H),6.00 (d, J = 4.3 Hz, 1H), 5.60 (q, J = 7.1 Hz, 1H), 4.51 (t, J = 4.6 Hz, 1H), 4.26(t, J = 4.6 Hz, 1H), 4.05 (tt, J = 1 0.1, 5.8 Hz, 2H), 3.88 (dd, J = 11.0, 6.2Hz, 1H), 2.17 (t, J = 20.4 Hz, 2H), 1. 53 (d, J = 6.7 Hz, 3H). C 19 H 24 ESIMS of ClFN5O9P2 [M+H] + , Calculated value 582.1, Measured value 5 82.1. [Example 94] Synthesis of [({[(2R,3S,4R,5R)-5-(6-chloro-4-{[(1S)-1-(3-fluorophenyl)ethyl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0319] The title compound was synthesized in the same manner as in Example 87. 1 H NMR (400 MHz, DMSO-d6) δ 9.17(d, J = 7.9 Hz, 1H), 8.31 (s, 1H), 7.50 -7.30 (m, 1H), 7.22 (d, J = 8.2 Hz, 2 H), 7.06 (td, J = 8.7, 2.5 Hz, 1H), 6.00(d, J = 4.2 Hz, 1H), 5.41 (t, J = 7.3 H z, 1H), 4.48 (t, J = 4.7 Hz, 1H), 4.26 (t,J = 4.8 Hz, 1H), 4.05 (dq, J = 11.7, 6.5 Hz, 2H), 3.88 (dt, J = 11.2, 6.2 Hz,1H), 2.17 (t, J = 20.5 Hz, 2H), 1.53 (d , J = 7.0 Hz, 3H). C 19 H 24 ESIMS of ClFN5O9P2 [M+H] + , Calculated value 582.1, Measured value 582.1 . [Example 95] Synthesis of [({[(2R,3S,4R,5R)-5-(6-chloro-4-{[(1R)-1-(3-fluorophenyl)ethyl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0320] The title compound was synthesized in the same manner as in Example 87. 1H NMR (400 MHz, DMSO-d6) δ 9.18(d, J = 7.9 Hz, 1H), 8.31 (t, J = 0.9 Hz,1H), 7.43 - 7.32 (m, 1H), 7.23 (d, J = 8.8 Hz, 2H), 7.07 (t, J = 8.6 Hz, 1H),6.00 (d, J = 4.3 Hz, 1H), 5.42 (t, J = 7.3 Hz, 1H), 4.51 (t, J = 4.5 Hz, 1H),4.26 (t, J = 4.7 Hz, 1H), 4.11 - 3.98(m, 2H), 3.88 (t, J = 8.6 Hz, 1H), 2.17 (t, J= 20.5 Hz, 2H), 1.52 (d, J = 7.0Hz, 3H).C 19 H 24 ESIMS of ClFN5O9P2 [M+H] + , Calculated value 582.1, Measured value 582.1. [Example 96] Synthesis of [({[(2R,3S,4R,5R)-5-(6-chloro-4-{[(1S)-1-(4-fluorophenyl)ethyl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0321] The title compound was synthesized in the same manner as in Example 87. 1 H NMR (400 MHz, DMSO-d6) δ 9.16(d, J = 7.9 Hz, 1H), 8.30 (d, J = 1.2 Hz,1H), 7.42 (dd, J = 8.4, 5.4 Hz, 2H), 7.15 (td, J = 8.9, 1.2 Hz, 2H), 6.00 (d, J= 4.2 Hz, 1H), 5.40 (t, J = 7.3 Hz,1H), 4.48 (t, J = 4.8 Hz, 1H), 4.25 (t, J =4.5 Hz, 1H), 4.18 - 3.95 (m, 2H), 3. 95 - 3.82 (m, 1H), 2.16 (t, J = 20.4 Hz,2H), 1.52 (d, J = 7.2 Hz, 3H). C 19 H 24 C ESIMS [M+H] of lFN5O9P2 + , Calculated value 582.1, Measured value 582.1. [Example 97] Synthesis of [({[(2R,3S,4R,5R)-5-(6-chloro-4-{[(1R)-1-(4-fluorophenyl)ethyl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0322] The title compound was synthesized in the same manner as in Example 87. 1 H NMR (400 MHz, DMSO-d6) δ 9.16(d, J = 7.9 Hz, 1H), 8.30 (t, J = 0.9 Hz,1H), 7.42 (dt, J = 6.1, 3.2 Hz, 2H), 7.23 - 7.08 (m, 2H), 6.00 (d, J = 4.3 Hz,1H), 5.40 (t, J = 7.2 Hz, 1H), 4.50(t, J = 4.5 Hz, 1H), 4.26 (t, J = 4.7 Hz,1H), 4.15 - 3.98 (m, 2H), 3.87 (q, J = 8.1, 5.5Hz, 1H), 2.16 (t, J = 20.4Hz,2H), 1.52 (d, J = 6.9Hz, 3H).C 19 H 24 Cl ESIMS [M+H] of FN5O9P2 + , Calculated value 582.1, Measured value 582.1. [Example 98] Synthesis of [({[(2R,3S,4R,5R)-5-(6-chloro-4-{[(2-chlorophenyl)methyl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0323] The title compound was synthesized in the same manner as in Example 87. 1 H NMR (400 MHz, DMSO-d6) δ 9.29(s, 1H), 8.29 (d, J = 1.9 Hz, 1H), 7.48(dd, J = 5.9, 3.1 Hz, 1H), 7.43 (d, J = 5.9 Hz, 1H), 7.32 (dt, J = 6.6, 2.5 Hz,2H), 6.07 - 6.00 (m, 1H), 4.84 - 4.69 (m, 2H), 4.51 (d, J = 5.0 Hz, 1H), 4.27 (s,1H), 4.06 (s, 2H), 3.89 (s, 1H), 2.1 6 (t, J = 20.6 Hz, 2H). C 18 H 22 ESIMS of Cl2N5O9P2 [M+H] + , Calculated value 584.0, Measured value 5 84.1. [Example 99] Synthesis of [({[(2R,3S,4R,5R)-5-(6-chloro-4-{[(2-chlorophenyl)methyl](methyl)amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0324] The title compound was synthesized in the same manner as in Example 87. 1 H NMR (400 MHz, DMSO-d6) δ 8.41(s, 1H), 7.49 (d, J = 7.4 Hz, 1H), 7.38 -7.24 (m, 2H), 7.16 (d, J = 7.6 Hz, 1 H), 6.08 (bs, 1H), 5.04 (bs, 2H), 4.50 (d,J = 30.8 Hz, 1H), 4.24 (d, J = 39.5 H z, 1H), 4.06 (s, 2H), 3.89 (s, 1H), 3.37(d, J = 54.8 Hz, 3H), 2.15 (t, J = 20.8 Hz, 2H). C 19 H 24 ESIMS of Cl2N5O9P2 [M+H] + , Calculated value 598.0, Measured value 598.1. [Example 100] Synthesis of [({[(2R,3S,4R,5R)-5-(6-chloro-4-{[2-(2-chlorophenyl)ethyl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0325] The title compound was synthesized in the same manner as in Example 87. 1H NMR (400 MHz, DMSO-d6) δ 8.95(t, J = 5.6 Hz, 1H), 8.28 - 8.10 (m, 1H),7.48 - 7.39 (m, 1H), 7.33 (d, J = 6. 4 Hz, 1H), 7.29 - 7.18 (m, 2H), 6.00 (d, J= 4.0 Hz, 1H), 4.50 (t, J = 4.5 Hz, 1 H), 4.25 (t, J = 4.5 Hz, 1H), 4.06 (d, J =14.9 Hz, 2H), 3.87 (t, J = 5.8 Hz, 1H ), 3.69 (q, J = 6.8 Hz, 2H), 3.10 - 3.00(m, 2H), 2.15 (t, J = 20.4 Hz, 2H). C1 9H 24 ESIMS of Cl2N5O9P2 [M+H] + , Calculated value 598.0, Measured value 598.2. [Example 101] Synthesis of [({[(2R,3S,4R,5R)-5-{4-[benzyl(methyl)amino]-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl}3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0326] The title compound was synthesized in the same manner as in Example 87. 1 H NMR (400 MHz, DMSO-d6) δ 8.35(s, 1H), 7.30 (dd, J = 20.4, 7.5 Hz, 5H),6.07 (bs, 1H), 4.99 (bs, 1H), 4.53 ( bs, 1H), 4.28 (bs, 1H), 4.05 (s, 3H), 3.88(s, 1H), 3.37 - 3.24 (m, 3H), 2.14 (t , J = 20.9 Hz, 3H). C19 H 25 ESIMS of ClN5O9P2 [M+H] + , Calculated value 564.1, Measured value 564.1 . [Example 102] Synthesis of [({[(2R,3S,4R,5R)-5-{6-chloro-4-[cyclopentyl(methyl)amino]-1H-pyrazolo[3,4-d]pyrimidin-1-yl}3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0327] The title compound was synthesized in the same manner as in Example 87. 1 H NMR (400 MHz, DMSO-d6) δ 8.31(s, 1H), 6.06 (s, 1H), 4.51 (d, J = 4.7Hz, 1H), 4.28 (d, J = 5.1 Hz, 1H), 4.1 4 - 3.96 (m, 2H), 3.88 (s, 1H), 3.21 (s,3H), 2.14 (t, J = 19.9 Hz, 2H), 1.67 (b s, 8H). C 17 H 27 ESIMS of ClN5O9P2 [M+H] + , Calculated value 542.1, Measured value 542.2. [Example 103] Synthesis of [({[(2R,3S,4R,5R)-5-[6-chloro-4-(methylamino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0328] The title compound was synthesized in the same manner as in Example 87. 1H NMR (400 MHz, DMSO-d6) δ 8.82(s, 1H), 8.16 (s, 1H), 6.01 (bs, 1H),4.50 (d, J = 5.9 Hz, 1H), 4.26 (bs, 1H), 4.05 (bs, 2H), 3.88 (bs, 1H), 2.95 (d, J =4.6 Hz, 3H), 2.15 (t, J = 20.4 Hz,2H).C 12 H 19 ESIMS of ClN5O9P2 [M+H] + , Calculated value 474.0, Measured value 474.2. [Example 104] Synthesis of [({[(2R,3S,4R,5R)-5-(6-chloro-4-{[(1R)-2,2,2-trifluoro-1-phenylethyl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0329] The title compound was synthesized in the same manner as in Example 87. 1 H NMR (400 MHz, DMSO-d6) δ 9.82(d, J = 9.3 Hz, 1H), 8.51 (s, 1H), 7.65(d, J = 7.2 Hz, 2H), 7.56 - 7.31 (m, 3 H), 6.33 (p, J = 8.8 Hz, 1H), 6.04 (d, J =4.2 Hz, 1H), 4.50 (t, J = 4.4 Hz, 1H) , 4.26 (t, J = 4.6 Hz, 1H), 4.16 - 4.00 (m,2H), 3.90 (dd, J = 10.6, 5.8 Hz, 1H) , 2.18 (t, J = 20.5 Hz, 2H).C 19 H 21 ESIMS of ClF3N5O9P2 [M-H] -, Calculated value 616.1, Actual Measurement: 616.2. [Example 105] Synthesis of [({[(2R,3S,4R,5R)-5-(6-chloro-4-{[(3S)-oxolan-3-yl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0330] The title compound was synthesized in the same manner as in Example 87. 1 H NMR (400 MHz, DMSO-d6) δ 8.95(d, J = 6.4 Hz, 1H), 8.26 (s, 1H), 6.01(d, J = 4.2 Hz, 1H), 4.69 - 4.59 (m, 1 H), 4.50 (t, J = 4.2 Hz, 1H), 4.26 (t, J =4.5 Hz, 1H), 4.15 - 3.99 (m, 2H), 3.9 5 - 3.81 (m, 3H), 3.74 (d, J = 7.9 Hz, 1H),3.67 - 3.58 (m, 1H), 2.35 - 2.06 (m, 3H), 1.98 - 1.80 (m, 1H). C 15 H 22 ClNO 10 ESIMS of P2 [MH] - , Calculated value 528.1, Measured value 528.2. [Example 106] Synthesis of [({[(2R,3S,4R,5R)-5-[6-chloro-4-(cyclopentylamino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-3,4-dihydroxyoxolan-2-yl]methoxy}({[(propan-2-yloxy)carbonyl]oxy}methoxy)phosphoryl)methyl]({[(propan-2-yloxy)carbonyl]oxy}methoxy)phosphinic acid [ka]

[0331] The title compound was synthesized in the same manner as in Example 69. 1 H NMR (400 MHz, DMSO-d6) δ 8.71- 8.60 (m, 1H), 8.26 - 8.15 (m, 1H), 6.02- 5.96 (m, 1H), 5.60 - 5.38 (m, 5H), 4.87 - 4.68 (m, 2H), 4.51 - 4.37 (m, 2H),4.33 - 3.79 (m, 5H), 2.74 - 2.53 (m,2H), 2.07 - 1.89 (m, 2H), 1.79 - 1.42 (m,7H), 1.27 - 1.12 (m, 12H).C 26 H 40 ClNO 15 ESI MS of P2 [M−H] - , Calculated value 758.2, Measured value 758.3. [Example 107] Synthesis of [({[(2R,3S,4R,5R)-5-(6-chloro-4-{[(1S)-1-phenylethyl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}({[(propan-2-yloxy)carbonyl]oxy}-methoxy)phosphoryl)methyl]({[(propan-2-yloxy)carbonyl]oxy}methoxy)phosphinic acid [ka]

[0332] The title compound was synthesized in the same manner as in Example 69. 1 H NMR (400 MHz, DMSO-d6) δ 9.17(d, J = 7.9 Hz, 1H), 8.32 - 8.27 (m, 1H),7.42 - 7.28 (m, 4H), 7.26 - 7.20 (m, 1H), 6.01 (d, J = 3.7 Hz, 1H), 5.58 - 5.32(m, 6H), 4.84 - 4.69 (m, 2H), 4.49 - C 29 H 40 ClNO 15 ESIMS of P2 [MH] - , Calculated value 794.2, Measured value 7 94.2. [Example 108] Synthesis of [({[(2R,3R,4S,5R)-5-[4-(benzylamino)-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid Growth [ka]

[0333] Step a: 4,6-Dichloro-1H-pyrazolo[3,4-d]pyrimidine (1.0 g, 5.3 mmol) was dissolved in anhydrous CHCN (10 mL) to give cyclopentylamine (478 mg, 5.6 mmol, 1.05 equiv) was added followed by TEA (779 μL, 5.6 mmol, 1.05 equiv). The mixture was stirred overnight at room temperature, then anhydrous Cs2CO3 (3.4 g, 10.6 mmol, 2 equiv) and bromide (2.2 g, 5.3 mmol) were added. The reaction mixture was stirred overnight at room temperature, then the solvent was evaporated. The crude residue was dissolved in MeOH (20 mL) and anhydrous K2CO3 (2.2 g, 15.9 mmol, 3 equiv) was added. The mixture was stirred overnight at room temperature, evaporated, and purified by column chromatography (SiO2, He x→100% EtOAc) to give first product B (800 mg, 41%) and then product A (600 mg, 30%).15 H 20 ESIMS of ClFN5O3 [M+H] + , Calculated value 372.1, Measured value 372.2.

[0334] Step b: The phosphonylation step was carried out in a similar manner to Example 1 using product B from step a: 1 HNMR (400 MHz, DMSO-d6)δ 8.76 (d, J = 7.2 Hz, 1H), 8.29 (s, 1H), 6.52 (d, J = 6.5 Hz, 1H), 5.50 - 5.29 (m,1H), 4.75 (dt, J = 18.7, 7.5 Hz, 1H), 4.43 (h, J = 6.9 Hz, 1H), 4.31 - 4.22 (m,1H), 4.18 - 4.05 (m, 1H), 4.04 - 3.92 (m, 1H), 2.20 (t, J = 20.5 Hz, 2H), 2.05 -1.93 (m, 2H), 1.80 - 1.46 (m, 6H). C 16 H 24 ESI MS [M+H] of ClFN5O8P2 + , Calculated value 530.1, Measured value 530.2. [Example 109] Synthesis of [({[(2R,3R,4S,5R)-5-(6-chloro-4-{[(1S)-1-phenylethyl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0335] The title compound was synthesized in the same manner as in Example 108. 1H NMR (400 MHz, DMSO-d6) δ 9.19(d, J = 8.1 Hz, 1H), 8.33 (s, 1H), 7.40(d, J = 7.9 Hz, 2H), 7.33 (t, J = 7. 5 Hz, 2H), 7.23 (t, J = 7.4 Hz, 1H), ),6.34 (dd, J = 14.3, 4.6 Hz, 1H), 5.39 (b s, 1H), 5.31 - 5.12 (m, 1H), 5.14 (bs, 1H),4.48 (dt, J = 18.5, 4.5 Hz, 1H), 4.1 7 (s, 3H), 4.01 (d, J = 5.2 Hz, 2H), 2.24(t, J = 20.4 Hz, 3H), 1.51 (d, J = 7.0 Hz, 3H). C 19 H 24 ESIMS of ClFN5O8P2 [M+H] + , Calculated value 566.1, Measured value 566.1. [Example 110] Synthesis of [({[(2R,3R,4S,5R)-5-(6-chloro-4-{[(1R)-1-phenylethyl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0336] The title compound was synthesized in the same manner as in Example 108. 1 H NMR (400 MHz, DMSO-d6) δ 9.18(d, J = 8.0 Hz, 1H), 8.33 (d, J = 1.2Hz, 1H), 7.40 (d, J = 7.9 Hz, 2H), 7.3 3 (t, J = 7.3 Hz, 2H), 7.24 (t, J = 7.6 Hz,1H), 6.50 (d, J = 6.5 Hz, 1H), 5.51 - 5.23 (m, 2H), 4.82 - 4.66 (m, 1H), 4.22(bs, 1H), 4.13 - 4.02 (m, 1H), 3.94 (b s, 1H), 2.17 (t, J = 20.5 Hz, 2H), 1.53 (d,J = 7.1 Hz, 3H).C 19 H 24 ClFN5O8P2 ESI MS [M+H] + , Calculated value 566.1, Measured value 566.2. [Example 111] Synthesis of [({[(2R,3R,4S,5R)-5-(6-chloro-4-{[(1S)-1-(2-fluorophenyl)ethyl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0337] The title compound was synthesized in the same manner as in Example 108. 1 H NMR (400 MHz, DMSO-d6) δ 9.27(d, J = 7.6 Hz, 1H), 8.36 (s, 1H), 7.48- 7.40 (m, 1H), 7.37 - 7.25 (m, 1H), 7.22 - 7.13 (m, 2H), 6.51 (d, J = 6.6 Hz,1H), 5.59 (p, J = 7.1 Hz, 1H), 5.49 - 5.26 (m, 1H), 4.74 (dt, J = 18.4, 7.6 Hz,1H), 4.30 - 4.17 (m, 1H), 4.15 - 4.02 (m, 1H), 3.99 - 3.90 (m, 1H), 2.17 (t, J =20.5 Hz, 2H), 1.54 (d, J = 7.0 Hz,3H). C 19 H 23ESIMS of ClF2N5O8P2 [M+H] + , Calculated value 584.1, Measured value 584.2. [Example 112] Synthesis of [({[(2R,3R,4S,5R)-5-(6-chloro-4-{[(1R)-1-(2-fluorophenyl)ethyl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0338] The title compound was synthesized in the same manner as in Example 108. 1 H NMR (400 MHz, DMSO-d6) δ 9.37- 9.16 (m, 1H), 8.36 (d, J = 3.4 Hz,1H), 7.44 (d, J = 8.4 Hz, 1H), 7.29 (t, J = 7.0 Hz, 1H), 7.18 (dt, J = 10.6, 5.6Hz, 2H), 6.51 (t, J = 4.8 Hz, 1H),5.60 (t, J = 6.8 Hz, 1H), 5.53 - 5.22 (m, 1H),4.84 - 4.64 (m, 1H), 4.31 - 4.16(m, C 19 H 23 ESIMS of ClF2N5O8P2 [M+H] + , Calculated value 584.1, Actual measurement Value 584.2. [Example 113] Synthesis of [({[(2R,3R,4S,5R)-5-(6-chloro-4-{[(1S)-1-(3-fluorophenyl)ethyl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0339] The title compound was synthesized in the same manner as in Example 108. 1 H NMR (400 MHz, DMSO-d6) δ 9.21(d, J = 7.8 Hz, 1H), 8.33 (s, 1H), 7.46- 7.30 (m, 1H), 7.27 - 7.16 (m, 2H), 7.12 - 7.00 (m, 1H), 6.51 (d, J = 6.5 Hz,1H), 5.49 - 5.26 (m, 2H), 4.74 (dt, J = 18.4, 7.6 Hz, 1H), 4.29 - 4.18 (m, 1H),4.14 - 4.02 (m, 1H), 4.00 - 3.89 (m,1H), 2.17 (t, J = 20.5 Hz, 2H), 1.54 (d, J= 7.0 Hz, 3H).C 19 H 23 E of ClF2N5O8P2 SI MS [M+H] + , Calculated value 584.1, Measured value 584.2. [Example 114] Synthesis of [({[(2R,3R,4S,5R)-5-(6-chloro-4-{[(1R)-1-(2-fluorophenyl)ethyl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0340] The title compound was synthesized in the same manner as in Example 108. 1 H NMR (400 MHz, DMSO-d6) δ 9.21(d, J = 8.0 Hz, 1H), 8.34 (s, 1H), 7.42- 7.32 (m, 1H), 7.27 - 7.20 (m, 2H), 7.12 - 7.03 (m, 1H), 6.51 (d, J = 6.5 Hz,1H), 5.49 - 5.29 (m, 2H), 4.74 (dt, J = 18.8, 7.8 Hz, 1H), 4.29 - 4.18 (m, 1H),4.15 - 4.03 (m, 1H), 3.99 - 3.90 (m,1H), 2.17 (t, J = 20.5 Hz, 2H), 1.54 (d, J= 7.0 Hz, 3H).C 19 H 23 E of ClF2N5O8P2 SI MS [M+H] + , Calculated value 584.1, Actual value 584.2. [Example 115] Synthesis of [({[(2R,3R,4S,5R)-5-(6-chloro-4-{[(1S)-1-(4-fluorophenyl)ethyl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0341] The title compound was synthesized in the same manner as in Example 108. 1 H NMR (400 MHz, DMSO-d6) δ 9.22(d, J = 7.9 Hz, 1H), 8.34 (s, 1H), 7.45(dd, J = 8.7, 5.6 Hz, 2H), 7.17 (t, J = 8.9 Hz, 2H), 6.52 (d, J = 6.5 Hz, 1H),5.53 - 5.27 (m, 2H), 4.75 (dt, J = 18 .7, 7.6 Hz, 1H), 4.31 - 4.20 (m, 1H), 4.17- 4.04 (m, 1H), 3.97 (dd, J = 7.5, 3. C 19 H 23 ClF2N5O8P ESIMS of 2 [M+H] + , Calculated value 584.1, Measured value 584.2. [Example 116] Synthesis of [({[(2R,3R,4S,5R)-5-(6-chloro-4-{[(1R)-1-(4-fluorophenyl)ethyl]amino}-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0342] The title compound was synthesized in the same manner as in Example 108. 1 H NMR (400 MHz, DMSO-d6) δ 9.21(d, J = 7.9 Hz, 1H), 8.34 (s, 1H), 7.54- 7.37 (m, 2H), 7.17 (t, J = 8.8 Hz, 2H), 6.52 (d, J = 6.6 Hz, 1H), 5.60 - 5.23(m, 2H), 4.76 (dt, J = 18.7, 7.6 Hz, 1H), 4.24 (dt, J = 7.4, 4.8 Hz, 1H), 4.09(dt, J = 10.9, 7.4 Hz, 1H), 3.96 (dt, C 19 H 23 ESIMS of ClF2N5O8P2 [M+H] + , Calculated value 584.1, Measured value 584.2. [Example 117] Synthesis of [({[(2R,3S,4R,5R)-5-[5-chloro-7-(cyclopentylamino)-3H-imidazo[4,5-b]pyridin-3-yl]-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0343] Step a: To a solution of 5,7-dichloroimidazo[4,5-b]pyridine (376 mg, 2 mmol) in MeCN (14 mL) at room temperature, N,O-bis(trimethylsilyl)acetamide (0.523 mL, 2.14 mmol) was added dropwise, and the reaction mixture was heated at 85° C. for 1 hour. The mixture was cooled to room temperature, and a solution of beta-D-ribofuranose 1,2,3,5-tetraacetate (726 mg, 2.28 mmol) and trimethylsilyl trifluoromethanesulfonate (0.471 mL, 2.60 mmol) in MeCN (7 mL) was added dropwise, successively. The reaction mixture was heated at 85° C. for 4 hours. The mixture was cooled, saturated aqueous sodium bicarbonate (50 mL) was added, followed by extraction three times with EtOAc (100 mL), drying over sodium sulfate, and concentration.

[0344] Step b: To the residue was added dioxane (2 mL) and cyclopentylamine (0.987 mL, 10 mmol). The mixture was heated at 100° C. for 16 h. The reaction mixture was loaded onto silica gel and purified by silica gel chromatography (0-10% MeOH in DCM) to give the desired product as a brown solid (298 mg, 40%).

[0345] Step c: The title compound was synthesized in a similar manner to Example 1 as a white solid (10 mg; 6% J). 1 HNMR (400 MHz, DMSO-d6)δ 8.40 (s, 1H), 7.12 (d, J = 7.3 Hz, 1H), 6 .40 (s, 1H), 5.91 (d, J = 5.8 Hz, 1H), 4.53(t, J = 5.4 Hz, 1H), 4.23 - 4.18 (m, C 17 H 24 ESIMS of ClN4O9P2 [M-H] - , calculated value 525. 1, Actual value 525.2. [Example 118] Synthesis of [({[(2R,3R,4S,5R)-5-[5-chloro-7-(cyclopentylamino)-3H-imidazo[4,5-b]pyridin-3-yl]-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0346] Step a: To a solution of 5,7-dichloroimidazo[4,5-b]pyridine (564 mg, 3 mmol) in MeCN (18 mL) at room temperature was added sodium hydride (130 mg, 3.24 mmol, 60% suspension in oil). The reaction mixture was stirred at room temperature for 30 minutes. A solution of 2,3,5-tri-O-benzoyl-D-ribofuranosyl bromide in MeCN (4 mL) was added at room temperature, and the reaction mixture was stirred at room temperature for 14 hours. The reaction mixture was quenched by the addition of methanol (5 mL) and sodium bicarbonate (5 g), filtered through Celite, and concentrated.

[0347] Step b: 1) To the residue was added dioxane (5 mL) and cyclopentylamine (1.48 mL, 15 mmol). The mixture was heated at 100° C. for 20 hours. The reaction mixture was cooled to room temperature.

[0348] 2) Potassium carbonate (4 g) and methanol (20 mL) were added at room temperature, and the reaction mixture was stirred at room temperature for 1 hour. Excess solvent was removed in vacuo, and the crude residue was purified by silica gel chromatography (0-15% MeOH in DCM) to give the desired product as a brown solid (499 mg, 45%).

[0349] Step c: In a similar manner to Example 1, the title compound was synthesized as a white solid (26 mg; 10%). 1 HNMR (400 MHz, DMSO-d6)δ 8.24 (d, J = 2.3 Hz, 1H), 7.19 (br s, 1 H), 6.42 (dd, J = 15.4, 4.4 Hz, 1H), 6.42(s, 1H), 5.23 (dt, J = 52.4, 4.1 Hz, 1 H), 4.58 - 4.44 (m, 1H), 4.19 (t, J = 6.1Hz, 2H), 4.08 - 3.99 (m, 1H), 2.27 (t, C 17 H 23 ESIMS of ClFN4O8P2 [M-H] - , Calculated value 527.1, Measured value 527.2. [Example 119] Synthesis of [({[(2R,3S,4R,5R)-5-[6-chloro-4-(cyclopentylamino)-1H-pyrazolo[3,4-b]pyridin-1-yl]-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]-phosphonic acid [ka]

[0350] Step a: Ethyl (ethoxymethylene)cyanoacetate (50.5 g, 299.0 mmol) was dissolved in absolute EtOH (350 mL), and then the product hydrazine (50 g, 328.9 mmol, 1.1 equiv.) was added. The reaction mixture was stirred under reflux overnight, and then the solvent was evaporated. The solid residue was washed with MTBE to give a white solid (55.5 g, 63%). 14 H 18 ESI MS of N3O3 [M+H] + ,Calculated value 276.1, Measured value 276.2.

[0351] Step b: Diethyl malonate (90 mL, 0.59 mol, 4 eq.) was dissolved in absolute EtOH (300 mL) and cooled to 0 °C (ice bath). A 21% solution of NaOEt in EtOH (220 mL, 0.59 mol, 4 eq.) was added dropwise (within 10 min), then the cooling bath was removed and the reaction was stirred at room temperature for 15 min. The solid product from step a (40.4 g, 147 mmol) was added in small portions (within 2 min), and the reaction mixture was stirred under reflux for 5 days, then the solvent was evaporated. The residue was diluted with HO (1.2 L) and neutralized to pH ∼5 with AcOH. The product was filtered off, washed with HO (200 mL) and dried under vacuum (48.4 g, 96%). C 17 H 18 ESI MS of N3O5 [M+H] + ,Calculated value 344.1, Measured value 344.2.

[0352] Step c: The product from step b (48.4 g, 141.1 mmol) was dissolved in 15% aqueous NaOH (500 mL) and stirred under reflux for 5 hours. The solution was cooled to 0° C. and the pH was adjusted to approximately 5. The white solid was filtered off and washed with HO (100 mL). , and dried under vacuum (38 g, quantitative). 14 H 14 ESIMS of N3O3 [M+H] + , calculated value 272.1, Actual value: 272.2.

[0353] Step d: A mixture of the product from step c (38 g, 140.2 mmol) and phenylphosphonic acid dichloride (79.5 mL, 560.8 mmol, 4 equiv.) was stirred at 170° C. for 7 h, then cooled to approximately 80° C. and poured onto vigorously stirred ice. A brown gum precipitated and turned to a solid upon extensive stirring. The ice-cooled mixture was diluted with concentrated aqueous NH The mixture was neutralized with HCl and the product was extracted with CH2Cl2 (2 x 400 mL). The combined organic layers were dried over MgSO4, filtered, and the solvent was evaporated to give the product, which was further purified. Used without further preparation (24g, 55%). 14 H 12 ESIMS of Cl2N3O [M+H] + , calculated value 308.0 , Actual measured value 308.1.

[0354] Step e: The product from step d (22 g, 71.4 mmol) was dissolved in TFA (75 mL) and stirred at 60 °C for 12 h, then cooled and poured into H2O (600 mL). The solid was filtered off, washed with saturated NaHCO3, then H2O, and dried under vacuum. ESI MS [M+H] + , Calculated value 188.0, Actual value 188.1.

[0355] Step f: The step f product was synthesized in a similar manner to Example 87. 1 H NMR (400 MHz, DMSO-d6) δ8.55 (s, 1H), 7.72 (s,1H), 6.48 (d, J = 3.0 Hz, 1H), 5.90 - 5.83 (m, 1H) , 5.67 - 5.61 (m, 1H), 4.46 - 4.38 (m, 1H),4.33 (ddd, J = 12.1, 3.5, 1.2 Hz, 1H ), 4.05 (ddd, J = 12.2, 5.1, 1.2 Hz, 1H),2.09 (s, 3H), 2.06 (s, 3H), 1.96 (s, 3 H).C 17 H 18 ESIMS of Cl2N3O7 [M+H] + , Calculated value 446.0, Measured value 446.1.

[0356] Step g: The step g product was synthesized in the same manner as in Example 87. 16 H 22 ESIMS of ClN4O4 [M+H] + , Calculated value 369.1, Measured value 369.2.

[0357] Step h: The title compound was synthesized in the same manner as in Example 87. 1 H NMR (400 MHz, DMSO-d6) δ8.27 (s, 1H), 7.66 (d, J = 6.7 Hz, 1H),6.22 (s, 1H), 6.08 (d, J = 4.2 Hz, 1H), 4.51 (t, J = 4.7 Hz, 1H), 4.26 (t, J =5.1 Hz, 1H), 4.17 - 3.83 (m, 4H), 2. 17 (t, J = 20.5 Hz, 2H), 2.06 - 1.92 (m,2H), 1.77 - 1.45 (m, 6H). C 17 H 26 ClNO ESI MS of 9P2 [M+H] + , Calculated value 527.1, Measured value 527.2. [Example 120] Synthesis of [({[(2R,3S,4R,5R)-5-(6-chloro-4-{[(1S)-1-phenylethyl]amino}-1H-pyrazolo[3,4-b]pyridin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]-phosphonic acid [ka]

[0358] The title compound was synthesized in the same manner as in Example 119. 1 H NMR (400 MHz, DMSO-d6) δ 8.38(s, 1H), 8.20 (d, J = 7.2 Hz, 1H), 7.42- 7.36 (m, 2H), 7.35 - 7.27 (m, 2H), 7.24 - 7.18 (m, 1H), 6.08 - 5.97 (m,2H),4.85 (s, 1H), 4.50 (t, J = 4.5 Hz, 1H), 4.25 (t, J = 4.8 Hz, 1H), 4.14 - 3.97(m,2H), 3.93 - 3.81 (m, 1H), 2.17 (t, J = 20.5 Hz, 2H), 1.52 (d, J = 6.2 Hz, 3H).C 20 H 26 ESIMS of ClN4O9P2 [M+H] + , total Calculated value 563.1, measured value 563.2. [Example 121] Synthesis of [({[(2R,3S,4R,5R)-5-(6-chloro-4-{[(1R)-1-phenylethyl]amino}-1H-pyrazolo[3,4-b]pyridin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]-phosphonic acid [ka]

[0359] The title compound was synthesized in the same manner as in Example 119. 1 H NMR (400 MHz, DMSO-d6) δ 8.38(s, 1H), 8.20 (d, J = 7.2 Hz, 1H), 7.44- 7.35 (m, 2H), 7.35 - 7.28 (m, 2H), 7.25 - 7.17 (m, 1H), 6.12 - 5.93 (m, 2H),4.85 (s, 1H), 4.57 - 4.48 (m, 1H),4.25 (t, J = 4.9 Hz, 1H), 4.12 - 3.95 (m,2H), 3.91 - 3.79 (m, 1H), 2.17 (t, J =2 0.5 Hz, 2H), 1.51 (d, J = 6.6 Hz, 3H).C 20 H 26 ESIMS of ClN4O9P2 [M+H] + , calculated value 5 63.1, actual value 563.2. [Example 122] Synthesis of [({[(2R,3S,4R,5R)-5-(6-chloro-4-{[(1S)-1-(2-fluorophenyl)ethyl]amino}-1H-pyrazolo[3,4-b]pyridin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0360] The title compound was synthesized in the same manner as in Example 119. 1 H NMR (400 MHz, DMSO-d6) δ 8.38(s, 1H), 8.23 ​​(d, J = 6.9 Hz, 1H), 7.42- 7.34 (m, 1H), 7.33 - 7.09 (m, 3H), 6.06 (d, J = 4.3 Hz, 1H), 5.97 (s, 1H),5.04 (s, 1H), 4.53 - 4.47 (m, 1H), 4.25 C 20 H 25 ESIMS of ClFN4O9P2 [M+H] +, calculated value 58 1.1, actual value 581.2. [Example 123] [({[(2R,3S,4R,5R)-5-(6-chloro-4-{[(1S)-1-( Synthesis of 4-fluorophenyl)ethyl]amino}-1H-pyrazolo[3,4-b]pyridin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0361] The title compound was synthesized in the same manner as in Example 119. 1 H NMR (400 MHz, DMSO-d6) δ 8.36(s, 1H), 8.18 (d, J = 7.2 Hz, 1H), 7.46- 7.39 (m, 2H), 7.19 - 7.10 (m, 2H), 6.13 - 5.99 (m, 2H), 4.89 (s, 1H), 4.53 -4.46 (m, 1H), 4.25 (t, J = 4.8 Hz,1H), 4.12 - 3.97 (m, 2H), 3.92 - 3.81 (m,1H), 2.18 (t, J = 20.5 Hz, 2H), 1.50(d, J = 7.3 Hz, 3H). C 20 H 25 ESIMS of ClFN4O9P2 [M+H] + Calculated value: 581.1, Measured value: 581.2. [Example 124] Synthesis of [({[(2R,3R,4S,5R)-5-[6-chloro-4-(cyclopentylamino)-1H-pyrazolo[3,4-b]pyridin-1-yl]-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0362] Step a: 4,6-dichloro-1H-pyrazolo[3,4 To a mixture of [-b]pyridine (2.1 g, 11.1 mmol) and bromide (4.7 g, 11.1 mmol) was added CsCO (4.3 g, 13.3 mmol, 1.2 equiv.), and the reaction mixture was stirred at room temperature overnight. Evaporation onto silica gel and purification by column chromatography (SiO, Hex → Hex: EtOAc, 2:8) gave a white solid (2.5 g). , 42%). 1 H NMR (400 MHz, DMSO-d6) δ 8.53 (s, 1H), 8.05(d, J = 8.4Hz , 2H), 7.91 (d, J = 8.4 Hz, 2H), 7.75 -7.65(m, 2H), 7.65 - 7.51 (m, 3H), 7.49 - 7.41 (m, 2H), 6.96 (d, J = 6.6 Hz, 1H),6.45 (dt, J = 15.8, 7.2 Hz, 1H), 6.19 - 5.97 (m, 1H), 4.78 - 4.53 (m, 3H).C 25 H 19 ESIMS of Cl2FN3O5 [M+H] + , calculated value 53 0.1, actual value 530.2.

[0363] Step b: A mixture of the product from step a (500 mg, 0.94 mmol), cyclopentylamine (84 mg, 0.99 mmol, 1.05 equiv.), and TEA (138 μL, 0.99 mmol, 1.05 equiv.) in absolute EtOH (5 mL) was placed in a pressure vial and heated at 110 °C for 2 days. After cooling to room temperature, KCO (262 mg, 1.9 mmol, 2 equiv.) was added and the reaction mixture was stirred overnight. Evaporation onto silica gel and purification by column chromatography (SiO, Hex → 100% EtOAc) gave a white solid (170 mg, 49 %) was obtained. 1HNMR (400 MHz, DMSO-d6) δ 8.28 (s, 1H), 7.66 (d, J= 6.9 Hz, 1H), 6.53 (d, J = 6.6 Hz, 1H), 6.23 (s, 1H),5.80 (d, J = 5.7 Hz, 1H), 5.45 - 5.23(m, 1H), 4.82 - 4.61 (m, 2H), 3.98 (s, 1H),3.80 - 3.50 (m, 3H), 2.09 - 1.89 (m, 2H), 1.76 - 1.46 (m, 6H). C 16 H 21 ESIMS of ClFN4O3 [M+H] + , Calculated value 371.1, Measured value 371.3.

[0364] Step c: The title compound was synthesized in the same manner as in Example 1. 1 H NMR (400 MHz, DMSO-d6) δ8.30 (s, 1H), 7.69 (d, J = 6.7 Hz, 1H),6.55 (d, J = 6.6 Hz, 1H), 6.23 (s, 1H) ), 5.48 - 5.25 (m, 1H), 4.77 (dt, J = 18.1,7.6 Hz, 1H), 4.28 - 4.18 (m, 1H), 4. 13 - 3.88 (m, 3H), 2.17 (t, J = 20.5 Hz,2H), 2.07 - 1.93 (m, 2H), 1.77 - 1.44 ( m, 6H). C 17 H 25 ESIMS of ClFN4O8P2 [M+H] + , Calculated value 529.1, Measured value 529.1. [Example 125] Synthesis of [({[(2R,3R,4S,5R)-5-(6-chloro-4-{[(1S)-1-phenylethyl]amino}-1H-pyrazolo[3,4-b]pyridin-1-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0365] The title compound was synthesized in the same manner as in Example 124. 1 H NMR (400 MHz, DMSO-d6) δ 8.40(s, 1H), 8.23 ​​(d, J = 7.1 Hz, 1H), 7.44- 7.37 (m, 2H), 7.37 - 7.28 (m, 2H), 7.26 - 7.16 (m, 1H), 6.54 (d, J = 6.5 Hz,1H), 6.04 (s, 1H), 5.36 (dt, J =53.5, 7.1 Hz, 1H), 4.93 - 4.67 (m, 2H), 4.27 -4.19 (m, 1H), 4.14 - 4.02 (m,1H), 3. 98 - 3.85 (m, 1H), 2.17 (t, J = 20.5 Hz,2H), 1.52 (d, J = 6.4 Hz, 3H). C 20 H 25 ESIMS of ClFN4O8P2 [M+H] + , Calculated value 565.1, Measured value 565.2. [Example 126] Synthesis of [({[(2R,3R,4S,5R)-5-(6-chloro-4-{[(1R)-1-phenylethyl]amino}-1H-pyrazolo[3,4-b]pyridin-1-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0366] The title compound was synthesized in the same manner as in Example 124. 1H NMR (400 MHz, DMSO-d6) δ 8.41(s, 1H), 8.22 (d, J = 7.1 Hz, 1H), 7.43- 7.36 (m, 2H), 7.36 - 7.27 (m, 2H), 7.26 - 7.17 (m, 1H), 6.54 (d, J = 6.5 Hz,1H), 6.04 (s, 1H), 5.37 (dt, J =53.7, 7.2 Hz, 1H), 4.96 - 4.67 (m, 2H), 4.27 -4.18 (m, 1H), 4.13 - 4.01 (m,1H), 3. 97 - 3.87 (m, 1H), 2.18 (t, J = 20.5 Hz,2H), 1.52 (d, J = 6.4 Hz, 3H). C 20 H 25 ESIMS of ClFN4O8P2 [M+H] + , Calculated value 565.1, Measured value 565.2. [Example 127] Synthesis of [({[(2R,3S,4R,5R)-5-(2-chloro-4-{[(1S)-1-(2-fluorophenyl)ethyl]amino}-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0367] Step a: 2,4-dichloro-7H-pyrrolo[2,3 To a mixture of [-d]pyrimidine (14.8 g, 78.9 mmol) and bromide (40 g, 118.3 mmol, 1.5 equiv.) was added CsCO (38.6 g, 118.3 mmol, 1.5 equiv.), and the reaction mixture was stirred at room temperature overnight. Evaporation onto silica gel and purification by column chromatography (SiO, Hex → Hex: EtOAc, 2:8) afforded a white solid. A solid (13.8 g, 39%) of C 17 H 18ESIMS of Cl2N3O7 [M+H] + , Calculated value 446.0, Actual value: 446.1.

[0368] Steps b and c were carried out in the same manner as in Example 1. 1 H NMR (400 MHz, DMSO-d6) δ8.38 (d, J = 7.9 Hz, 1H), 7.47 - 7.36 (m,2H), 7.32 - 7.23 (m, 1H), 7.20 - 7. 09 (m, 2H), 6.81 (s, 1H), 5.96 (d, J = 6.0Hz, 1H), 5.58 (t, J = 7.3 Hz, 1H), 4. 29 (t, J = 5.7 Hz, 1H), 4.14 - 3.96 (m,4H), 2.24 (t, J = 20.5 Hz, 2H), 1.51 (d, J = 6.9 Hz, 3H). C 20 H 25 ESIMS of ClFN4O9P2 [M+H] + , Calculated value 581.1, Measured value 581.2 . [Example 128] Synthesis of [({[(2R,3S,4R,5R)-5-(2-chloro-4-{[(1S)-1-(3-fluorophenyl)ethyl]amino}-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0369] The title compound was synthesized in the same manner as in Example 127. 1 H NMR (400 MHz, DMSO-d6) δ 8.34(d, J = 8.1 Hz, 1H), 7.42 - 7.30 (m,2H), 7.26 - 7.16 (m, 2H), 7.08 - 6.98 ( m, 1H), 6.77 (s, 1H), 5.97 (d, J = 6.0 Hz,1H), 5.45 - 5.33 (m, 1H), 4.29 (t, J = 5.5 Hz, 1H), 4.14 - 3.98 (m, 4H), 2.24(d, J = 20.5 Hz, 2H), 1.51 (d, J = 6.8 Hz, 3H). C 20 H 25 ESIMS of ClFN4O9P2 [M+H] + , Calculated value 581.1, Measured value 581.2. [Example 129] Synthesis of [({[(2R,3S,4R,5R)-5-(2-chloro-4-{[(1S)-1-(4-fluorophenyl)ethyl]amino}-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0370] The title compound was synthesized in the same manner as in Example 127. 1 H NMR (400 MHz, DMSO-d6) δ 8.32(d, J = 8.0 Hz, 1H), 7.46 - 7.34 (m,3H), 7.17 - 7.08 (m, 2H), 6.76 (s, 1H), 5.97 (d, J = 6.3 Hz, 1H), 5.44 - 5.33 (m,1H), 4.29 (t, J = 5.8 Hz, 1H), 4.14 - 3.97 (m, 4H), 2.24 (d, J = 20.5 Hz, 2H),1.50 (d, J = 6.9 Hz, 3H).C 20 H 25 ClFN ESIMS [M+H] of 4O9P2 + , Calculated value 581.1, Measured value 581.2. [Example 130] Synthesis of [({[(2R,3R,4S,5R)-5-(2-chloro-4-{[(1R)-1-phenylethyl]amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0371] The title compound was synthesized in the same manner as in Example 65. 1 H NMR (400 MHz, DMSO-d6) δ 8.36(d, J = 8.4 Hz, 1 H), 7.39 (d, J = 7.6Hz, 2 H), 7.35 - 7.23 (m, 3 H), 7.21 (t , J = 7.2 Hz, 1 H), 6.78 (s, 1 H), 6.44(dd, J = 15.5, 4.6 Hz, 1 H), 5.40 (t, J = 8.1 Hz, 1 H), 4.40 (dt, J = 18.8, 4.6 Hz,1 H), 4.13 (d, J = 6.7 Hz, 2 H), 3.9 5 (q, J = 5.1 Hz, 1 H), 2.22 (t, J = 20.3Hz, 2 H), 1.51 (d, J = 7.0 Hz, 3 H).C 209 H 23 ESI MS [M−H] of ClFN4O8P2 - , Calculated value 563.1, Measured value 563.2. [Example 131] Synthesis of [({[(2R,3R,4S,5R)-5-(2-chloro-4-{[(1S)-1-phenylethyl]amino}-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0372] The title compound was synthesized in the same manner as in Example 65. 1 H NMR (400 MHz, DMSO-d6) δ 8.37(d, J = 8.3 Hz, 1 H), 7.39 (d, J = 7.7Hz, 2 H), 7.31 (t, J = 7.5 Hz, 2 H), 7. 28 - 7.16 (m, 2 H), 6.79 (s, 1 H), 6.44(dd, J = 15.8, 4.5 Hz, 1 H), 5.39 (s, 1 H), 4.39 (dt, J = 18.7, 4.4 Hz, 1 H), 4.12(m, 2 H), 3.95 (q, J = 5.1 Hz, 1 H), 2.23 (t, J = 20.5 Hz, 2 H), 1.51 (d, J =7.0 Hz, 3 H).C 20 H 25 ESIMS of ClFN4O8P2 [M+H] + , Calculated value 565.1, Measured value 565.2. [Example 132] [({[(2R,3R,4S,5R)-5-(2-chloro-4-{[(1S)-1-(3-fluorophenyl)ethyl]amino}-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy Synthesis of [( ... [ka]

[0373] The title compound was synthesized in the same manner as in Example 65. 1 H NMR (400 MHz, DMSO-d6) δ 8.40(d, J = 8.0 Hz, 1 H), 7.40 - 7.16 (m, 4H), 7.03 (td, J = 8.7, 2.6 Hz, 1 H), 6 .78 (d, J = 3.9 Hz, 1 H), 6.45 (dd, J =15.7, 4.4 Hz, 1 H), 5.43 - 5.34 (m, 1 H) , 5.11 (dt, J = 52.7, 4.0 Hz, 1 H), 4.38(dq, J = 18.7, 4.5 Hz, 1 H), 4.18 - 4.0 6 (m, 1 H), 3.96 (q, J = 5.0 Hz, 1 H), 2.25(t, J = 20.5 Hz, 2 H), 1.51 (d, J = 6.9 Hz, 3 H). C 20 H 22 ESIMS of ClF2N4O8P2 [M-H] - , Calculated value 581.1, Measured value 581.2. [Example 133] Synthesis of [({[(2R,3R,4S,5R)-5-(2-chloro-4-{[(1R)-1-(4-fluorophenyl)ethyl]amino}-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0374] The title compound was synthesized in the same manner as in Example 65. 1 H NMR (400 MHz, DMSO-d6) δ 8.37(d, J = 8.1 Hz, 1 H), 7.42 (dd, J = 8.4,5.5 Hz, 2 H), 7.26 (t, J = 3.1 Hz, 1 H), 7.22 - 7.09 (m, 2 H), 6.77 (d, J = 3.6Hz, 1 H), 6.44 (dd, J = 15.5, 4.5 Hz, 1 H), 5.43 - 5.34 (m, 1 H), 5.14 (dt, J =52.7, 4.0 Hz, 1 H), 4.40 (dt, J =18.7, 4.3 Hz, 1 H), 4.20 - 4.02 (m, 2 H), 3.95(q, J = 5.1 Hz, 1 H), 2.24 (t, J= 2 0.5 Hz, 2 H), 1.50 (d, J = 7.0 Hz, 3 H).C 20 H 22 ESIMS of ClF2N4O8P2 [M-H] - , calculation Value 581.1, actual value 581.2. [Example 134] Synthesis of [({[(2R,3R,4S,5R)-5-(2-chloro-4-{[(1S)-1-(4-fluorophenyl)ethyl]amino}-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0375] The title compound was synthesized in the same manner as in Example 65. 1 H NMR (400 MHz, DMSO-d6) δ 8.38(d, J = 7.7 Hz, 1 H), 7.41 (dd, J = 8.6,5.5 Hz, 2 H), 7.26 (s, 1 H), 7.18 - 7 .08 (m, 2 H), 6.77 (s, 1 H), 6.44 (dd, J =15.6, 4.3 Hz, 1 H), 5.38 (s, 1 H), 5. 24 - 4.96 (m, 1 H), 4.45 - 4.34 (m, 1 H),4.13 (s, 2 H), 3.95 (d, J = 5.2 Hz, 1 H), 2.23 (t, J = 20.4 Hz, 2 H), 1.50 (d, J= 7.1 Hz, 3 H).C 20 H24 ClF2N4O8P2 ES I MS [M+H] + , Calculated value 583.1, Measured value 583.2. [Example 135] Synthesis of [({[(2R,3R,4S,5R)-5-(2-chloro-4-{[(1S)-1-(2-fluorophenyl)ethyl]amino}-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0376] The title compound was synthesized in the same manner as in Example 65. 1 H NMR (400 MHz, DMSO-d6) δ 8.46(d, J = 7.8 Hz, 1 H), 7.44 (t, J = 7.8Hz, 1 H), 7.28 (dt, J = 11.3, 4.5 Hz, 2 H), 7.17 (q, J = 8.1, 7.4 Hz, 2 H), 6.83(d, J = 3.9 Hz, 1 H), 6.47 (dd, J =15.7, 4.5 Hz, 1 H), 5.60 (t, J = 7.4 Hz, 1H), 5.13 (dt, J = 52.5, 4.2Hz, 1H),4 .41 (dt, J = 18.8, 4.4 Hz, 1 H), 4.14 (td,J = 12.0, 10.7, 5.7 Hz, 2 H), 3.97 (q , J = 5.0 Hz, 1 H), 2.27 (t, J = 20.5 Hz, 2H), 1.53 (d, J = 7.0 Hz, 3 H). 20 H 24 ESI MS [M+H] of ClF2N4O8P2 + , Calculated value 583.1, Measured value 583.2. [Example 136] Synthesis of [({[(2R,3R,4S,5R)-5-(2-chloro-4-{[(1R)-1-(2-fluorophenyl)ethyl]amino}-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0377] The title compound was synthesized in the same manner as in Example 65. 1 H NMR (400 MHz, DMSO-d6) δ 8.44(s, 1 H), 7.43 (t, J = 7.8 Hz, 1 H), 7.28(s, 2 H), 7.21 - 7.09 (m, 2 H), 6.44 (dd, J = 15.8, 4.7 Hz, 1 H), 5.59 (s, 1H), 5.14 (d, J = 52.8 Hz, 1 H), 4.40(d, J = 18.9 Hz, 1 H), 4.13 (s, 2 H), 3.95(d, J = 5.7 Hz, 1 H), 2.24 (t, J =20.7 Hz, 2 H), 1.51 (d, J = 6.9 Hz, 3 H).C 20 H 24 ESIMS of ClF2N4O8P2 [M+H] + , Calculated value 583.1, Measured value 583.2 [Example 137] Synthesis of [({[(2R,3R,4S,5R)-5-(2-chloro-4-{[(1R)-1-(3-fluorophenyl)ethyl]amino}-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0378] The title compound was synthesized in the same manner as in Example 65.1 H NMR (400 MHz, DMSO-d6) δ 8.39(d, J = 8.1 Hz, 1 H), 7.35 (td, J = 8.0,7.5, 6.0 Hz, 1 H), 7.31 - 7.17 (m, 3 H), 7.04 (td, J = 8.6, 2.5 Hz, 1 H), 6.81 -6.75 (m, 1 H), 6.45 (dd, J = 15.6, 4 .4 Hz, 1 H), 5.44 - 5.35 (m, 1 H), 5.14(dt, J = 52.8, 4.1 Hz, 1 H), 4.40 (dt, J = 18.8, 4.4 Hz, 1 H), 4.19 - 4.09 (m, 1H), 3.95 (q, J = 5.0 Hz, 1 H), 2.24 (t, J = 20.5 Hz, 2 H), 1.50 (d, J = 6.9 Hz, 3H).C 20 H 22 ESIMS of ClF2N4O8P2 [M-H] - , Calculated value 581.1, measured value 581.2. [Example 138] Synthesis of [({[(2R,3S,4R,5R)-5-[6-chloro-4-(cyclopentylamino)-1H-imidazo[4,5-c]pyridin-1-yl]-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0379] Step a: To a solution of β-D-ribofuranose-1,2,3,5-tetraacetate (4.07 g, 12.8 mmol) and 4,6-dichloro-1H-imidazo[4,5-c]pyridine (2.0 g, 10.6 mmol) in ACN (64 mL) was added TMS-OTf (4.6 mL, 25.6 mmol) via syringe. DBU (1.9 mL, 12.8 mmol) was then added dropwise, and the reaction was stirred at room temperature for 2 h. The reaction was cooled to 0 °C and poured into a cold saturated solution of NaHCO3. The mixture was transferred to a separatory funnel and extracted with DCM (3x). The combined organic layers were dried over MgSO4 and concentrated to dryness. The crude material (1.4 g) was further C 17 H 17 ESIMS of Cl2N3O7 [M+H] + , Calculated value 446.0, Measured value 446.1.

[0380] Step b: To a screw-top flask containing the crude dichloride (1.4 g) was added cyclopentylamine (7 mL). The vial was sealed and heated to 80 °C overnight. The reaction was cooled to room temperature and concentrated to dryness under reduced pressure. The crude product was reconstituted in DCM and purified by column chromatography (SiO, 0-15% MeOH / DCM) to give the desired product (352 mg). Got it. C 16 H 21 ESI MS of ClN4O4 [M+H] + ,Calculated value 368.1, Measured value 369.2.

[0381] Step C: The title compound was synthesized in the same manner as in Example 1. 1 H NMR (400 MHz, DMSO-d6) δ8.33 (s, 1H), 7.05 (s, 1H), 6.88 (s, 1H),5.77 (d, J = 5.9 Hz, 1H), 4.43 (br. C 17 H 25 ESIMS of ClN4O9P2 [M-H] - , Calculated value 525.1, Actual value 525.2. [Example 139] Synthesis of [({[(2R,3R,4S,5R)-5-[6-chloro-4-(cyclopentylamino)-1H-imidazo[4,5-c]pyridin-1-yl]-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0382] Step a: A solution of 4,6-dichloro-1H-imidazo[4,5-c]pyridine (2.0 g, 10.6 mmol) and 2-deoxy-2-fluoro-D-arabinofuranosyl bromide 3,5-dibenzoate (4.95 g, 11.7 mmol; CAS: 97614-44-3) in 50 mL of acetonitrile was treated with CsCO (4.16 g, 12.8 mmol). The mixture was stirred at room temperature for 3 hours, then diluted with ethyl acetate and washed with water and brine. The organic layer was dried over MgSO and concentrated under reduced pressure. The resulting crude product was further purified by HPLC. Used without further purification. 25 H 18 ESIMS of Cl2FN3O5 [M+H] + , Calculated value 530.1, Measured value 530.2.

[0383] Step b: To a screw-top flask containing the crude dichloride (3.5 g, 6.6 mmol) was added cyclopentylamine (18 mL). The vial was sealed and heated to 80 °C overnight. The reaction was cooled to room temperature and concentrated to dryness under reduced pressure. The crude product was reconstituted in DCM and purified by column chromatography (SiO2, 0-15% MeOH / DCM) to give the desired product. The product was obtained. 16 H 20 ESI MS of ClFN4O3 [M+H] + ,Calculated value 371.1, Measured value 371.2.

[0384] Step c: The title compound was synthesized in the same manner as in Example 1. 1 H NMR (400 MHz, DMSO-d6) δ8.22 (d, J = 1.8 Hz, 1H), 7.06 (s, 2H),6.98 - 6.82 (m, 1H), 6.37 (dd, J = 15 .9, 4.4 Hz, 1H), 5.21 (dt, J = 52.4, 3.8Hz, 1H), 4.53 - 4.33 (m, 2H), 4.21 (t, J = 5.8 Hz, 2H), 4.00 (q, J = 4.9 Hz, 1H),2.28 (t, J = 20.4 Hz, 2H), 1.93 (s, 2 H), 1.74 - 1.47 (m, 7H). C 17 H 24 ESIMS of ClFN4O8P2 [M-H] - , Calculated value 527.1, Measured value 5 27.2. [Example 140] Synthesis of [({[(2R,3R,4S,5R)-5-{6-chloro-4-[cyclopentyl(methyl)amino]-1H-imidazo[4,5-c]pyridin-1-yl}-4-fluoro-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)methyl]phosphonic acid [ka]

[0385] The title compound was synthesized in the same manner as in Example 139. 1 H NMR (400 MHz, DMSO-d6) δ 8.24(d, J = 1.8 Hz, 1H), 6.98 (d, J = 0.7Hz, 1H), 6.39 (dd, J = 15.4, 4.4 Hz, 1 H), 5.80 (p, J = 7.7 Hz, 1H), 5.41 - 5.03(m, 1H), 4.43 (ddd, J = 19.9, 5.4, 3.5 C 18 H 26 ClFN4O8P2のESIMS [MH] - , calculated value 5 41.1, measured value 541.2.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

[0386] Biological Examples Materials and Methods Where indicated, the following general materials and methods were used or can be used in the examples below:

[0387] Standard methods in molecular biology are described in the scientific literature (e.g., Sambrooka nd Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Pres. s, Cold Spring Harbor, NY, and Ausubel, et al. (2001)CurrentProtocols in Mole cular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY (which , cloning and DNA mutagenesis in bacterial cells (Volume 1), cloning in mammalian cells and yeast (Volume 2), expression of glycoconjugates and proteins (Volume 3), and bioinformatics (Volume 4).

[0388] These scientific publications describe methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, as well as chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and protein glycosylation (see, e.g., Coliganet al., (2000)Current Protocols in ProteinScience, Vol.1-2, John Wiley and Sons, Inc., NY).

[0389] For example, software packages and databases are available for determining antigen fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments (e.g., GCG Wisconsin Package (Accelrys, Inc., S San Diego, CA); and DeCypher® (TimeLogic Corp., Crystal Bay, NV) see).

[0390] This literature is replete with assays and other experimental techniques that can serve as a basis for the evaluation of the compounds described herein.

[0391] Inhibition of ecto-5'-nucleotidase (CD73) activity Compounds were evaluated to measure ecto-5'-nucleotidase (CD73) inhibitory activity. Briefly, CHO-K1 cells stably transfected with human CD73 were cultured in a 500-well platelet-free medium (LakePharma, Belmont, CA). A) was generated using molecular cloning of human CD73 (http: / / www.uniprot.org / uniprot / P21589) and a mammalian transient expression vector (P21589.1). After antibiotic selection in CD OptiCHO cell medium (Invitrogen, catalog no. 12681-011) containing 5 μg / mL puromycin and 200 μg / mL hygromycin B, a suspension pool of CHO-CD73 cells was collected and frozen in 7.5% DMSO in antibiotic-free cell medium.

[0392] On the day of the experiment, thaw one vial of CHO-CD73 cells and resuspend them in 20 mM HEPES, pH 7.4, 137 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl, The cells were suspended in assay medium consisting of 4.2 mM NaHCO3 and 0.1% glucose. To test the ability of compounds to inhibit CD73 enzymatic activity, 2 μL of 500 μM compound dissolved in DMSO (50×) was added to a 96-well polystyrene plate containing 58 μL of assay buffer. 20 μL of CHO-CD73 cells in assay buffer was then added to the assay plate, followed by 20 μL of 125 μM AMP (adenosine 5'-monophosphate monohydrate) in assay buffer. Final assay conditions consisted of 2500 cells per well in 2% DMSO and 25 μM AMP substrate. After 50 minutes of incubation (37°C and 5% CO2) and centrifugation at 225 × g for 5 minutes, 8 cells were cultured. 0 μL of the supernatant was transferred to a 96-well Spectra plate (PerkinElmer, Cat. No. 6005640) pre-dispensed with 20 μL of PiColorLock Gold colorimetric assay reagent (Thermo, Cat. No. 30 300 30). The amount of inorganic phosphate was determined by reading the absorbance at 620 nm using an EnVision Multilabel plate reader (PerkinElmer). The enzymatic activity of CD73 was measured by The activity was based on the amount of phosphate produced. Percent activity was calculated based on DMSO and no cell control wells. Compound IC 50 Values ​​are calculated using GraphPadPrism software. The percent activity in the serotonin-dependent agonist was determined by four-parameter nonlinear regression fitting.

[0393] Pharmacodynamic and pharmacokinetic evaluation Pharmacodynamic assays can be based on measuring CD73-mediated serum levels of adenosine. Adenosine levels can be determined by HPLC analysis, and serum compound levels can sometimes be determined in the same HPLC experiment. It can be determined.

[0394] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Upon reading the foregoing, it is expected that variations of the disclosed embodiments may become apparent to those skilled in the art, who will be able to employ such variations as they see fit. Indeed, it is intended that the invention may be practiced otherwise than as specifically described herein, and the invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, the invention includes any combination of the above-described elements in all possible variations thereof unless otherwise indicated herein or clearly contradicted by context.

[0395] All publications, patent applications, accession numbers, and other references cited herein are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0396] According to a preferred embodiment of the present invention, for example, the following is provided: (Section 1) formula: [ka] or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein Each R 1 is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted aryl -C(R 2 R 2 )-OC(O)-OR 3 are independently selected from the group consisting of , or two R 1 groups optionally joined to form a 5- to 7-membered ring; Each R 2 are independently selected from the group consisting of H and optionally substituted C1-C6 alkyl be; Each R 3 is selected from the group consisting of H, C1-C6 alkyl, and optionally substituted aryl Independently selected; R 5 is selected from the group consisting of H and optionally substituted C1-C6 alkyl; X is O; A is, [ka] selected from the group consisting of: Het is [ka] selected from the group consisting of where the wavy line indicates the point of attachment to the remainder of the compound, and where: R a are H, NH2, and NHR 7 , NHC(O)R 7 , N.R. 7 R 7 , R 7 , O.H., S.R. 7 , and OR 7 selected from the group consisting of: R b is H, halogen, NH2, NHR 7 , N.R. 7 R 7 , R 7 , OH, and OR 7 A group consisting of Selected from; R c and R d is H, halogen, haloalkyl, NH2, NHR 7 , N.R. 7 R 7 , R 7 , O.H. , OR 7 , S.R. 7 , SO2R 7 , -X 1 -NH2, -X 1 -NHR 7 , -X 1 -NR 7 R7 , -X 1 -OH, -X 1 -OR 7 , -X 1 -SR 7 , and -X 1 -SO2R 7 Independently selected from the group consisting of be; R e and R f are independently selected from the group consisting of H, halogen, and optionally substituted C1-C6 alkyl; each X 1 is a C1-C4 alkylene; and Each R 7 is an optionally substituted C1-C 10 Alkyl, optionally substituted C-C 10 Alkenyl, optionally substituted C-C 10 alkynyl, optionally substituted C3-C7 cycloalkyl, optionally substituted C3-C7 cycloalkylC1-C4 alkyl, optionally substituted 4-7 membered cycloheteroalkyl, optionally substituted 4-7 membered cycloheteroalkyl C1-C4 alkyl, optionally substituted aryl, optionally substituted aryl C1-C4 alkyl, optionally substituted aryl C2-C4 alkenyl, two R independently selected from the group consisting of optionally substituted aryl C2-C4 alkynyl, optionally substituted heteroaryl, optionally substituted heteroaryl C1-C4 alkyl, optionally substituted heteroaryl C1-C4 alkenyl, and optionally substituted heteroaryl C2-C4 alkynyl, and optionally bonded to a nitrogen atom; 7 The groups are joined together to form a 4- to 7-membered heterocyclic ring that is optionally fused to an aryl ring. [to achieve]. (Section 2) A, [ka] Item 1, or a pharmaceutically acceptable salt, hydrate, or solvate thereof. (Section 3) Het, [ka] Item 1. The compound according to item 1 above, selected from the group consisting of: (Section 4) formula: [ka] Item 1. The compound according to item 1, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, having the formula: (Section 5) R a However, NH2, NHR 7 , N.R. 7 R 7 , S.R. 7 , and OR 7 The above is selected from the group consisting of Item 5. The compound according to item 4, or a pharmaceutically acceptable salt, hydrate, or solvate thereof. (Section 6) R c But halogen, R 7 , OR 7 , S.R. 7 , SO2R 7 , -X 1 -NH2, -X 1 -NHR 7 , -X 1 -NR 7 R 7 , -X 1 -OH, -X 1 -OR 7 , -X 1 -SR 7 , and -X 1 -SO2R 7 from Item 5. The compound according to item 4 above, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, selected from the group consisting of: (Section 7) R eThe compound according to the above item 4, wherein is H, or a pharmaceutically acceptable salt or hydrate thereof; Or a solvate. (Section 8) formula: [ka] or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein Each R 1 is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted aryl -C(R 2 R 2 )-OC(O)-OR 3 are independently selected from the group consisting of , or two R 1 groups optionally joined to form a 5- to 7-membered ring; Each R 2 are independently selected from the group consisting of H and optionally substituted C1-C6 alkyl be; Each R 3 is selected from the group consisting of H, C1-C6 alkyl, and optionally substituted aryl Independently selected; R 5 is selected from the group consisting of H and optionally substituted C1-C6 alkyl; X is selected from the group consisting of O, CH2, and S; A is, [ka] each of which is selected from the group consisting of 1 to 5 R 6 optionally substituted with a substituent; where n is an integer from 0 to 3; Z is CH2, CHR 6 , N.R. 6 selected from the group consisting of: Each R 6are independently selected from the group consisting of H, CH3, OH, CN, F, optionally substituted C1-C6 alkyl, and OC(O)-C1-C6 alkyl; and optionally, two R on adjacent ring vertices 6 The groups are joined together to form at least one heterocyclic ring vertex. forming a 5-6 membered ring with atoms; Het is [ka] selected from the group consisting of where the wavy line indicates the point of attachment to the remainder of the compound, and where: R a are H, NH2, and NHR 7 , NHC(O)R 7 , N.R. 7 R 7 , R 7 , O.H., S.R. 7 , and OR 7 selected from the group consisting of: R b is H, halogen, NH2, NHR 7 , N.R. 7 R 7 , R 7 , OH, and OR 7 A group consisting of Selected from; R c and R d is H, halogen, haloalkyl, NH2, NHR 7 , N.R. 7 R 7 , R 7 , O.H. , OR 7 , S.R. 7 , SO2R 7 , -X 1 -NH2, -X 1 -NHR 7 , -X 1 -NR 7 R 7 , -X 1 -OH, -X 1 -OR 7 , -X 1 -SR 7 , and -X1 -SO2R 7 Independently selected from the group consisting of be; R e and R f are independently selected from the group consisting of H, halogen, and optionally substituted C1-C6 alkyl; each X 1 is a C1-C4 alkylene; and Each R 7 is an optionally substituted C1-C 10 Alkyl, optionally substituted C-C 10 Alkenyl, optionally substituted C-C 10 alkynyl, optionally substituted C3-C7 cycloalkyl, optionally substituted C3-C7 cycloalkylC1-C4 alkyl, two R independently selected from the group consisting of optionally substituted 4- to 7-membered cycloheteroalkyl, optionally substituted 4- to 7-membered cycloheteroalkyl C1-C4 alkyl, optionally substituted aryl, optionally substituted aryl C1-C4 alkyl, optionally substituted aryl C2-C4 alkenyl, optionally substituted aryl C2-C4 alkynyl, optionally substituted heteroaryl, optionally substituted heteroaryl C1-C4 alkyl, optionally substituted heteroaryl C1-C4 alkenyl, optionally substituted heteroaryl C2-C4 alkynyl, and optionally bonded to a nitrogen atom; 7 The groups are joined together to form a 4- to 7-membered heterocyclic ring that is optionally fused to an aryl ring. but; However, the compound may be such that the combination of X, A, and Het is: [ka] {where R g is H or two R g groups are linked to form an acetonide; and (i)R c and R e is hydrogen and R a-OEt, -OCH2Ph, -SCH2Ph, -NH2, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl -N-ethylamino, phenylamino, benzylamino, 1-phenylethylamino, 2-phenylethylamino, N-benzyl-N-ethylamino, N-benzyl-N-methylamino, dibenzylamino, 4-aminobenzylamino, 2-chlorobenzylamino, 3-chlorobenzylamino, 4-chlorobenzylamino, 4-hydroxybenzylamino, 4-methoxybenzylamino, 4-nitrobenzylamino, or 4-sulfamoylbenzylamino; or (ii)R c is hydrogen and R a is -NH2 and R e is bromo, chloro, aminomethyl, or thioethyl; or (iii)R c is hydrogen and R a is benzylamino, and R e is bromo; or (iv)R c is amino and R e is hydrogen, and R q is -NH2, dimethylamino, diethylamino, benzylamino, or N-benzyl-N-methylamino; or (v)R c is chloro and R e is hydrogen and R a is —NH2, benzylamino, 2-chlorobenzylamino, 1-phenylethylamino, (S)-1-phenylethylamino, (R)-1-phenylethylamino, or N-benzyl-N-methylamino; or (vi)R c is iodine and R e is hydrogen, and R a is -NH2, benzylamino, or N-benzyl-N-methylamino; or (vii)R a is amino and R e is hydrogen, and R cPiperazinyl, thioarylin cyclohexylethylthio} other than compounds that give the (Section 9) A is [ka] (This is 1 to 5 R 6 Item 9. The compound according to item 8, wherein: (Section 10) A, [ka] Item 10. The compound according to item 9, selected from the group consisting of: (Section 11) Het, [ka] Item 9. The compound according to item 8, wherein (Section 12) Het is a compound of the formula: [ka] Item 10. The compound according to item 9, having the formula: (Section 13) R c 13. The compound according to item 12, wherein is other than hydrogen. (Section 14) below: [ka] (where each R g are independently selected from the group consisting of H and C(O)—C1-C6 alkyl. 14. The compound according to claim 13, having a formula selected from the group consisting of: (Section 15) Item 15. The compound according to item 14, wherein X is oxygen. (Section 16) R e 16. The compound according to item 15, wherein is hydrogen. (Section 17) R g 17. The compound according to item 16, wherein is hydrogen. (Section 18) R 5 The compound according to item 8, wherein is H. (Section 19) R 5 The compound according to the above item 8, wherein is H and X is O. (Section 20) R 5 is H, X is O, and each R 1 The compound according to item 8, wherein is H. (Section 21) Het, [ka] The compound according to item 8, selected from: (Section 22) R 5 is H, X is O, and each R 1 22. The compound according to item 21, wherein is H. (Section 23) R 5 is H, X is O, and each R 1 is H and R e is H and R a However, NH2, NHR 7 , and N(R 7 22. The compound according to claim 21, wherein the compound is selected from the group consisting of: (Section 24) R 5 is H, X is O, and each R 1 is H and R e is H and R c is other than H and R a NHR 7 22. The compound according to item 21, wherein (Section 25) formula: [ka] Item 15. The compound according to item 14, having the formula: (Section 26) formula: [ka] Item 15. The compound according to item 14, having the formula: (Section 27) formula: [ka] 26. The compound according to item 25, having the formula: (Section 28) formula: [ka] 27. The compound according to item 26, having the formula: (Section 29) The compound according to item 8 above, selected from the compounds in Table 1. (Section 30) A pharmaceutical composition comprising the compound according to item 8 above and a pharmaceutically acceptable excipient. (Section 31) A method for treating a disease, disorder, or condition mediated at least in part by CD73, comprising administering to a subject in need thereof an effective amount of the compound described in paragraph 8 above. (Section 32) 32. The method according to claim 31, wherein the compound is administered in an amount effective to reverse or stop the progression of CD73-mediated immunosuppression. (Section 33) 32. The method according to claim 31, wherein the disease, disorder, or condition is cancer. (Section 34) The cancer may be of the prostate, colon, rectum, pancreas, cervix, stomach, endometrium, brain, liver, bladder, ovary, testicle, head, neck, skin (including melanoma and basal carcinoma), mesothelial lining ng), white blood cell (including lymphoma and leukemia), esophageal, breast, muscle, connective tissue, lung (including small cell lung cancer and non-small cell carcinoma), adrenal gland, thyroid, kidney, or bone cancer; or glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma (including Kaposi's sarcoma), choriocarcinoma, basal cell carcinoma of the skin, and testicular seminoma. (Section 35) 34. The method according to item 33, wherein the cancer is selected from the group consisting of melanoma, colon cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, leukemia, brain cancer, lymphoma, ovarian cancer, or Kaposi's sarcoma. (Section 36) The disease, disorder, or condition is selected from the group consisting of rheumatoid arthritis, renal failure, lupus, asthma, psoriasis, colitis, pancreatitis, allergies, fibrosis, anemia, fibromyalgia, Alzheimer's disease, congestive heart failure, stroke, aortic stenosis, arteriosclerosis, osteoporosis, Parkinson's disease, infections, Crohn's disease, and ulcerative colitis. 32. The method according to item 31, wherein the immune-related disease, disorder, or condition is selected from the group consisting of colitis, allergic contact dermatitis and other eczema, systemic sclerosis, and multiple sclerosis. (Section 37) A combination comprising a compound according to paragraph 8 above and at least one additional therapeutic agent. (Section 38) 38. The combination according to paragraph 37, wherein the at least one additional therapeutic agent is a chemotherapeutic agent, an immune and / or inflammatory modulating agent, an antihypercholesterolemic agent, or an anti-infective agent. (Section 39) 38. The combination of claim 37, wherein at least one additional therapeutic agent is an immune checkpoint inhibitor. (Section 40) A kit comprising the compound according to item 8 above and at least one additional therapeutic agent. (Section 41) 41. The kit according to item 40, wherein the at least one additional therapeutic agent is a chemotherapeutic agent, an immune and / or inflammatory modulating agent, a hypercholesterolemic agent, or an anti-infective agent. (Section 42) 41. The kit according to paragraph 40, wherein at least one additional therapeutic agent is an immune checkpoint inhibitor. (Section 43) A method for treating cancer in a subject, comprising administering to the subject effective amounts of the compound described in paragraph 8 above and an immune checkpoint inhibitor. (Section 44) Item 44. The method according to item 43, wherein the administration is carried out before, simultaneously with, or after radiation therapy. (Section 45) 44. The method according to claim 43, wherein the compound and the immune checkpoint inhibitor are administered in combination. (Section 46) 44. The method of claim 43, wherein the compound and the immune checkpoint inhibitor are administered sequentially. (Section 47) 44. The method according to claim 43, wherein the compound is administered after the immune checkpoint inhibitor. (Section 48) 44. The method according to claim 43, wherein the compound is administered before the immune checkpoint inhibitor. (Section 49) 44. The combination, kit, or method according to any one of paragraphs 39, 42, and 43, wherein the immune checkpoint inhibitor is selected from the group consisting of ipilimumab, nivolumab, and lambrolizumab. (Section 50) A method for treating cancer in a subject, comprising administering to the subject effective amounts of the compound described in paragraph 29 and an immune checkpoint inhibitor.

Claims

[Claim 1] The invention as set forth in the drawings.

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